U.S. patent application number 16/354873 was filed with the patent office on 2019-09-05 for peptidomimetic macrocycles and uses thereof.
The applicant listed for this patent is AILERON THERAPEUTICS, INC.. Invention is credited to Manuel Aivado, David Allen Annis, Yong Chang, Hubert Chen, Karen Olson, Chris Viau.
Application Number | 20190269753 16/354873 |
Document ID | / |
Family ID | 55582021 |
Filed Date | 2019-09-05 |
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United States Patent
Application |
20190269753 |
Kind Code |
A1 |
Chen; Hubert ; et
al. |
September 5, 2019 |
PEPTIDOMIMETIC MACROCYCLES AND USES THEREOF
Abstract
Methods for treating solid tumor, determined to lack a p53
deactivation mutation, in a subject are provided. Also provided are
peptidomimetic macrocycles for use in treatment of a solid tumor,
determined to lack a p53 deactivation mutation, in a subject.
Inventors: |
Chen; Hubert; (San Diego,
CA) ; Annis; David Allen; (Cambridge, MA) ;
Chang; Yong; (Acton, MA) ; Aivado; Manuel;
(Chester Springs, PA) ; Olson; Karen; (Waltham,
MA) ; Viau; Chris; (Mashpee, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AILERON THERAPEUTICS, INC. |
Watertown |
MA |
US |
|
|
Family ID: |
55582021 |
Appl. No.: |
16/354873 |
Filed: |
March 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14864801 |
Sep 24, 2015 |
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16354873 |
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62054861 |
Sep 24, 2014 |
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62213831 |
Sep 3, 2015 |
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62216670 |
Sep 10, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 19/08 20180101;
C12Q 1/6886 20130101; A61P 17/00 20180101; A61P 35/00 20180101;
A61P 21/00 20180101; C12Q 2600/156 20130101; A61P 1/16 20180101;
A61P 25/00 20180101; A61K 9/0019 20130101; A61K 38/12 20130101;
A61P 15/00 20180101; C12Q 2600/158 20130101 |
International
Class: |
A61K 38/12 20060101
A61K038/12; C12Q 1/6886 20060101 C12Q001/6886; A61K 9/00 20060101
A61K009/00 |
Claims
1. A method of treating a cancer in a subject in need thereof, the
method comprising: administering to the subject a
therapeutically-effective amount of a p53 activator, and a
therapeutically-effective amount of another anticancer agent;
wherein the p53 activator reduces a side effect associated with the
other anticancer agent.
2. The method of claim 1, wherein the therapeutically-effective
amount of the p53 activator and the therapeutically-effective
amount of the other anticancer agent synergize.
3. The method of claim 1, wherein the therapeutically-effective
amount of the other anticancer agent is lower than is a
therapeutically-effective amount of the other anticancer agent in
absence of the p53 activator.
4. The method of claim 1, wherein the side effect is toxicity.
5. The method of claim 1, wherein the p53 activator and the other
anticancer agent are administered in a common pharmaceutical
composition.
6. The method of claim 1, wherein the p53 activator and the other
anticancer agent are administered in separate pharmaceutical
compositions.
7. The method of claim 1, wherein the p53 activator and the other
anticancer agent are administered concurrently.
8. The method of claim 1, wherein the p53 activator and the other
anticancer agent are administered sequentially.
9. The method of claim 1, wherein the other anticancer agent is a
hormonal agent, aromatase inhibitor, selective estrogen receptor
modulator (SERM), estrogen receptor antagonist, chemotherapeutic
agent, microtubule disassembly blocker, antimetabolite,
topoisomerase inhibitor, DNA crosslinker or damaging agent,
anti-antigenic agent, VEGF antagonist, receptor antagonist,
integrin antagonist, vascular targeting agent (VTA), or vascular
disrupting agent (VDA).
10. The method of claim 1, wherein the other anticancer agent is a
class I (camptotheca) topoisomerase inhibitor, topotecan,
irinotecan, rubitecan, belotecan, a class II (podophyllum)
topoisomerase inhibitor, etoposide, VP-16, teniposide,
anthracyclines, doxorubicin, epirubicin, Doxil, aclarubicin,
amrubicin, daunorubicin, idarubicin, pirarubicin, valrubicin, and
zorubicin, anthracenediones, mitoxantrone, or pixantrone.
11. The method of claim 1, wherein the side effect is related to a
hematopoietic organ tissue.
12. The method of claim 1, wherein the side effect is
hematologic.
13. The method of claim 1, wherein the side effect is
thrombocytopenia, neutropenia, or febrile neutropenia.
14. The method of claim 1, wherein the p53 activator binds to MDM2
and/or MDMX.
15. The method of claim 1, wherein the p53 activator disrupts an
interaction between p53 and MDM2 and/or MDMX.
16. The method of claim 1, wherein the p53 activator is
administered in a dosage of about 0.5-20 mg per kilogram body
weight of the subject.
17. The method of claim 1, wherein the therapeutically-effective
amount of the p53 activator is about 0.04 mg, 0.08 mg, 0.16 mg,
0.32 mg, 0.64 mg, 1.28 mg, 3.56 mg, 7.12 mg, or 14.24 mg of the p53
activator per kilogram body mass of the subject.
18. The method of claim 1 wherein the administering is over a
period of 0.25-2.0 h.
19. The method of claim 1, wherein the p53 activator comprises an
amino acid sequence which is at least about 60% identical to an
amino acid sequence in any of Table 3, Table 3a, Table 3b, and
Table 3c, wherein the peptidomimetic macrocycle has the formula:
##STR00076## wherein: each A, C, D and E is independently an amino
acid; each B is independently an amino acid, ##STR00077##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or [--NH-L.sub.3-];
each R.sub.1 and R.sub.2 is independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or forms
a macrocycle-forming linker L' connected to the alpha position of
one of said D or E amino acids; each R.sub.3 independently is
hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or
heterocycloaryl, optionally substituted with R.sub.5; each L and L'
is independently a macrocycle-forming linker of the formula
-L.sub.1-L.sub.2-; each L.sub.1, L.sub.2, and L.sub.3 is
independently alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, cycloarylene,
heterocycloarylene, or [--R.sub.4--K--R.sub.4--].sub.n, each being
optionally substituted with R.sub.5; each R.sub.4 is independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene; each K is
independently O, S, SO, SO.sub.2, CO, CO.sub.2, or CONR.sub.3; each
R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.E, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent; each R.sub.6 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a
fluorescent moiety, a radioisotope or a therapeutic agent; each
R.sub.7 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue; each R.sub.8 is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or
heterocycloaryl, optionally substituted with R.sub.5, or part of a
cyclic structure with an E residue; each v is independently an
integer; each w is independently an integer from 3-1000; u is an
integer from 1-10; each x, y and z is independently an integer from
0-10; and each n is independently an integer from 1-5.
20. The method of claim 1, wherein the p53 activator induces cell
cycle arrest in the subject.
21. The method of claim 1, wherein the cancer is non-small cell
lung cancer.
Description
CROSS REFERENCE
[0001] This application is a Continuation Application of U.S.
application Ser. No. 14/864,801, filed Sep. 24, 2015, which claims
the benefit of U.S. Provisional Application No. 62/054,861, filed
Sep. 24, 2014, U.S. Provisional Application No. 62/213,831, filed
Sep. 3, 2015 and U.S. Provisional Application No. 62/216,670, filed
Sep. 10, 2015, each of which is incorporated herein by reference in
its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 22, 2015, is named 35224-804-302_SL.txt and is 1,199,023
bytes in size.
BACKGROUND OF THE INVENTION
[0003] The human transcription factor protein p53 induces cell
cycle arrest and apoptosis in response to DNA damage and cellular
stress, and thereby plays a critical role in protecting cells from
malignant transformation. The E3 ubiquitin ligase MDM2 (also known
as HDM2 or human double minute 2) negatively regulates p53 function
through a direct binding interaction that neutralizes the p53
transactivation activity, leads to export from the nucleus of p53
protein, and targets p53 for degradation via the
ubiquitylation-proteasomal pathway. Loss of p53 activity, either by
deletion, mutation, or MDM2 overexpression, is the most common
defect in human cancers. Tumors that express wild type p53 are
vulnerable to pharmacologic agents that stabilize or increase the
concentration of active p53. In this context, inhibition of the
activities of MDM2 has emerged as a validated approach to restore
p53 activity and resensitize cancer cells to apoptosis in vitro and
in vivo. MDMX (also known as MDM4, HDM4 or human double minute 4)
has more recently been identified as a similar negative regulator
of p53, and studies have revealed significant structural homology
between the p53 binding interfaces of MDM2 and MDMX.MDMX has also
been observed to be overexpressed in human tumors. The p53-MDM2 and
p53-MDMX protein-protein interactions are mediated by the same
15-residue alpha-helical transactivation domain of p53, which
inserts into hydrophobic clefts on the surface of MDM2 and MDMX.
Three residues within this domain of WT p53 (F19, W23, and L26) are
essential for binding to MDM2 and MDMX.
[0004] There remains a considerable need for methods for treating
solid tumor. Provided herein are compounds capable of binding to
and modulating the activity of p53, MDM2 and/or MDMX. Also provided
herein are pharmaceutical formulations comprising p53-based
peptidomimetic macrocycles that modulate an activity of p53. Also
provided herein are pharmaceutical formulations comprising
p53-based peptidomimetic macrocycles that inhibit the interactions
between p53, MDM2 and/or MDMX proteins.
[0005] Further, provided herein are methods for treating diseases
including but not limited to solid tumors and other
hyperproliferative diseases.
SUMMARY OF THE INVENTION
[0006] In one aspect, the disclosure provides a method of treating
solid tumor determined to lack a p53 deactivating mutation, in a
human subject, wherein the method comprises administering to the
human subject a therapeutically effective amount of a
peptidomimetic macrocycle or a pharmaceutically acceptable salt
thereof, wherein the peptidomimetic macrocycle binds to MDM2 and/or
MDMX proteins. In some embodiments, the peptidomimetic macrocycle
disrupts the interaction between p53 and MDM2 and MDMX.
[0007] In another aspect, the disclosure provides a method of
treating a solid tumor that lacks a p53 deactivating mutation, in a
human subject in need thereof wherein the method comprises
administering to the human subject a pharmaceutical composition
comprising a therapeutically effective amount of a peptidomimetic
macrocycle or a therapeutically equivalent amount of a
pharmaceutically acceptable salt thereof, wherein the
peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.
[0008] In another aspect, the disclosure provides a method of
treating a solid tumor that has a p53 deactivating mutation in a
p53 gene, in a human subject in need thereof wherein the method
comprises administering to the human subject a pharmaceutical
composition comprising a therapeutically effective amount of a
peptidomimetic macrocycle or a therapeutically equivalent amount of
a pharmaceutically acceptable salt thereof, wherein the
peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.
[0009] In another aspect, the disclosure provides a method of
treating a solid tumor in a human subject in need thereof, wherein
the method comprises administering to the human subject a
pharmaceutical composition comprising a therapeutically effective
amount of a peptidomimetic macrocycle or a therapeutically
equivalent amount of a pharmaceutically acceptable salt thereof,
wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX
proteins and wherein the solid tumor is not negative for p53
protein expression (such as solid tumors that express wild-type p53
protein or mutated p53 protein with partial functionality).
[0010] In another aspect, the disclosure provides a method of
treating a solid tumor in a human subject in need thereof, wherein
the method comprises administering to the human subject a
pharmaceutical composition comprising a therapeutically effective
amount of a peptidomimetic macrocycle or a therapeutically
equivalent amount of a pharmaceutically acceptable salt thereof,
wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX
proteins and wherein the solid tumor expresses a p53 protein with a
gain of function mutation (such as a super apoptotic p53).
[0011] In another aspect, the disclosure provides a method of
treating a solid tumor in a human subject in need thereof, wherein
the method comprises administering to the human subject a
pharmaceutical composition comprising a therapeutically effective
amount of a peptidomimetic macrocycle or a therapeutically
equivalent amount of a pharmaceutically acceptable salt thereof,
wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX
proteins and wherein the solid tumor express a p53 protein with a
mutation that causes a partial loss of function.
[0012] In another aspect, the disclosure provides a method of
treating a solid tumor a human subject in need thereof wherein the
method comprises administering to the human subject a
pharmaceutical composition comprising a therapeutically effective
amount of a peptidomimetic macrocycle or a therapeutically
equivalent amount of a pharmaceutically acceptable salt thereof,
wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX
proteins and wherein cells in the solid tumor express p53 from only
a single genomic copy of the p53 gene (for example where the cells
have a copy loss mutation, e.g., are haploinsufficient).
[0013] In another aspect, the disclosure provides a method of
treating a solid tumor a human subject in need thereof wherein the
method comprises administering to the human subject a
pharmaceutical composition comprising a therapeutically effective
amount of a peptidomimetic macrocycle or a therapeutically
equivalent amount of a pharmaceutically acceptable salt thereof,
wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX
proteins and wherein the solid tumor express a p53 protein with one
or more silent mutations.
[0014] In another aspect, the disclosure provides a method of
treating a solid tumor a human subject in need thereof wherein the
method comprises administering to the human subject a
pharmaceutical composition comprising a therapeutically effective
amount of a peptidomimetic macrocycle or a therapeutically
equivalent amount of a pharmaceutically acceptable salt thereof,
wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX
proteins and wherein cells in the solid tumor are negative for p53
expression.
[0015] In another aspect, the disclosure provides a method of
treating a solid tumor that has a p53 deactivating mutation in a
p53 gene, in a human subject in need thereof wherein the method
comprises administering to the human subject a pharmaceutical
composition comprising a therapeutically effective amount of a
peptidomimetic macrocycle or a therapeutically equivalent amount of
a pharmaceutically acceptable salt thereof, wherein the
peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins and
wherein cells in the solid tumor have the p53 deactivating mutation
in one copy of the p53 gene. In some embodiments, the cells in the
solid tumor have a second p53 deactivating mutation in a second
copy of a p53 gene. In some embodiments, the p53 deactivating
mutation in one copy of the p53 gene is the same as the second p53
deactivating mutation in the second copy of a p53 gene. In some
embodiments, the p53 deactivating mutation in one copy of the p53
gene is different from the second p53 deactivating mutation in the
second copy of a p53 gene.
[0016] In some embodiments, the p53 deactivating mutation in the
p53 gene results in the lack of p53 protein expression from the p53
gene or in expression of partial a p53 protein with partial loss of
function. In some embodiments, the second p53 deactivating mutation
in the second copy of a p53 gene results in the lack of p53 protein
expression from the p53 gene or in expression of partial a p53
protein with partial loss of function.
[0017] In some embodiments of the methods described herein, the
cells of the solid tumor have at least one mutation in a copy of a
p53 gene, wherein the mutation eliminates or reduces the or
activity of a p53 protein expressed from the copy of the p53 gene,
as compared to wild type p53 expressed from a copy of a non-mutated
p53 gene.
[0018] In another aspect, the disclosure provides a method of
treating a solid tumor in a human subject in need thereof wherein
the method comprises administering to the human subject a
pharmaceutical composition comprising a therapeutically effective
amount of a peptidomimetic macrocycle or a therapeutically
equivalent amount of a pharmaceutically acceptable salt thereof,
wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX
proteins.
[0019] In some embodiments, the peptidomimetic macrocycle used in
the various methods described herein is a peptidomimetic macrocycle
that disrupts the interaction between p53 and MDM2 and MDMX.
[0020] In some embodiments, the various methods described herein
further comprise determining the lack of the p53 deactivating
mutation in the solid tumor prior to the administration of the
pharmaceutical composition. In some embodiments, the determining
the lack of the p53 deactivating mutation comprises confirming the
presence of wild type p53 in the solid tumor.
[0021] In some embodiments, the various methods described herein
further comprise determining a presence of a p53 gain of function
mutation in the solid tumor.
[0022] In some embodiments, the various methods described herein
further comprise determining a presence of a deactivating mutation
of p53 in the solid tumor.
[0023] In some embodiments, the various methods described herein
further comprise determining a presence of a copy loss mutation of
p53 in the solid tumor.
[0024] In some embodiments, the various methods described herein
further comprise determining a presence of a partial loss of
function mutation of P53 in the solid tumor.
[0025] In some embodiments, the methods described herein can
further comprise confirming the lack of the p53 deactivating
mutation in the solid tumor, prior to the administration of the
peptidomimetic macrocycle. For example, confirming the presence of
wild type p53 in the solid tumor.
[0026] In some embodiments, the methods described herein can
further comprise confirming a presence of a p53 gain of function
mutation in the solid tumor.
[0027] In some embodiments, the methods described herein can
further comprise confirming a presence of a deactivating mutation
of p53 in the solid tumor.
[0028] In some embodiments, the methods described herein can
further comprise confirming a presence of a copy loss mutation of
p53 in the solid tumor.
[0029] In some embodiments, the methods described herein can
further comprise confirming a presence of a partial loss of
function mutation of P53 in the solid tumor.
[0030] In various embodiments, the determining or confirming is
performed within 3 years, 2 years, within 1 year, within 1-12
months, within 1-3 months, within 1 month, or within 21 days prior
to the administration of the peptidomimetic macrocycle.
[0031] In various embodiments, the treatment methods provided
herein can result in re-activation of the p53 pathway, decreased
tumor cell proliferation, increased p53 protein, increased p21,
and/or increased apoptosis in the human subject.
[0032] The peptidomimetic macrocycle can be administered two or
three times a week, for example two times a week. In some examples,
the peptidomimetic macrocycle is administered once every 2 or 3
weeks. In other examples the peptidomimetic macrocycle is
administered once every 1 or 2 weeks. In some embodiments, the
peptidomimetic macrocycle is administered on days 1, 8 and 15 of a
28-day cycle. In other examples the peptidomimetic macrocycle is
administered once every week. In some examples, a dose of the
pharmaceutical composition is administered on days 1, 4, 8 and 11
of a 21-day cycle.
[0033] The amount of the peptidomimetic macrocycle administered is
about 0.5-20 mg per kilogram body weight of the human subject, for
example 0.5-10 mg per kilogram body weight of the human subject. In
some embodiments, the amount of the peptidomimetic macrocycle
administered is about 0.04 mg, 0.08 mg, 0.16 mg, 0.32 mg, 0.64 mg,
1.28 mg, 3.56 mg, 7.12 mg, or 14.24 mg per kilogram body weight of
the human subject. In some examples, the amount of the
peptidomimetic macrocycle administered is about 1.25 mg, 2.5 mg,
5.0 mg 10.0 mg, or 20.0 mg per kilogram body weight of the human
subject and the peptidomimetic macrocycle is administered two times
a week. In other examples, the amount of the peptidomimetic
macrocycle administered is about 1.25 mg, 2.5 mg, 5.0 mg or 10.0 mg
per kilogram body weight of the human subject and the
peptidomimetic macrocycle is administered two times a week. In
other examples, 0.32 mg, 0.64 mg, 1.25 mg 2.5 or 5.0 mg per
kilogram body weight of the human subject and the pharmaceutical
composition is administered two times a week. In some examples,
about 0.32 mg, 0.64 mg, 1.25 mg 2.5 or 5.0 mg per kilogram body
weight of the human subject and the pharmaceutical composition is
administered on day 1, 4, 8, 11 of a 21-day cycle. In some
examples, about 0.16 mg, 0.32 mg, 0.64 mg, 1.25 mg, 2.5, 5.0 mg or
10 mg per kilogram body weight of the human subject and the
pharmaceutical composition is administered on day 1, 8, and 15 of a
28 day cycle.
[0034] In other examples, the amount of the peptidomimetic
macrocycle administered is about 1.25 mg, 2.5 mg, 5.0 mg, 10.0 mg,
or 20.0 mg per kilogram body weight of the human subject and the
peptidomimetic macrocycle is administered once a week. In some
examples, the amount of the peptidomimetic macrocycle administered
is about 1.25 mg, 2.5 mg, 5.0 mg or 10.0 mg per kilogram body
weight of the human subject and the peptidomimetic macrocycle is
administered once a week.
[0035] In some examples, the amount of the peptidomimetic
macrocycle administered is about 1.25 mg, 2.5 mg, 5.0 mg 10.0 mg,
or 20.0 mg per kilogram body weight of the human subject and the
peptidomimetic macrocycle is administered once daily, three, five
or seven times in a seven day period. For example, the
peptidomimetic macrocycle is administered once daily intravenously,
seven times in a seven day period.
[0036] In some examples, the amount of the peptidomimetic
macrocycle administered is about 1.25 mg, 2.5 mg, 5.0 mg, or 10.0
mg per kilogram body weight of the human subject and the
peptidomimetic macrocycle is administered once daily three, five or
seven times in a seven day period. For example, the peptidomimetic
macrocycle is administered once daily intravenously, seven times in
a seven day period.
[0037] The peptidomimetic macrocycle can be administered gradually
over a period of 0.25-12 h, for example over a period of 0.25 h,
0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, or
12 h. In some examples, the peptidomimetic macrocycle is
administered over a period of 0.25-2.0 h. In some embodiments, the
peptidomimetic macrocycle is gradually administered over a period
of 1 h. In other embodiments, the peptidomimetic macrocycle is
gradually administered over a period of 2 h.
[0038] The method provided herein can result in reduction of tumor
volume. For example, treatment according to methods provided herein
can result in about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,
50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% reduction in
tumor volume within a period of 1 month after treatment initiation.
In some example, the treatment results in at least 60%, 65%, 70%,
75%, 80%, 85%, 90%, or 95% reduction in tumor volume within a
period of 1 month after treatment initiation. In some examples, the
treatment results in about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%,
55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% reduction
in tumor volume within a period of 1 year after treatment
initiation. In some embodiments, the treatment results in at least
60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% reduction in tumor volume
within a period of 1 year after treatment initiation. In some
examples, the treatment results in about 95%, 90%, 85%, 80%, 75%,
70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or
5% reduction in tumor volume within a period of 6 months after
treatment initiation. In some examples, the treatment results in at
least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% reduction in tumor
volume within a period of 6 months after treatment initiation. In
some examples, the treatment results in about 95%, 90%, 85%, 80%,
75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,
10%, or 5% reduction in tumor volume within a period of 3 months
after treatment initiation. In some examples, the treatment results
in at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% reduction in
tumor volume within a period of 3 months after treatment
initiation. In some embodiments, the solid tumor is a stable
disease. In some embodiments, the solid tumor is a progressive
disease.
[0039] In some embodiments, the methods provided herein can result
in an increased survival time of the human subject as compared to
the expected survival time of the human subject if the human
subject was not treated with the peptidomimetic macrocycle. In some
examples, the increase in the survival time of the human subject is
at least 30 days, at least 3 months, at least 6 months or at least
1 year.
[0040] The in vivo circulating half-life of the peptidomimetic
macrocycle is about 1 h-12 h, for example about 1 h, 2 h, 3 h, 4 h,
5 h, 6 h, 7 h, 8 h, 9 h, 10 h or 12 h. In some examples, the in
vivo circulating half-life of the peptidomimetic macrocycle is
about 4 h, about 6 h.
[0041] The biological tissue half-life of the peptidomimetic
macrocycle is about 1 h-12 h, for example about 1 h, 2 h, 3 h, 4 h,
5 h, 6 h, 7 h, 8 h, 9 h, 10 h or 12 h. In some examples, the
biological tissue half-life of the peptidomimetic macrocycle is
about 10 h.
[0042] In some embodiments, the human subject treated in accordance
to the methods of the disclosure is refractory and/or intolerant to
one or more other treatment of the solid tumor. In some
embodiments, the human subject has had at least one unsuccessful
prior treatment and/or therapy of the solid tumor.
[0043] In some embodiments, the solid tumor expresses wild-type p53
protein.
[0044] The solid tumor treated by the methods of the disclosure is
selected from a group consisting of pancreatic cancer, bladder
cancer, colon cancer, liver cancer, colorectal cancer, breast
cancer, prostate cancer, renal cancer, hepatocellular cancer, lung
cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal
cancer, head and neck cancer, melanoma, neuroendocrine cancers, CNS
cancers, brain tumors, bone cancer, skin cancer, ocular tumor,
rectal cancer, choriocarcinoma (tumor of the placenta), sarcoma and
soft tissue cancer, testicular cancer, gall bladder cancer, and
biliary cancer. In some embodiments, the solid tumor is selected
from a group consisting of bladder cancer, bone cancer, breast
cancer, cervical cancer, CNS cancer, colon cancer, ocular tumor,
renal cancer, liver cancer, lung cancer, pancreatic cancer,
choriocarcinoma (tumor of the placenta), prostate cancer, sarcoma,
skin cancer, soft tissue cancer, gastric cancer, gall bladder
cancer, biliary cancer, renal cancer, or neuroendocrine cancer. The
ocular tumor can be choroidal nevus, choroidal melanoma, choroidal
metastasis, choroidal hemangioma, choroidal osteoma, iris melanoma,
uveal melanoma, melanocytoma, metastasis retinal capillary
hemangiomas, congenital hypertrophy of the RPE, RPE adenoma or
retinoblastoma. In some embodiments, the solid tumor is selected
from non-small cell lung cancer, small-cell lung cancer, colon
cancer, CNS cancer, melanoma, ovarian cancer, renal cancer,
prostate cancer and breast cancer. In some embodiments, the solid
tumor is breast cancer. In some embodiments, the solid tumor is
gall bladder cancer. In some embodiments, the solid tumor is the
solid tumor is biliary cancer. In some embodiments, the solid tumor
is neuroendocrine cancer. In some embodiments, the solid tumor is
bone cancer. In some embodiments, the solid tumor is osteosarcoma.
In some embodiments, the solid tumor is skin cancer. In some
embodiments, the solid tumor is melanoma.
[0045] In some examples, the solid tumor treated by the methods of
the disclosure is not a HPV positive cancer.
[0046] In some examples, the solid tumor treated by the methods of
the disclosure is not HPV positive cervical cancer, HPV positive
anal cancer or HPV positive head and neck cancer, such as
oropharyngeal cancers.
[0047] In some embodiments, the peptidomimetic macrocycle is
administered intravenously.
[0048] In some embodiments, the methods of the disclosure further
comprise administering in addition to the peptidomimetic macrocycle
or pharmaceutically acceptable salt thereof, a therapeutically
effective amount of at least one additional therapeutic agent
and/or therapeutic procedure to the human subject.
[0049] In some embodiments, the human subject exhibits a complete
response to the treatment. In some embodiments, the human subject
exhibits a partial response to the treatment.
[0050] In some embodiments, the methods of the disclosure further
comprise determining clinical activity of the administered
peptidomimetic macrocycle. The clinical activity can be determined
by an imaging method selected from a group consisting of computed
tomography (CT), magnetic resonance imaging (MRI), and bone
scanning.
[0051] The methods of the disclosure can further comprise obtaining
a biological sample from the human subject at one or more specific
time-points and analyzing the biological sample with an analytical
procedure. The biological samples can be used for biomarker
assessment, pharmacokinetic assessment, immunogenicity assays
and/or pharmacodynamic assessment. The pharmacokinetic assessment
can comprise studying the level of the peptidomimetic macrocycle
and/or its metabolites in the biological sample at the specific
time-points. The pharmacodynamic assessment can comprise studying
the level of p53, MDM2, MDMX, p21 and/or caspase in the biological
sample at the specific time-points.
[0052] The analytical procedure can be selected from a group
comprising blood chemistry analysis, chromosomal translocation
analysis, needle biopsy, tissue biopsy, fluorescence in situ
hybridization, laboratory biomarker analysis, immunohistochemistry
staining method, flow cytometry, or a combination thereof. The
methods can further comprise tabulating and/or plotting results of
the analytical procedure. The one or more specific time-points can
comprise a time-point before the administration of the
peptidomimetic macrocycle to the human subject. The one or more
specific time-points can comprise a time-point after the
administration of the peptidomimetic macrocycle to the human
subject. The one or more specific time-points can comprise a
time-point before and a time-point after the administration of the
peptidomimetic macrocycle to the human subject. The one or more
specific time-points comprise multiple time-points before and after
the administration of the peptidomimetic macrocycle to the human
subject. The method can further comprise comparing the biological
samples collected before and after the administration of the
peptidomimetic macrocycle to the human subject or comparing the
biological samples collected at the multiple time-points. The
biological sample can be a blood sample or a tumor specimen.
[0053] The method of the disclosure can further comprise selecting
and/or identifying at least one target lesion in the human subject
prior to the administration of the peptidomimetic macrocycle to the
human subject. The methods can also comprise measuring cumulative
diameters at one or more specific time-points, wherein the
cumulative diameter is a sum of the diameters of the at least one
target lesions at the specific time-point. The one or more specific
time-points, can comprise a time-point after the treatment. The
methods can also comprise measuring a baseline sum diameter,
wherein the baseline sum diameter is a sum of the diameters of the
at least one target lesions prior to the administration of the
pharmaceutical composition to the human subject. In some examples,
the treatment in accordance to the methods of the disclosure
results in disappearance of the least one target lesion. In some
embodiments, after the treatment all pathological lymph nodes in
the human subject exhibit a reduction in short axis to less than 10
mm. In some examples, the cumulative diameter at the time-point
after the treatment is at least 30% less than the baseline sum
diameter. In some examples, the treatment results in neither a
sufficient increase nor a sufficient decrease in the cumulative
diameters at the one or more specific time-points, taking as
reference the baseline sum diameter.
[0054] In some examples, the peptidomimetic macrocycle is not an
inhibitor of cytochrome P450 isoforms. In some examples, the
treatment results in essentially no dose-limiting thrombocytopenia.
In some examples, the treatment causes essentially no adverse
effects in a normal-hematopoietic organ and/or tissue. In some
examples, the treatment results in essentially no adverse event in
the human subject that can be possibly, probably, or definitely
related to the administration of the peptidomimetic macrocycle. In
some examples, the treatment results in essentially no serious
adverse event in the human subject that can be probably, probably,
or definitely related to the administration of the peptidomimetic
macrocycle.
[0055] The lack of p53 deactivation mutation can be determined by
any known method known in the art. In some examples, the lack of
p53 deactivation mutation can be determined by DNA sequencing of
the nucleic acid encoding the p53 protein. In some examples, the
lack of p53 deactivation mutation can be determined by RNA array
based testing. In some examples, the lack of p53 deactivation
mutation can be determined by RNA analysis. In some examples, the
lack of p53 deactivation mutation can be determined by polymerase
chain reaction (PCR).
[0056] In some embodiments, the p53 deactivating mutation can
comprise mutations in DNA-binding domain of the protein. In some
embodiments, the p53 deactivating mutation can comprise missense
mutation. In some embodiments, the p53 deactivating mutation is a
dominant deactivating mutation. In some embodiments, the p53
deactivating mutation comprises one or more mutations selected from
a groups consisting of V173L, R175H, G245C, R248W, R249S and R273H.
In some embodiments, the p53 deactivating mutation comprises one or
more of mutations shown in Table 1a. In some embodiments, the p53
gain of function mutation comprises one or more of mutations shown
in Table 1b.
[0057] In another aspect, the disclosure provides a method of
treating solid tumor in a human subject determined to lack a p53
deactivating mutation, wherein the method comprises administering
to the human subject 0.5-20 mg, for example 0.5-10 mg of a
peptidomimetic macrocycle per kilogram body weight of the human
subject or a pharmaceutically acceptable salt thereof on days 1, 8
and 15 of a 28-day cycle. In some embodiments, the amount of the
peptidomimetic macrocycle entered on day 8 and/or day 15 is greater
than the amount of the peptidomimetic macrocycle entered on day 1.
In some embodiments, the peptidomimetic macrocycle entered on day 8
and/or day 15 is equal than the amount of the peptidomimetic
macrocycle entered on day 1. In some embodiments, t the
peptidomimetic macrocycle entered on day 1 and/or day 8 is greater
than the amount of the peptidomimetic macrocycle entered on day 15.
In some embodiments, an equal amount of the peptidomimetic
macrocycle is administered on days 1, 8 and 15. In some
embodiments, the 28-day cycle is repeated 2 or 3 times.
[0058] In another aspect, the disclosure provides a method of
treating solid tumor in a human subject, wherein the method
comprises administering to the human subject 0.32-10 mg of a
peptidomimetic macrocycle per kilogram body weight of the human
subject or a pharmaceutically acceptable salt thereof on days 1, 4,
8 and 11 of a 21-day cycle. In some embodiments, the solid tumor is
determined to lack a p53 deactivating mutation. In some
embodiments, 0.32 mg of the peptidomimetic macrocycle per kilogram
body weight of the human subject or the pharmaceutically acceptable
salt thereof is administered on days 1, 4, 8 and 11 respectively of
a 21-day cycle. In some embodiments, 0.64 mg of the peptidomimetic
macrocycle per kilogram body weight of the human subject or the
pharmaceutically acceptable salt thereof is administered on days 1,
4, 8 and 11 respectively of a 21-day cycle. In some embodiments,
1.25 mg of the peptidomimetic macrocycle per kilogram body weight
of the human subject or the pharmaceutically acceptable salt
thereof is administered on days 1, 4, 8 and 11 respectively of a
21-day cycle. In some embodiments, 2.5 mg of the peptidomimetic
macrocycle per kilogram body weight of the human subject or the
pharmaceutically acceptable salt thereof is administered on days 1,
4, 8 and 11 respectively of a 21-day cycle. In some embodiments,
5.0 mg of the peptidomimetic macrocycle per kilogram body weight of
the human subject or the pharmaceutically acceptable salt thereof
is administered on days 1, 4, 8 and 11 respectively of a 21-day
cycle.
[0059] In various embodiments, the peptidomimetic macrocycle used
in the methods described herein comprises an amino acid sequence
which is at least about 60%, 70%, 80%, 90%, or 95% identical to an
amino acid sequence in any of Table 3, Table 3a, Table 3b, and
Table 3c, wherein the peptidomimetic macrocycle has the
formula:
##STR00001##
wherein: each A, C, D and E is independently an amino acid; each B
is independently an amino acid,
##STR00002##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or [--NH-L.sub.3-];
each R.sub.1 and R.sub.2 is independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or forms
a macrocycle-forming linker L' connected to the alpha position of
one of said D or E amino acids; each R.sub.3 independently is
hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or
heterocycloaryl, optionally substituted with R.sub.5; each L and L'
is independently a macrocycle-forming linker of the formula
-L.sub.1-L.sub.2-; each L.sub.1, L.sub.2, and L.sub.3 is
independently alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, cycloarylene,
heterocycloarylene, or [--R.sub.4--K--R.sub.4--].sub.n, each being
optionally substituted with R.sub.5; each R.sub.4 is independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene; each K is
independently O, S, SO, SO.sub.2, CO, CO.sub.2, or CONR.sub.3; each
R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent; each R.sub.6 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a
fluorescent moiety, a radioisotope or a therapeutic agent; each
R.sub.7 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue; each R.sub.8 is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or
heterocycloaryl, optionally substituted with R.sub.5, or part of a
cyclic structure with an E residue; each v is independently an
integer; each w is independently an integer from 3-1000; u is an
integer from 1-10; each x, y and z is independently an integer from
0-10; and each n is independently an integer from 1-5.
[0060] In various embodiments, the peptidomimetic macrocycle used
in the methods described herein has formula:
##STR00003##
wherein: each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-Hiss-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.sub.10-
-X.sub.11-Ser.sub.12 (SEQ ID NO: 8) or
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 9), where each
X.sub.4 and X.sub.11 is independently an amino acid; each D and E
is independently an amino acid; each R.sub.1 and R.sub.2 are
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or
substituted with halo-; or forms a macrocycle-forming linker L'
connected to the alpha position of one of said D or E amino acids;
each L or L' is independently a macrocycle-forming linker each
R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent; each R.sub.6 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a
fluorescent moiety, a radioisotope or a therapeutic agent; each
R.sub.7 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue; each R.sub.8 is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or
heterocycloaryl, optionally substituted with R.sub.5, or part of a
cyclic structure with an E residue; v is an integer from 1-1000; w
is an integer from 0-1000.
[0061] In some embodiments, at least one of the macrocycle-forming
linker in the Formulas described herein has a formula
-L.sub.1-L.sub.2-, wherein
each L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5; each R.sub.4 is independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
arylene, or heteroarylene; each K is independently O, S, SO,
SO.sub.2, CO, CO.sub.2, or CONR.sub.3; each R.sub.3 is
independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5;
and each n is independently an integer from 1-5.
[0062] In some embodiments, at least one of the macrocycle-forming
linker in the Formulas described herein each w is independently an
integer from 3-1000, for example 3-500, 3-200, 3-100, 3-50, 3-30,
3-20, or 3-10.
[0063] In some embodiments, Xaa5 is Glu or an amino acid analog
thereof.
[0064] In some embodiments, each E is independently Ala (alanine),
Ser (serine) or an analog thereof.
[0065] In some embodiments, [D]v is -Leu.sub.1-Thr.sub.2.
[0066] In some embodiments, w is 3-10. In some embodiments, w is
3-6. In some embodiments, w is 6-10. In some embodiments, w is
6.
[0067] In some embodiments, v is 1-10. In some embodiments, v is
2-10. In some embodiments, v is 2-5. In some embodiments, v is
2.
[0068] In some embodiments, each L.sub.1, L.sub.2 and L.sub.3 in
the Formulas described herein are independently alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, or heterocycloarylene, each
being optionally substituted with R.sub.5.
[0069] In some embodiments, each L.sub.1, L.sub.2 and L.sub.3 are
independently alkylene or alkenylene.
[0070] In some embodiments, L is alkylene, alkenylene, or
alkynylene. In some embodiments, L is alkylene. In some
embodiments, L is C3-C16 alkylene. In some embodiments, L is
C10-C14 alkylene.
[0071] In some embodiments, each R.sub.1 and R.sub.2 in the
Formulas described herein are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-. In some
embodiments, R.sub.1 and R.sub.2 are H. In some embodiments, each
R.sub.1 and R.sub.2 are independently alkyl. In some embodiments,
R.sub.1 and R.sub.2 are methyl.
[0072] In some embodiments, x+y+z in the Formulas described herein
is 6.
[0073] In some embodiments, u in the Formulas described herein is
1.
[0074] The some embodiments, peptidomimetic macrocycles comprise at
least one amino acid which is an amino acid analog. In some
embodiments, the peptidomimetic macrocycle is chosen from the
peptidomimetic macrocycles shown in Table 3c.
[0075] In one aspect, the disclosure provides a method of
identifying one or more solid tumor biomarkers in a human subject
lacking a p53 deactivating mutation, comprising administering to
the human subject a therapeutically effective amount of a
peptidomimetic macrocycle described herein. In some examples, the
biomarkers are selected from a group comprising p53 status, MDM2
expression level and MDMX expression level.
[0076] In the various embodiments, the pharmaceutical composition
comprises the pharmaceutically acceptable salt of the
peptidomimetic macrocycle. In some embodiments, the
pharmaceutically acceptable salt is a sodium salt, potassium salt
or calcium salt. In some embodiments, the pharmaceutically
acceptable salt is a sodium salt.
INCORPORATION BY REFERENCE
[0077] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0078] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0079] FIG. 1. Shows human wild type P53 protein sequence (SEQ ID
NO: 1501).
[0080] FIG. 2. Shows an exemplary dose level and Dose Regimen.
[0081] FIG. 3. Shows an exemplary dosing overview.
[0082] FIG. 4. Shows the amount of Aileron peptide-1 administered
for each dose level (DL) and dose regimen.
[0083] FIG. 5. Shows an exemplary dose escalation strategy of the
disclosure.
[0084] FIG. 6. Shows one way Aileron peptide-1 was designed to
inhibit both MDMX and MDM2, to reactivate WT p53.
[0085] FIG. 7. Shows potential indications of Aileron peptide-1
(from orphan indications or large market opportunities).
[0086] FIG. 8. Shows the effect of Aileron peptide-1 across a
variety of different cancers.
[0087] FIG. 9. Shows the effect of Aileron peptide-1 administered
by an intravenous, or IV, injection in an MDMX-driven MCF-7 breast
cancer xenograft model
[0088] FIG. 10. Shows dose escalation based on a "3+3" dose
escalation design.
[0089] FIGS. 11a and 11b Show drug concentration (measured or
projected) in dose levels for Cohorts.
[0090] FIG. 12. Shows the pharmacokinetic model of Aileron
peptide-1 which shows a 2-compartment, parallel nonlinear
Michaelis-Menten clearance and linear elimination.
[0091] FIG. 13. Shows dose dependent increase of MIC-1.
[0092] FIG. 14. Shows that patients who have completed at least two
cycles of treatment have stable disease. Aileron peptide-1 shows a
stable disease rate.
[0093] FIG. 15. Shows that Aileron peptide 1 shows on target
activation of p21 and p53 in patient blood cells.
DETAILED DESCRIPTION OF THE INVENTION
[0094] While preferred embodiments of the present disclosure have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
disclosure. It should be understood that various alternatives to
the embodiments of the disclosure described herein can be employed
in practicing the disclosure. It is intended that the following
claims define the scope of the disclosure and that methods and
structures within the scope of these claims and their equivalents
be covered thereby.
Definitions
[0095] As used herein, the term "macrocycle" refers to a molecule
having a chemical structure including a ring or cycle formed by at
least 9 covalently bonded atoms.
[0096] As used herein, the term "peptidomimetic macrocycle" or
"crosslinked polypeptide" refers to a compound comprising a
plurality of amino acid residues joined by a plurality of peptide
bonds and at least one macrocycle-forming linker which forms a
macrocycle between a first naturally-occurring or
non-naturally-occurring amino acid residue (or analog) and a second
naturally-occurring or non-naturally-occurring amino acid residue
(or analog) within the same molecule. Peptidomimetic macrocycle
include embodiments where the macrocycle-forming linker connects
the .alpha. carbon of the first amino acid residue (or analog) to
the .alpha. carbon of the second amino acid residue (or analog).
The peptidomimetic macrocycles optionally include one or more
non-peptide bonds between one or more amino acid residues and/or
amino acid analog residues, and optionally include one or more
non-naturally-occurring amino acid residues or amino acid analog
residues in addition to any which form the macrocycle. A
"corresponding uncrosslinked polypeptide" when referred to in the
context of a peptidomimetic macrocycle is understood to relate to a
polypeptide of the same length as the macrocycle and comprising the
equivalent natural amino acids of the wild-type sequence
corresponding to the macrocycle.
[0097] As used herein, the term "helical stability" refers to the
maintenance of a helical structure by a peptidomimetic macrocycle
as measured by circular dichroism or NMR. For example, in some
embodiments, a peptidomimetic macrocycle exhibits at least a 1.25,
1.5, 1.75 or 2-fold increase in .alpha.-helicity as determined by
circular dichroism compared to a corresponding uncrosslinked
macrocycle.
[0098] The term "amino acid" refers to a molecule containing both
an amino group and a carboxyl group. Suitable amino acids include,
without limitation, both the D- and L-isomers of the
naturally-occurring amino acids, as well as non-naturally occurring
amino acids prepared by organic synthesis or other metabolic
routes. The term amino acid, as used herein, includes, without
limitation, .alpha.-amino acids, natural amino acids, non-natural
amino acids, and amino acid analogs.
[0099] The term ".alpha.-amino acid" refers to a molecule
containing both an amino group and a carboxyl group bound to a
carbon which is designated the .alpha.-carbon.
[0100] The term ".beta.-amino acid" refers to a molecule containing
both an amino group and a carboxyl group in a 3 configuration.
[0101] The term "naturally occurring amino acid" refers to any one
of the twenty amino acids commonly found in peptides synthesized in
nature, and known by the one letter abbreviations A, R, N, C, D, Q,
E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
[0102] The following table shows a summary of the properties of
natural amino acids:
TABLE-US-00001 3- 1- Side- Side-chain Letter Letter chain charge
Hydropathy Amino Acid Code Code Polarity (pH 7.4) Index Alanine Ala
A nonpolar neutral 1.8 Arginine Arg R polar positive -4.5
Asparagine Asn N polar neutral -3.5 Aspartic acid Asp D polar
negative -3.5 Cysteine Cys C polar neutral 2.5 Glutamic acid Glu E
polar negative -3.5 Glutamine Gln Q polar neutral -3.5 Glycine Gly
G nonpolar neutral -0.4 Histidine His H polar positive(10%) -3.2
neutral(90%) Isoleucine Ile I nonpolar neutral 4.5 Leucine Leu L
nonpolar neutral 3.8 Lysine Lys K polar positive -3.9 Methionine
Met M nonpolar neutral 1.9 Phenylalanine Phe F nonpolar neutral 2.8
Proline Pro P nonpolar neutral -1.6 Serine Ser S polar neutral -0.8
Threonine Thr T polar neutral -0.7 Tryptophan Trp W nonpolar
neutral -0.9 Tyrosine Tyr Y polar neutral -1.3 Valine Val V
nonpolar neutral 4.2
[0103] "Hydrophobic amino acids" include small hydrophobic amino
acids and large hydrophobic amino acids. "Small hydrophobic amino
acid" are glycine, alanine, proline, and analogs thereof. "Large
hydrophobic amino acids" are valine, leucine, isoleucine,
phenylalanine, methionine, tryptophan, and analogs thereof. "Polar
amino acids" are serine, threonine, asparagine, glutamine,
cysteine, tyrosine, and analogs thereof. "Charged amino acids" are
lysine, arginine, histidine, aspartate, glutamate, and analogs
thereof.
[0104] The term "amino acid analog" refers to a molecule which is
structurally similar to an amino acid and which can be substituted
for an amino acid in the formation of a peptidomimetic macrocycle.
Amino acid analogs include, without limitation, .beta.-amino acids
and amino acids where the amino or carboxy group is substituted by
a similarly reactive group (e.g., substitution of the primary amine
with a secondary or tertiary amine, or substitution of the carboxy
group with an ester).
[0105] The term "non-natural amino acid" refers to an amino acid
which is not one of the twenty amino acids commonly found in
peptides synthesized in nature, and known by the one letter
abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W,
Y and V. Non-natural amino acids or amino acid analogs include,
without limitation, structures according to the following:
##STR00004## ##STR00005## ##STR00006## ##STR00007## ##STR00008##
##STR00009##
[0106] Amino acid analogs include .beta.-amino acid analogs.
Examples of .beta.-amino acid analogs include, but are not limited
to, the following: cyclic .beta.-amino acid analogs;
.beta.-alanine; (R)-.beta.-phenylalanine;
(R)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid;
(R)-3-amino-4-(1-naphthyl)-butyric acid;
(R)-3-amino-4-(2,4-dichlorophenyl)butyric acid;
(R)-3-amino-4-(2-chlorophenyl)-butyric acid;
(R)-3-amino-4-(2-cyanophenyl)-butyric acid;
(R)-3-amino-4-(2-fluorophenyl)-butyric acid;
(R)-3-amino-4-(2-furyl)-butyric acid;
(R)-3-amino-4-(2-methylphenyl)-butyric acid;
(R)-3-amino-4-(2-naphthyl)-butyric acid;
(R)-3-amino-4-(2-thienyl)-butyric acid;
(R)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid;
(R)-3-amino-4-(3,4-dichlorophenyl)butyric acid;
(R)-3-amino-4-(3,4-difluorophenyl)butyric acid;
(R)-3-amino-4-(3-benzothienyl)-butyric acid;
(R)-3-amino-4-(3-chlorophenyl)-butyric acid;
(R)-3-amino-4-(3-cyanophenyl)-butyric acid;
(R)-3-amino-4-(3-fluorophenyl)-butyric acid;
(R)-3-amino-4-(3-methylphenyl)-butyric acid;
(R)-3-amino-4-(3-pyridyl)-butyric acid;
(R)-3-amino-4-(3-thienyl)-butyric acid;
(R)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid;
(R)-3-amino-4-(4-bromophenyl)-butyric acid;
(R)-3-amino-4-(4-chlorophenyl)-butyric acid;
(R)-3-amino-4-(4-cyanophenyl)-butyric acid;
(R)-3-amino-4-(4-fluorophenyl)-butyric acid;
(R)-3-amino-4-(4-iodophenyl)-butyric acid;
(R)-3-amino-4-(4-methylphenyl)-butyric acid;
(R)-3-amino-4-(4-nitrophenyl)-butyric acid;
(R)-3-amino-4-(4-pyridyl)-butyric acid;
(R)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid;
(R)-3-amino-4-pentafluoro-phenylbutyric acid;
(R)-3-amino-5-hexenoic acid; (R)-3-amino-5-hexynoic acid;
(R)-3-amino-5-phenylpentanoic acid; (R)-3-amino-6-phenyl-5-hexenoic
acid; (S)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid;
(S)-3-amino-4-(1-naphthyl)-butyric acid;
(S)-3-amino-4-(2,4-dichlorophenyl)butyric acid;
(S)-3-amino-4-(2-chlorophenyl)-butyric acid;
(S)-3-amino-4-(2-cyanophenyl)-butyric acid;
(S)-3-amino-4-(2-fluorophenyl)-butyric acid;
(S)-3-amino-4-(2-furyl)-butyric acid;
(S)-3-amino-4-(2-methylphenyl)-butyric acid;
(S)-3-amino-4-(2-naphthyl)-butyric acid;
(S)-3-amino-4-(2-thienyl)-butyric acid;
(S)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid;
(S)-3-amino-4-(3,4-dichlorophenyl)butyric acid;
(S)-3-amino-4-(3,4-difluorophenyl)butyric acid;
(S)-3-amino-4-(3-benzothienyl)-butyric acid;
(S)-3-amino-4-(3-chlorophenyl)-butyric acid;
(S)-3-amino-4-(3-cyanophenyl)-butyric acid;
(S)-3-amino-4-(3-fluorophenyl)-butyric acid;
(S)-3-amino-4-(3-methylphenyl)-butyric acid;
(S)-3-amino-4-(3-pyridyl)-butyric acid;
(S)-3-amino-4-(3-thienyl)-butyric acid;
(S)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid;
(S)-3-amino-4-(4-bromophenyl)-butyric acid;
(S)-3-amino-4-(4-chlorophenyl)-butyric acid;
(S)-3-amino-4-(4-cyanophenyl)-butyric acid;
(S)-3-amino-4-(4-fluorophenyl)-butyric acid;
(S)-3-amino-4-(4-iodophenyl)-butyric acid;
(S)-3-amino-4-(4-methylphenyl)-butyric acid;
(S)-3-amino-4-(4-nitrophenyl)-butyric acid;
(S)-3-amino-4-(4-pyridyl)-butyric acid;
(S)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid;
(S)-3-amino-4-pentafluoro-phenylbutyric acid;
(S)-3-amino-5-hexenoic acid; (S)-3-amino-5-hexynoic acid;
(S)-3-amino-5-phenylpentanoic acid; (S)-3-amino-6-phenyl-5-hexenoic
acid; 1,2,5,6-tetrahydropyridine-3-carboxylic acid;
1,2,5,6-tetrahydropyridine-4-carboxylic acid;
3-amino-3-(2-chlorophenyl)-propionic acid;
3-amino-3-(2-thienyl)-propionic acid;
3-amino-3-(3-bromophenyl)-propionic acid;
3-amino-3-(4-chlorophenyl)-propionic acid;
3-amino-3-(4-methoxyphenyl)-propionic acid;
3-amino-4,4,4-trifluoro-butyric acid; 3-aminoadipic acid;
D-.beta.-phenylalanine; .beta.-leucine; L-.beta.-homoalanine;
L-.beta.-homoaspartic acid .gamma.-benzyl ester;
L-.beta.-homoglutamic acid .delta.-benzyl ester;
L-.beta.-homoisoleucine; L-.beta.-homoleucine;
L-.beta.-homomethionine; L-.beta.-homophenylalanine;
L-.beta.-homoproline; L-.beta.3-homotryptophan;
L-.beta.-homovaline;
L-N.omega.-benzyloxycarbonyl-.beta.-homolysine;
N.omega.-L-.beta.-homoarginine;
O-benzyl-L-.beta.-homohydroxyproline; O-benzyl-L-.beta.-homoserine;
O-benzyl-L-.beta.-homothreonine; O-benzyl-L-.beta.-homotyrosine;
.gamma.-trityl-L-.beta.-homoasparagine; (R)-.beta.-phenylalanine;
L-.beta.-homoaspartic acid .gamma.-t-butyl ester;
L-.beta.-homoglutamic acid .delta.-t-butyl ester;
L-N.omega.-.beta.-homolysine;
N.delta.-trityl-L-.beta.-homoglutamine;
N.omega.-2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl-L-.beta.-homo-
arginine; O-t-butyl-L-.beta.-homohydroxy-proline;
O-t-butyl-L-.beta.-homoserine; O-t -butyl-L-.beta.-homothreonine;
O-t-butyl-L-.beta.-homotyrosine; 2-aminocyclopentane carboxylic
acid; and 2-aminocyclohexane carboxylic acid.
[0107] Amino acid analogs include analogs of alanine, valine,
glycine or leucine. Examples of amino acid analogs of alanine,
valine, glycine, and leucine include, but are not limited to, the
following: .alpha.-methoxyglycine; .alpha.-allyl-L-alanine;
.alpha.-aminoisobutyric acid; .alpha.-methyl-leucine;
.beta.-(1-naphthyl)-D-alanine; .beta.-(1-naphthyl)-L-alanine;
.beta.-(2-naphthyl)-D-alanine; .beta.-(2-naphthyl)-L-alanine;
.beta.-(2-pyridyl)-D-alanine; .beta.-(2-pyridyl)-L-alanine;
.beta.-(2-thienyl)-D-alanine; .beta.-(2-thienyl)-L-alanine;
.beta.-(3-benzothienyl)-D-alanine;
.beta.-(3-benzothienyl)-L-alanine; .beta.-(3-pyridyl)-D-alanine;
.beta.-(3-pyridyl)-L-alanine; .beta.-(4-pyridyl)-D-alanine;
.beta.-(4-pyridyl)-L-alanine; .beta.-chloro-L-alanine;
.beta.-cyano-L-alanin; .beta.-cyclohexyl-D-alanine;
.beta.-cyclohexyl-L-alanine; .beta.-cyclopenten-1-yl-alanine;
.beta.-cyclopentyl-alanine;
.beta.-cyclopropyl-L-Ala-OH.dicyclohexylammonium salt;
.beta.-t-butyl-D-alanine; .beta.-t-butyl-L-alanine;
.gamma.-aminobutyric acid; L-.alpha.,.beta.-diaminopropionic acid;
2,4-dinitro-phenylglycine; 2,5-dihydro-D-phenylglycine;
2-amino-4,4,4-trifluorobutyric acid; 2-fluoro-phenylglycine;
3-amino-4,4,4-trifluoro-butyric acid; 3-fluoro-valine;
4,4,4-trifluoro-valine; 4,5-dehydro-L-leu-OH.dicyclohexylammonium
salt; 4-fluoro-D-phenylglycine; 4-fluoro-L-phenylglycine;
4-hydroxy-D-phenylglycine; 5,5,5-trifluoro-leucine; 6-aminohexanoic
acid; cyclopentyl-D-Gly-OH.dicyclohexylammonium salt;
cyclopentyl-Gly-OH.dicyclohexylammonium salt;
D-.alpha.,.beta.-diaminopropionic acid; D-.alpha.-aminobutyric
acid; D-.alpha.-t-butylglycine; D-(2-thienyl)glycine;
D-(3-thienyl)glycine; D-2-aminocaproic acid; D-2-indanylglycine;
D-allylglycine.dicyclohexylammonium salt; D-cyclohexylglycine;
D-norvaline; D-phenylglycine; .beta.-aminobutyric acid;
.beta.-aminoisobutyric acid; (2-bromophenyl)glycine;
(2-methoxyphenyl)glycine; (2-methylphenyl)glycine;
(2-thiazoyl)glycine; (2-thienyl)glycine;
2-amino-3-(dimethylamino)-propionic acid;
L-.alpha.,.beta.-diaminopropionic acid; L-.alpha.-aminobutyric
acid; L-.alpha.-t-butylglycine; L-(3-thienyl)glycine;
L-2-amino-3-(dimethylamino)-propionic acid; L-2-aminocaproic acid
dicyclohexyl-ammonium salt; L-2-indanylglycine;
L-allylglycine.dicyclohexyl ammonium salt; L-cyclohexylglycine;
L-phenylglycine; L-propargylglycine; L-norvaline;
N-.alpha.-aminomethyl-L-alanine; D-.alpha.,.gamma.-diaminobutyric
acid; L-.alpha.,.gamma.-diaminobutyric acid;
.beta.-cyclopropyl-L-alanine;
(N-.beta.-(2,4-dinitrophenyl))-L-.alpha.,.beta.-diaminopropionic
acid;
(N-.beta.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-.alpha.,.b-
eta.-diaminopropionic acid;
(N-.beta.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-.alpha.,.b-
eta.-diaminopropionic acid;
(N-.beta.-4-methyltrityl)-L-.alpha.,.beta.-diaminopropionic acid;
(N-.beta.-allyloxycarbonyl)-L-.alpha.,.beta.-diaminopropionic acid;
(N-.gamma.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-.alpha.,.-
gamma.-diaminobutyric acid;
(N-.gamma.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-.alpha.,.-
gamma.-diaminobutyric acid;
(N-.gamma.-4-methyltrityl)-D-.alpha.,.gamma.-diaminobutyric acid;
(N-.gamma.-4-methyltrityl)-L-.alpha.,.gamma.-diaminobutyric acid;
(N-.gamma.-allyloxycarbonyl)-L-.alpha.,.gamma.-diaminobutyric acid;
D-.alpha.,.gamma.-diaminobutyric acid; 4,5-dehydro-L-leucine;
cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH; D-allylglycine;
D-homocyclohexylalanine; L-1-pyrenylalanine; L-2-aminocaproic acid;
L-allylglycine; L-homocyclohexylalanine; and
N-(2-hydroxy-4-methoxy-Bzl)-Gly-OH.
[0108] Amino acid analogs include analogs of arginine or lysine.
Examples of amino acid analogs of arginine and lysine include, but
are not limited to, the following: citrulline;
L-2-amino-3-guanidinopropionic acid; L-2-amino-3-ureidopropionic
acid; L-citrulline; Lys(Me).sub.2-OH; Lys(N.sub.3)--OH;
N.delta.-benzyloxycarbonyl-L-omithine; No-nitro-D-arginine;
No-nitro-L-arginine; .alpha.-methyl-omithine;
2,6-diaminoheptanedioic acid; L-omithine;
(N.delta.-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-D-omithine;
(N.delta.-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-L-omithine;
(N.delta.-4-methyltrityl)-D-omithine;
(N.delta.-4-methyltrityl)-L-omithine; D-omithine; L-omithine;
Arg(Me)(Pbf)-OH; Arg(Me).sub.2-OH (asymmetrical); Arg(Me).sub.2-OH
(symmetrical); Lys(ivDde)-OH; Lys(Me).sub.2-OH.HCl; Lys(Me3)-OH
chloride; No-nitro-D-arginine; and N.omega.-nitro-L-arginine.
[0109] Amino acid analogs include analogs of aspartic or glutamic
acids. Examples of amino acid analogs of aspartic and glutamic
acids include, but are not limited to, the following:
.alpha.-methyl-D-aspartic acid; .alpha.-methyl-glutamic acid;
.alpha.-methyl-L-aspartic acid; .gamma.-methylene-glutamic acid;
(N-.gamma.-ethyl)-L-glutamine;
[N-.alpha.-(4-aminobenzoyl)]-L-glutamic acid; 2,6-diaminopimelic
acid; L-.alpha.-aminosuberic acid; D-2-aminoadipic acid;
D-.alpha.-aminosuberic acid; .alpha.-aminopimelic acid;
iminodiacetic acid; L-2-aminoadipic acid;
threo-.beta.-methyl-aspartic acid; .gamma.-carboxy-D-glutamic acid
.gamma.,.gamma.-di-t-butyl ester; .gamma.-carboxy-L-glutamic acid
.gamma.,.gamma.-di-t-butyl ester; Glu(OAll)-OH; L-Asu(OtBu)-OH; and
pyroglutamic acid.
[0110] Amino acid analogs include analogs of cysteine and
methionine. Examples of amino acid analogs of cysteine and
methionine include, but are not limited to, Cys(famesyl)-OH,
Cys(famesyl)-OMe, .alpha.-methyl-methionine,
Cys(2-hydroxyethyl)-OH, Cys(3-aminopropyl)-OH,
2-amino-4-(ethylthio)butyric acid, buthionine,
buthioninesulfoximine, ethionine, methionine methylsulfonium
chloride, selenomethionine, cysteic acid,
[2-(4-pyridyl)ethyl]-DL-penicillamine,
[2-(4-pyridyl)ethyl]-L-cysteine, 4-methoxybenzyl-D-penicillamine,
4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D-penicillamine,
4-methylbenzyl-L-penicillamine, benzyl-D-cysteine,
benzyl-L-cysteine, benzyl-DL-homocysteine, carbamoyl-L-cysteine,
carboxyethyl-L-cysteine, carboxymethyl-L-cysteine,
diphenylmethyl-L-cysteine, ethyl-L-cysteine, methyl-L-cysteine,
t-butyl-D-cysteine, trityl-L-homocysteine, trityl-D-penicillamine,
cystathionine, homocystine, L-homocystine,
(2-aminoethyl)-L-cysteine, seleno-L-cystine, cystathionine,
Cys(StBu)-OH, and acetamidomethyl-D-penicillamine.
[0111] Amino acid analogs include analogs of phenylalanine and
tyrosine. Examples of amino acid analogs of phenylalanine and
tyrosine include 3-methyl-phenylalanine, 3-hydroxyphenylalanine,
.alpha.-methyl-3-methoxy-DL-phenylalanine,
.alpha.-methyl-D-phenylalanine, .alpha.-methyl-L-phenylalanine,
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,
2,4-dichloro-phenylalanine, 2-(trifluoromethyl)-D-phenylalanine,
2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine,
2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine,
2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine,
2-cyano-L-phenylalanine, 2-fluoro-D-phenylalanine,
2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine,
2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine,
2-nitro-L-phenylalanine, 2;4;5-trihydroxy-phenylalanine,
3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine,
3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine,
3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine,
3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine,
3,5,3'-triiodo-L-thyronine, 3,5-diiodo-D-tyrosine,
3,5-diiodo-L-tyrosine, 3,5-diiodo-L-thyronine,
3-(trifluoromethyl)-D-phenylalanine,
3-(trifluoromethyl)-L-phenylalanine, 3-amino-L-tyrosine,
3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine,
3-chloro-D-phenylalanine, 3-chloro-L-phenylalanine,
3-chloro-L-tyrosine, 3-cyano-D-phenylalanine,
3-cyano-L-phenylalanine, 3-fluoro-D-phenylalanine,
3-fluoro-L-phenylalanine, 3-fluoro-tyrosine,
3-iodo-D-phenylalanine, 3-iodo-L-phenylalanine, 3-iodo-L-tyrosine,
3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine,
3-methyl-L-phenylalanine, 3-nitro-D-phenylalanine,
3-nitro-L-phenylalanine, 3-nitro-L-tyrosine,
4-(trifluoromethyl)-D-phenylalanine,
4-(trifluoromethyl)-L-phenylalanine, 4-amino-D-phenylalanine,
4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine,
4-benzoyl-L-phenylalanine,
4-bis(2-chloroethyl)amino-L-phenylalanine, 4-bromo-D-phenylalanine,
4-bromo-L-phenylalanine, 4-chloro-D-phenylalanine,
4-chloro-L-phenylalanine, 4-cyano-D-phenylalanine,
4-cyano-L-phenylalanine, 4-fluoro-D-phenylalanine,
4-fluoro-L-phenylalanine, 4-iodo-D-phenylalanine,
4-iodo-L-phenylalanine, homophenylalanine, thyroxine,
3,3-diphenylalanine, thyronine, ethyl-tyrosine, and
methyl-tyrosine.
[0112] Amino acid analogs include analogs of proline. Examples of
amino acid analogs of proline include, but are not limited to,
3,4-dehydro-proline, 4-fluoro-proline, cis-4-hydroxy-proline,
thiazolidine-2-carboxylic acid, and trans-4-fluoro-proline.
[0113] Amino acid analogs include analogs of serine and threonine.
Examples of amino acid analogs of serine and threonine include, but
are not limited to, 3-amino-2-hydroxy-5-methylhexanoic acid,
2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutanoic
acid, 2-amino-3-methoxybutanoic acid,
4-amino-3-hydroxy-6-methylheptanoic acid,
2-amino-3-benzyloxypropionic acid, 2-amino-3-benzyloxypropionic
acid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutanoic
acid, and .alpha.-methylserine.
[0114] Amino acid analogs include analogs of tryptophan. Examples
of amino acid analogs of tryptophan include, but are not limited
to, the following: .alpha.-methyl-tryptophan;
.beta.-(3-benzothienyl)-D-alanine;
.beta.-(3-benzothienyl)-L-alanine; 1-methyl-tryptophan;
4-methyl-tryptophan; 5-benzyloxy-tryptophan; 5-bromo-tryptophan;
5-chloro-tryptophan; 5-fluoro-tryptophan; 5-hydroxy-tryptophan;
5-hydroxy-L-tryptophan; 5-methoxy-tryptophan;
5-methoxy-L-tryptophan; 5-methyl-tryptophan; 6-bromo-tryptophan;
6-chloro-D-tryptophan; 6-chloro-tryptophan; 6-fluoro-tryptophan;
6-methyl-tryptophan; 7-benzyloxy-tryptophan; 7-bromo-tryptophan;
7-methyl-tryptophan; D-1,2,3,4-tetrahydro-norharman-3-carboxylic
acid; 6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid;
7-azatryptophan; L-1,2,3,4-tetrahydro-norharman-3-carboxylic acid;
5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan.
[0115] In some embodiments, amino acid analogs are racemic. In some
embodiments, the D isomer of the amino acid analog is used. In some
embodiments, the L isomer of the amino acid analog is used. In
other embodiments, the amino acid analog comprises chiral centers
that are in the R or S configuration. In still other embodiments,
the amino group(s) of a .beta.-amino acid analog is substituted
with a protecting group, e.g., tert-butyloxycarbonyl (BOC group),
9-fluorenylmethyloxycarbonyl (FMOC), tosyl, and the like. In yet
other embodiments, the carboxylic acid functional group of a
.beta.-amino acid analog is protected, e.g., as its ester
derivative. In some embodiments the salt of the amino acid analog
is used.
[0116] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of a polypeptide without
abolishing or substantially altering its essential biological or
biochemical activity (e.g., receptor binding or activation). An
"essential" amino acid residue is a residue that, when altered from
the wild-type sequence of the polypeptide, results in abolishing or
substantially abolishing the polypeptide's essential biological or
biochemical activity.
[0117] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., K, R, H), acidic side
chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S,
T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M, W),
beta-branched side chains (e.g., T, V, I) and aromatic side chains
(e.g., Y, F, W, H). Thus, a predicted nonessential amino acid
residue in a polypeptide, for example, is replaced with another
amino acid residue from the same side chain family. Other examples
of acceptable substitutions are substitutions based on isosteric
considerations (e.g. norleucine for methionine) or other properties
(e.g. 2-thienylalanine for phenylalanine, or 6-Cl-tryptophan for
tryptophan).
[0118] The term "capping group" refers to the chemical moiety
occurring at either the carboxy or amino terminus of the
polypeptide chain of the subject peptidomimetic macrocycle. The
capping group of a carboxy terminus includes an unmodified
carboxylic acid (i.e. --COOH) or a carboxylic acid with a
substituent.
[0119] For example, the carboxy terminus can be substituted with an
amino group to yield a carboxamide at the C-terminus. Various
substituents include but are not limited to primary and secondary
amines, including pegylated secondary amines. Representative
secondary amine capping groups for the C-terminus include:
##STR00010##
[0120] The capping group of an amino terminus includes an
unmodified amine (ie --NH.sub.2) or an amine with a substituent.
For example, the amino terminus can be substituted with an acyl
group to yield a carboxamide at the N-terminus. Various
substituents include but are not limited to substituted acyl
groups, including C.sub.1-C.sub.6 carbonyls, C.sub.7-C.sub.30
carbonyls, and pegylated carbamates. Representative capping groups
for the N-terminus include, but are not limited to, 4-FBzl
(4-fluoro-benzyl) and the following:
##STR00011##
[0121] The term "member" as used herein in conjunction with
macrocycles or macrocycle-forming linkers refers to the atoms that
form or can form the macrocycle, and excludes substituent or side
chain atoms. By analogy, cyclodecane, 1,2-difluoro-decane and
1,3-dimethyl cyclodecane are all considered ten-membered
macrocycles as the hydrogen or fluoro substituents or methyl side
chains do not participate in forming the macrocycle.
[0122] The symbol "" when used as part of a molecular structure
refers to a single bond or a trans or cis double bond.
[0123] The term "amino acid side chain" refers to a moiety attached
to the .alpha.-carbon (or another backbone atom) in an amino acid.
For example, the amino acid side chain for alanine is methyl, the
amino acid side chain for phenylalanine is phenylmethyl, the amino
acid side chain for cysteine is thiomethyl, the amino acid side
chain for aspartate is carboxymethyl, the amino acid side chain for
tyrosine is 4-hydroxyphenylmethyl, etc. Other non-naturally
occurring amino acid side chains are also included, for example,
those that occur in nature (e.g., an amino acid metabolite) or
those that are made synthetically (e.g., an .alpha.,.alpha.
di-substituted amino acid).
[0124] The term ".alpha.,.alpha. di-substituted amino" acid refers
to a molecule or moiety containing both an amino group and a
carboxyl group bound to a carbon (the .alpha.-carbon) that is
attached to two natural or non-natural amino acid side chains.
[0125] The term "polypeptide" encompasses two or more naturally or
non-naturally-occurring amino acids joined by a covalent bond
(e.g., an amide bond). Polypeptides as described herein include
full length proteins (e.g., fully processed proteins) as well as
shorter amino acid sequences (e.g., fragments of
naturally-occurring proteins or synthetic polypeptide
fragments).
[0126] The term "first C-terminal amino acid" refers to the amino
acid which is closest to the C-terminus. The term "second
C-terminal amino acid" refers to the amino acid attached at the
N-terminus of the first C-terminal amino acid.
[0127] The term "macrocyclization reagent" or "macrocycle-forming
reagent" as used herein refers to any reagent which can be used to
prepare a peptidomimetic macrocycle by mediating the reaction
between two reactive groups. Reactive groups can be, for example,
an azide and alkyne, in which case macrocyclization reagents
include, without limitation, Cu reagents such as reagents which
provide a reactive Cu(I) species, such as CuBr, CuI or CuOTf, as
well as Cu(II) salts such as Cu(CO.sub.2CH.sub.3).sub.2,
CuSO.sub.4, and CuCl.sub.2 that can be converted in situ to an
active Cu(I) reagent by the addition of a reducing agent such as
ascorbic acid or sodium ascorbate. Macrocyclization reagents can
additionally include, for example, Ru reagents known in the art
such as Cp*RuCl(PPh.sub.3).sub.2, [Cp*RuCl].sub.4 or other Ru
reagents which can provide a reactive Ru(II) species. In other
cases, the reactive groups are terminal olefins. In such
embodiments, the macrocyclization reagents or macrocycle-forming
reagents are metathesis catalysts including, but not limited to,
stabilized, late transition metal carbene complex catalysts such as
Group VIII transition metal carbene catalysts. For example, such
catalysts are Ru and Os metal centers having a +2 oxidation state,
an electron count of 16 and pentacoordinated. In other examples,
catalysts have W or Mo centers. Various catalysts are disclosed in
Grubbs et al., "Ring Closing Metathesis and Related Processes in
Organic Synthesis" Acc. Chem. Res. 1995, 28, 446-452, U.S. Pat.
Nos. 5,811,515; 7,932,397; U.S. Application No. 2011/0065915; U.S.
Application No. 2011/0245477; Yu et al., "Synthesis of Macrocyclic
Natural Products by Catalyst-Controlled Stereoselective
Ring-Closing Metathesis," Nature 2011, 479, 88; and Peryshkov et
al., "Z-Selective Olefin Metathesis Reactions Promoted by Tungsten
Oxo Alkylidene Complexes," J. Am. Chem. Soc. 2011, 133, 20754. In
yet other cases, the reactive groups are thiol groups. In such
embodiments, the macrocyclization reagent is, for example, a linker
functionalized with two thiol-reactive groups such as halogen
groups. In some examples, the macrocyclization reagent include
palladium reagents, for example Pd(PPh.sub.3).sub.4,
Pd(PPh.sub.3).sub.2Cl.sub.2, Pd(dppe)Cl, Pd(dppp)Cl.sub.2, and
Pd(dppf)Cl.sub.2.
[0128] The term "halo" or "halogen" refers to fluorine, chlorine,
bromine or iodine or a radical thereof.
[0129] The term "alkyl" refers to a hydrocarbon chain that is a
straight chain or branched chain, containing the indicated number
of carbon atoms. For example, C.sub.1-C.sub.10 indicates that the
group has from 1 to 10 (inclusive) carbon atoms in it. In the
absence of any numerical designation, "alkyl" is a chain (straight
or branched) having 1 to 20 (inclusive) carbon atoms in it.
[0130] The term "alkylene" refers to a divalent alkyl (i.e.,
--R--).
[0131] The term "alkenyl" refers to a hydrocarbon chain that is a
straight chain or branched chain having one or more carbon-carbon
double bonds. The alkenyl moiety contains the indicated number of
carbon atoms. For example, C.sub.2-C.sub.10 indicates that the
group has from 2 to 10 (inclusive) carbon atoms in it. The term
"lower alkenyl" refers to a C.sub.2-C.sub.6 alkenyl chain. In the
absence of any numerical designation, "alkenyl" is a chain
(straight or branched) having 2 to 20 (inclusive) carbon atoms in
it.
[0132] The term "alkynyl" refers to a hydrocarbon chain that is a
straight chain or branched chain having one or more carbon-carbon
triple bonds. The alkynyl moiety contains the indicated number of
carbon atoms. For example, C.sub.2-C.sub.10 indicates that the
group has from 2 to 10 (inclusive) carbon atoms in it. The term
"lower alkynyl" refers to a C.sub.2-C.sub.6 alkynyl chain. In the
absence of any numerical designation, "alkynyl" is a chain
(straight or branched) having 2 to 20 (inclusive) carbon atoms in
it.
[0133] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon
bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of
each ring are substituted by a substituent. Examples of aryl groups
include phenyl, naphthyl and the like. The term "arylalkoxy" refers
to an alkoxy substituted with aryl.
[0134] "Arylalkyl" refers to an aryl group, as defined above,
wherein one of the aryl group's hydrogen atoms has been replaced
with a C.sub.1-C.sub.5 alkyl group, as defined above.
Representative examples of an arylalkyl group include, but are not
limited to, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl,
2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2-propylphenyl,
3-propylphenyl, 4-propylphenyl, 2-butylphenyl, 3-butylphenyl,
4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl, 4-pentylphenyl,
2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl,
2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl,
2-sec-butylphenyl, 3-sec-butylphenyl, 4-sec-butylphenyl,
2-t-butylphenyl, 3-t-butylphenyl and 4-t-butylphenyl.
[0135] "Arylamido" refers to an aryl group, as defined above,
wherein one of the aryl group's hydrogen atoms has been replaced
with one or more --C(O)NH.sub.2 groups. Representative examples of
an arylamido group include 2-C(O)NH.sub.2-phenyl,
3-C(O)NH.sub.2-phenyl, 4-C(O)NH.sub.2-phenyl,
2-C(O)NH.sub.2-pyridyl, 3-C(O)NH.sub.2-pyridyl, and
4-C(O)NH.sub.2-pyridyl,
[0136] "Alkylheterocycle" refers to a C.sub.1-C.sub.5 alkyl group,
as defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a heterocycle. Representative
examples of an alkylheterocycle group include, but are not limited
to, --CH.sub.2CH.sub.2-morpholine, --CH.sub.2CH.sub.2-piperidine,
--CH.sub.2CH.sub.2CH.sub.2-morpholine, and
--CH.sub.2CH.sub.2CH.sub.2-imidazole.
[0137] "Alkylamido" refers to a C.sub.1-C.sub.5 alkyl group, as
defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a --C(O)NH.sub.2 group.
Representative examples of an alkylamido group include, but are not
limited to, --CH.sub.2--C(O)NH.sub.2,
--CH.sub.2CH.sub.2--C(O)NH.sub.2,
--CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2CH(C(O)NH.sub.2)CH.sub.3,
--CH.sub.2CH(C(O)NH.sub.2)CH.sub.2CH.sub.3,
--CH(C(O)NH.sub.2)CH.sub.2CH.sub.3,
--C(CH.sub.3).sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.3--CH3, and
--CH.sub.2--CH.sub.2--NH--C(O)--CH.dbd.CH.sub.2.
[0138] "Alkanol" refers to a C.sub.1-C.sub.5 alkyl group, as
defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a hydroxyl group.
Representative examples of an alkanol group include, but are not
limited to, --CH.sub.2OH, --CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2CH.sub.2 CH.sub.2CH.sub.2OH,
--CH.sub.2CH(OH)CH.sub.3, --CH.sub.2CH(OH)CH.sub.2CH.sub.3,
--CH(OH)CH.sub.3 and --C(CH.sub.3).sub.2CH.sub.2OH.
[0139] "Alkylcarboxy" refers to a C.sub.1-C.sub.5 alkyl group, as
defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a --COOH group.
Representative examples of an alkylcarboxy group include, but are
not limited to, --CH.sub.2COOH, --CH.sub.2CH.sub.2COOH,
--CH.sub.2CH.sub.2CH.sub.2COOH,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2COOH, --CH.sub.2CH(COOH)CH.sub.3,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2COOH,
--CH.sub.2CH(COOH)CH.sub.2CH.sub.3, --CH(COOH)CH.sub.2CH.sub.3 and
--C(CH.sub.3).sub.2CH.sub.2COOH.
[0140] The term "cycloalkyl" as employed herein includes saturated
and partially unsaturated cyclic hydrocarbon groups having 3 to 12
carbons, preferably 3 to 8 carbons, and more preferably 3 to 6
carbons, wherein the cycloalkyl group additionally is optionally
substituted. Some cycloalkyl groups include, without limitation,
cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,
cyclohexenyl, cycloheptyl, and cyclooctyl.
[0141] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring are
substituted by a substituent. Examples of heteroaryl groups include
pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl,
thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the
like.
[0142] The term "heteroarylalkyl" or the term "heteroaralkyl"
refers to an alkyl substituted with a heteroaryl. The term
"heteroarylalkoxy" refers to an alkoxy substituted with
heteroaryl.
[0143] The term "heteroarylalkyl" or the term "heteroaralkyl"
refers to an alkyl substituted with a heteroaryl. The term
"heteroarylalkoxy" refers to an alkoxy substituted with
heteroaryl.
[0144] The term "heterocyclyl" refers to a nonaromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2 or 3 atoms of each ring are
substituted by a substituent. Examples of heterocyclyl groups
include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl,
tetrahydrofuranyl, and the like.
[0145] The term "substituent" refers to a group replacing a second
atom or group such as a hydrogen atom on any molecule, compound or
moiety. Suitable substituents include, without limitation, halo,
hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl,
aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido,
carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups.
[0146] In some embodiments, the compounds disclosed herein contain
one or more asymmetric centers and thus occur as racemates and
racemic mixtures, single enantiomers, individual diastereomers and
diastereomeric mixtures. All such isomeric forms of these compounds
are included unless expressly provided otherwise. In some
embodiments, the compounds disclosed herein are also represented in
multiple tautomeric forms, in such instances, the compounds include
all tautomeric forms of the compounds described herein (e.g., if
alkylation of a ring system results in alkylation at multiple
sites, the disclosure includes all such reaction products). All
such isomeric forms of such compounds are included unless expressly
provided otherwise. All crystal forms of the compounds described
herein are included unless expressly provided otherwise.
[0147] As used herein, the terms "increase" and "decrease" mean,
respectively, to cause a statistically significantly (i.e.,
p<0.1) increase or decrease of at least 5%.
[0148] As used herein, the recitation of a numerical range for a
variable is intended to convey that the variable is equal to any of
the values within that range. Thus, for a variable which is
inherently discrete, the variable is equal to any integer value
within the numerical range, including the end-points of the range.
Similarly, for a variable which is inherently continuous, the
variable is equal to any real value within the numerical range,
including the end-points of the range. As an example, and without
limitation, a variable which is described as having values between
0 and 2 takes the values 0, 1 or 2 if the variable is inherently
discrete, and takes the values 0.0, 0.1, 0.01, 0.001, or any other
real values .gtoreq.0 and .ltoreq.2 if the variable is inherently
continuous.
[0149] As used herein, unless specifically indicated otherwise, the
word "or" is used in the inclusive sense of "and/or" and not the
exclusive sense of "either/or."
[0150] The term "on average" represents the mean value derived from
performing at least three independent replicates for each data
point.
[0151] The term "biological activity" encompasses structural and
functional properties of a macrocycle. Biological activity is, for
example, structural stability, alpha-helicity, affinity for a
target, resistance to proteolytic degradation, cell penetrability,
intracellular stability, in vivo stability, or any combination
thereof.
[0152] The term "binding affinity" refers to the strength of a
binding interaction, for example between a peptidomimetic
macrocycle and a target. Binding affinity can be expressed, for
example, as an equilibrium dissociation constant ("K.sub.D"), which
is expressed in units which are a measure of concentration (e.g. M,
mM, .mu.M, nM etc). Numerically, binding affinity and K.sub.D
values vary inversely, such that a lower binding affinity
corresponds to a higher K.sub.D value, while a higher binding
affinity corresponds to a lower K.sub.D value. Where high binding
affinity is desirable, "improved" binding affinity refers to higher
binding affinity and therefore lower K.sub.D values.
[0153] The term "in vitro efficacy" refers to the extent to which a
test compound, such as a peptidomimetic macrocycle, produces a
beneficial result in an in vitro test system or assay. In vitro
efficacy can be measured, for example, as an "IC.sub.50" or
"EC.sub.50" value, which represents the concentration of the test
compound which produces 50% of the maximal effect in the test
system.
[0154] The term "ratio of in vitro efficacies" or "in vitro
efficacy ratio" refers to the ratio of IC.sub.50 or EC.sub.50
values from a first assay (the numerator) versus a second assay
(the denominator). Consequently, an improved in vitro efficacy
ratio for Assay 1 versus Assay 2 refers to a lower value for the
ratio expressed as IC.sub.50(Assay 1)/IC.sub.50(Assay 2) or
alternatively as EC.sub.50(Assay 1)/EC.sub.50(Assay 2). This
concept can also be characterized as "improved selectivity" in
Assay 1 versus Assay 2, which can be due either to a decrease in
the IC.sub.50 or EC.sub.50 value for Target 1 or an increase in the
value for the IC.sub.50 or EC.sub.50 value for Target 2.
[0155] The term "solid tumor" or "solid cancer" as used herein
refers to tumors that usually do not contain cysts or liquid areas.
Solid tumors as used herein include sarcomas, carcinomas and
lymphomas. In various embodiments leukemia (cancer of blood) is not
solid tumor.
[0156] The term "adverse event" (AE) as used herein includes any
noxious, pathological, or unintended change in anatomical,
physiological, or metabolic functions as indicated by physical
signs, symptoms, and/or laboratory changes occurring in any phase
of the clinical study whether or not temporally associated with the
administration of study medication and whether or not considered
related to the study medication. This definition includes an
exacerbation of pre-existing medical conditions or events,
intercurrent illnesses, hypersensitivity reactions, drug
interactions, or clinically significant laboratory findings. An AE
does not include the following: (i) medical or surgical procedures,
e.g., tooth extraction, transfusion, surgery (The medical condition
that leads to the procedure is to be recorded as an AE); (ii)
pre-existing conditions or procedures present or detected at the
start of the study that do not worsen; (iii) hospitalization for
elective surgeries or for other situations in which an untoward
medical event has not occurred; (iv) abnormal laboratory value,
unless it is clinically significant according to the Investigator,
requires intervention, or results in a delay, discontinuation or
change in the dose of study drug; (v) overdose of study drug or
concomitant medication unaccompanied by signs/symptoms; if
sign/symptoms occur, the final diagnosis should be recorded as an
AE; (vi) pregnancy by itself, unless a complication occurs during
pregnancy leading to hospitalization; in this case, the medical
condition that leads to the hospitalization is to be recorded as
the AE; and (vii) significant worsening of the disease under
investigation which is captured as an efficacy parameter in this
study and, thus, is not recorded as an AE.
[0157] The term serious adverse event (SAE) as used herein refers
to an adverse event that results in any of the following outcomes:
(i) death; (ii) life-threatening adverse experience (i.e.,
immediate risk of death from the event as it occurred; this does
not include an adverse event that, had it occurred in a more
serious form, might have caused death); (iii) persistent or
significant disability/incapacitation; (iv) hospitalization or
prolongation of existing hospitalization; and (v) congenital
anomaly/birth defect. Important medical events that may not result
in death, be life-threatening, or require hospitalization can be
considered serious when, based on medical judgment, they can
jeopardize the patient or may require medical or surgical
intervention to prevent one of the outcomes listed in this
definition. Hospitalizations due to the underlying disease will not
be reported as an SAE unless there is reason to suspect a causal
relationship with the study drug.
[0158] An AE or suspected adverse reaction is considered
"unexpected" (referred to as Unexpected Adverse Event (UAE) if it
is not listed in the peptidomimetic macrocycle Investigator's
Brochure or is not listed at the specificity or severity that has
been observed; or, is not consistent with the risk information
described in the protocol or elsewhere. For example, under this
definition, hepatic necrosis would be unexpected (by virtue of
greater severity) if the Investigator's Brochure referred only to
elevated hepatic enzymes or hepatitis. Similarly, cerebral
thromboembolism and cerebral vasculitis would be unexpected (by
virtue of greater specificity) if the Investigator's Brochure
listed only cerebral vascular accidents. "Unexpected," as used in
this definition, also refers to AEs or suspected adverse reactions
that are mentioned in the Investigator's Brochure as occurring with
a class of drugs or as anticipated from the pharmacological
properties of the peptidomimetic macrocycle but are not
specifically mentioned as occurring with the peptidomimetic
macrocycle.
[0159] A "Dose-Limiting Toxicity" (DLT) as used herein is defined
as any Grade .gtoreq.3 AE that is considered to be possibly,
probably, or definitely related to the study drug, with the
following exceptions: (1) for nausea, emesis, diarrhea, rash, or
mucositis, only Grade .gtoreq.3 AE that do not respond within 48
hours to standard supportive/pharmacological treatment will be
considered DLT; (2) for electrolyte imbalances, only Grade
.gtoreq.3 AE that do not respond to correction within 24 hours will
be considered DLT. In addition, specific hematologic DLTs are
defined as: [0160] (i) Thrombocytopenia--Grade 4 of any duration,
Grade 3 for .gtoreq.7 days, or Grade 3 associated with clinically
significant bleeding; [0161] (ii) Neutropenia--Grade 4 for
.gtoreq.3 days, or any Grade .gtoreq.3 febrile neutropenia
[0162] The above criteria can be used to make individual patient
determinations regarding dose reductions, interruptions or
discontinuation throughout the course of the trial, but DLTs
occurring during Cycle 1 will be used to inform safety and
tolerability assessments for dose escalation decisions.
[0163] The "Maximum Tolerated Dose" (MTD) as used herein is defined
as the dose at which .ltoreq.1 of 6 patients experiences a
treatment-related toxicity that qualifies as a DLT, with the next
higher dose having .gtoreq.2 of up to 6 patients experiencing a
DLT. The MTD may not be established until all patients enrolled in
the cohort have completed Cycle 1, discontinued treatment or had a
dose reduction. Previously established tolerability of a dose level
will be reevaluated if DLTs are observed in later cycles.
[0164] "Measurable disease" (MD) as used herein is defined by the
presence of at least one measurable lesion.
[0165] Measurable lesions are defined as those that can be
accurately measured in at least one dimension [longest diameter
(LD) in the plane of measurement to be recorded] with a minimum
size of: 10 mm by CT scan (CT scan slice thickness no greater than
5 mm), 10 mm caliper measurement by clinical exam (lesions which
cannot be accurately measured with calipers can be recorded as
non-measurable), or 20 mm by chest x-ray.
[0166] "Malignant lymph nodes" is to be considered pathologically
enlarged and measurable if the lymph node is .gtoreq.15 mm in short
axis when assessed by CT scan (CT scan slice thickness no greater
than 5 mm).
[0167] "Non-measurable Disease" as used herein include all other
lesions (or sites of disease) that are not measurable, including
small lesions (longest diameter <10 mm or pathological lymph
nodes with .gtoreq.10 to <15 mm short axis) are considered
non-measurable disease. Lesions considered truly non-measurable
include: leptomeningeal disease, ascites, pleural/pericardial
effusions, lymphangitis cutis/pulmonis, inflammatory breast
disease, abdominal masses/abdominal organomegaly identified by
physical exam and not followed by CT or MRI.
[0168] "Target lesions" as used herein include all measurable
lesions up to a maximum of two lesions per organ and five lesions
in total, representative of all involved organs identified as
target lesions and recorded and measured at baseline. Target
lesions can be selected on the basis of their size (lesions with
the longest diameter) and their suitability for accurate repeated
measurements (either by imaging techniques or clinically). A sum of
the diameters (longest for non-nodal lesions, short axis for nodal
lesions) for all target lesions can be calculated and reported as
the baseline sum diameters. The baseline sum diameters can be used
as reference by which to characterize the objective tumor
response.
[0169] "Non-target lesions" as used herein include all other
lesions (or sites of disease) including pathological lymph nodes
that are not target lesions. Non-target lesions can be identified
as non-target lesions and can also be recorded at baseline.
Measurements of these lesions may not be required and these lesions
can be followed as "present," "absent," or in rare cases
"unequivocal progression." In addition, it can be possible to
record multiple non-target lesions involving the same organ as a
single item on the case report form (e.g., `multiple enlarged
pelvic lymph nodes` or `multiple liver metastases`).
[0170] "Complete response" (CR) as used herein is defined as the
disappearance of all target lesions. Any pathological lymph nodes
(whether target or non-target) must have reduction in short axis to
<10 mm.
[0171] "Partial response (PR)" as used herein is defined as at
least a 30% decrease in the sum of diameters of target lesions,
taking as reference the baseline sum diameters.
[0172] "Progressive disease (PD)" as used herein is defined as at
least a 20% increase in the sum of diameters of target lesions,
taking as reference the smallest sum on study (this includes the
baseline sum if that is the smallest). In addition to the relative
increase of 20%, the sum must also demonstrate an absolute increase
of at least 5 mm. The appearance of one or more new lesions can
also be considered progression.
[0173] "Stable disease" (SD) as used herein is defined as neither
sufficient shrinkage to qualify for PR nor sufficient increase to
qualify for PD, taking as reference the smallest sum diameters
while on study.
[0174] The term "subject" or "patient" encompasses mammals and
non-mammals. Examples of mammals include, but are not limited to,
humans; non-human primates such as chimpanzees, and other apes and
monkey species; farm animals such as cattle, horses, sheep, goats,
swine; domestic animals such as rabbits, dogs, and cats; laboratory
animals including rodents, such as rats, mice and guinea pigs, and
the like. Examples of non-mammals include, but are not limited to,
birds, fish and the like. In one embodiment of the methods and
compositions provided herein, the mammal is a human.
[0175] The term "haploinsufficient" means a condition that occurs
when a diploid organism has only a single functional copy of a gene
(with the other copy inactivated by mutation) and the single
functional copy does not produce enough of a gene product
(typically a protein) to bring about a wild-type condition, leading
to an abnormal or diseased state.
[0176] The term "silent mutation" as used herein silent mutation is
a type of mutation in the coding region of a gene that doesn't
actually change the amino acid sequence of the protein that is
made.
[0177] The details of one or more particular embodiments of the
invention are set forth in the accompanying drawings and the
description below. Other features, objects, and advantages of the
invention will be apparent from the description and drawings, and
from the claims.
Overview
[0178] In one aspect, the disclosure provides a method of treating
solid tumor in a subject. For example, the methods disclosed herein
can be used for treating solid tumors that are not p53 negative. In
some cases the methods disclosed herein can be used for treating
solid tumor which has been determined to lack a p53 deactivating
mutation. The methods of the disclosure can also be used to treat
solid tumors that express gain of function mutant p53 i.e. a super
apoptotic p53. In other examples, the methods of the disclosure are
useful in treating solid tumors, wherein the solid tumor expresses
p53 with a partial loss of function mutation, p53 with a copy loss
mutation, or p53 with one or more silent mutations. In some
example, the solid tumor expresses p53 with a copy loss mutation
and a deactivating mutation.
[0179] The method comprises administering to the subject a
pharmaceutical composition comprising a therapeutically effective
amount of a peptidomimetic macrocycle or a therapeutically
equivalent amount of a pharmaceutically acceptable salt thereof,
wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX
proteins. In some embodiments, the peptidomimetic macrocycle
disrupts the interaction between p53 and MDM2 and MDMX.
[0180] In another aspect, the disclosure provides a method of
treating solid tumor in a subject expressing wild type p53. The
method comprises administering to the subject a pharmaceutical
composition comprising a therapeutically effective amount of a
peptidomimetic macrocycle or a therapeutically equivalent amount of
a pharmaceutically acceptable salt thereof, wherein the
peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins. In
some embodiments, the peptidomimetic macrocycle disrupts the
interaction between p53 and MDM2 and MDMX.
[0181] In some embodiments the subject treated by the methods
disclosed herein is a human. In some embodiments, a subject treated
in accordance with the methods provided herein is a human who has
or is diagnosed with solid tumor lacking p53 deactivating mutation
and/or expressing wild type p53. In some embodiments, a subject
treated for solid tumor in accordance with the methods provided
herein is a human predisposed or susceptible to solid tumor lacking
p53 deactivating mutation and/or expressing wild type p53. In some
embodiments, a subject treated for solid tumor in accordance with
the methods provided herein is a human at risk of developing solid
tumor lacking p53 deactivating mutation and/or expressing wild type
p53. A p53 deactivating mutation in some examples can be a mutation
in DNA-binding domain of the p53 protein. In some examples the p53
deactivating mutation can be a missense mutation. In various
examples, the solid tumor can be determined to lack one or more p53
deactivating mutations selected from mutations at one or more of
residues R175, G245, R248, R249, R273, and R282. The lack of p53
deactivating mutation and/or the presence of wild type p53 in the
solid tumor can be determined by any suitable method known in art,
for example by sequencing, array based testing, RNA analysis and
amplifications methods like PCR.
[0182] In certain embodiments, the human subject is refractory
and/or intolerant to one or more other standard treatment of the
solid tumor known in art. In some embodiments, the human subject
has had at least one unsuccessful prior treatment and/or therapy of
the solid tumor.
[0183] In some embodiments, the subject treated in accordance with
the methods of the disclosure is a human who has or is diagnosed
with solid tumor that is not p53 negative. In some embodiments, the
subject treated in accordance with the methods of the disclosure is
a human who has or is diagnosed with solid tumor that express gain
of function mutant p53 i.e. a super apoptotic p53. In some
embodiments, the subject treated in accordance with the methods of
the disclosure is a human who has or is diagnosed with solid tumor
that expresses p53 with a partial loss of function mutation. In
some embodiments, the subject treated in accordance with the
methods provided herein is a human who has or is diagnosed with
solid tumor that expresses p53 with a copy loss mutation. In some
embodiments, the subject treated in accordance with the methods
provided herein is a human who has or is diagnosed with solid tumor
that expresses p53 with one or more silent mutations. In some
embodiments, the subject treated in accordance with the methods
provided herein is a human who has or is diagnosed with solid tumor
that expresses p53 with a copy loss mutation and a deactivating
mutation.
[0184] In some embodiments, the methods for treating solid tumor
provided herein inhibit, reduce, diminish, arrest, or stabilize a
tumor associated with the solid tumor. In some embodiments, the
methods for treating solid tumor provided herein inhibit, reduce,
diminish, arrest, or stabilize the blood flow, metabolism, or edema
in a tumor associated with the solid tumor or one or more symptoms
thereof. In some embodiments, the methods for treating solid tumor
provided herein cause the regression of a tumor, tumor blood flow,
tumor metabolism, or peritumor edema, and/or one or more symptoms
associated with the solid tumor. In some embodiments, the methods
for treating solid tumor provided herein maintain the size of the
tumor so that it does not increase, or so that it increases by less
than the increase of a tumor after administration of a standard
therapy as measured by conventional methods available to one of
skill in the art, such as ultrasound, CT Scan, MRI, dynamic
contrast-enhanced MRI, or PET Scan. In specific embodiments, the
methods for treating solid tumor provided herein decrease tumor
size. In some embodiments, the methods for treating solid tumor
provided herein reduce the formation of a tumor. In certain
embodiments, the methods for treating solid tumor provided herein
eradicate, remove, or control primary, regional and/or metastatic
tumors associated with the solid tumor. In some embodiments, the
methods for treating solid tumor provided herein decrease the
number or size of metastases associated with the solid tumor. In
some embodiments, the methods for treating solid tumor provided
herein result in complete response to the treatment. In some
embodiments, the methods for treating solid tumor provided herein
result in partial response to the treatment. In some embodiments,
the solid tumor treated by the methods disclosed herein is a stable
disease. In some embodiments, the solid tumor treated by the
methods disclosed herein is a progressive disease.
[0185] Solid tumor cancers that can be treated by the methods
provided herein include, but are not limited to, sarcomas,
carcinomas, and lymphomas. In specific embodiments, solid tumors
that can be treated in accordance with the methods described
include, but are not limited to, cancer of the breast, liver,
neuroblastoma, head, neck, eye, mouth, throat, esophagus,
esophagus, chest, bone, lung, kidney, colon, rectum or other
gastrointestinal tract organs, stomach, spleen, skeletal muscle,
subcutaneous tissue, prostate, breast, ovaries, testicles or other
reproductive organs, skin, thyroid, blood, lymph nodes, kidney,
liver, pancreas, and brain or central nervous system.
[0186] The peptidomimetic macrocycle can be any cross-linked
peptide, i.e. any peptide that comprises at least one
macrocycle-forming linker which forms a macrocycle between a first
amino acid residue (or analog) and a second amino acid residue. For
example, the peptidomimetic macrocycle can be a peptidomimetic
macrocycle capable of binding to the MDM2 and/or MDMX proteins. In
some embodiments, the peptidomimetic macrocycles can be a
peptidomimetic macrocycle of Formula I:
##STR00012##
wherein: each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-Hiss-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.sub.10-
-X.sub.11-Ser.sub.12 (SEQ ID NO: 8) or
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.2 (SEQ ID NO: 9), where each X is
an amino acid; each D and E is independently an amino acid; each
R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids; each L or L' is independently a macrocycle-forming each
R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent; each R.sub.6 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a
fluorescent moiety, a radioisotope or a therapeutic agent; each
R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or
heterocycloaryl, optionally substituted with R.sub.5, or part of a
cyclic structure with a D residue; each R.sub.8 is --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,
optionally substituted with R.sub.5, or part of a cyclic structure
with an E residue; each v is an integer from 1-1000, for example
1-500, 1-200, 1-100, 1-50, 1-30, 1-20, or 1-10; and each w is an
integer from 0-1000, for example 1-500, 1-200, 1-100, 1-50, 1-30,
1-20, or 1-10.
[0187] Administration of the a pharmaceutical composition
comprising a therapeutically effective amount of a peptidomimetic
macrocycle or a therapeutically equivalent amount of a
pharmaceutically acceptable salt thereof can be achieved by any
suitable means. For example the pharmaceutical composition can be
administered parenterally. For example, administration can be
intravenous, intra-arterial, intraosseous infusion, intra-muscular,
intracerebral, intracerebroventricular, intrathecal, or
subcutaneous. In some embodiments administration is performed
intravenously.
[0188] In some embodiments, the methods disclosed herein
additionally or optionally comprise evaluating the safety and/or
tolerability of a pharmaceutical composition comprising a
therapeutically effective amount of a peptidomimetic macrocycle or
a therapeutically equivalent amount of a pharmaceutically
acceptable salt thereof, of the disclosure in subjects with solid
tumors determined to lack a p53 deactivating mutation or with solid
tumors expressing wild-type (WT) p53 protein.
[0189] Also provided here in are methods to determine the dose
limiting toxicities (DLT) and the maximum tolerated dose (MTD) of
the peptidomimetic macrocycles disclosed herein in subjects with
solid tumors determined to lack a p53 deactivating mutation or with
solid tumors expressing wild-type (WT) p53 protein.
[0190] In some embodiments, the methods disclosed herein
additionally or optionally comprise the pharmacokinetic(PK)
analysis of the peptidomimetic macrocycles and/or its metabolites
in blood following single and/or multiple administration of the
peptidomimetic macrocycles to the subject.
[0191] In some embodiments, the methods disclosed herein
additionally or optionally comprise studying the effect of the
peptidomimetic macrocycles on pharmacodynamic biomarkers in tumor
biopsy samples (e.g., p21, caspase, MDM2) and blood samples (e.g.,
macrophage inhibitory cytokine-1 [MIC-1]), and assessing possible
correlation between these biomarkers and clinical response.
[0192] In some embodiments, the methods disclosed herein
additionally or optionally include steps to assess potential
patient biomarkers (e.g., p53 status, MDM2 and MDMX expression
levels), the effect of the peptidomimetic macrocycles treatment on
these biomarkers, and possible correlation between these biomarkers
and clinical response of the peptidomimetic macrocycles.
[0193] Also provided herein are methods to evaluate clinical
activity of the peptidomimetic macrocycles in subjects with
specific tumor types lacking a p53 deactivating mutation and/or
expressing WT p53 in the dose expansion phase.
Compound and Compositions
Peptidomimetic Macrocycles
[0194] In some embodiments, a peptidomimetic macrocycle has the
Formula (I):
##STR00013##
wherein: each A, C, and D is independently an amino acid; each B is
independently an amino acid,
##STR00014##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or [--NH-L.sub.3-];
each E is independently an amino acid selected from the group
consisting of Ala (alanine), D-Ala (D-alanine), Aib
(.alpha.-aminoisobutyric acid), Sar (N-methyl glycine), and Ser
(serine); each R.sub.3 is independently hydrogen, alkyl, alkenyl,
alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5; each R.sub.1 and R.sub.2 is independently
--H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or
substituted with halo-; or forms a macrocycle-forming linker L'
connected to the alpha position of one of said D or E amino acids;
each L and L' is independently a macrocycle-forming linker; each
L.sub.3 is independently alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,
heterocycloarylene, or [--R.sub.4--K--R.sub.4--].sub.n, each being
optionally substituted with R.sub.5; each R.sub.4 is independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene; each K is
independently O, S, SO, SO.sub.2, CO, CO.sub.2, or CONR.sub.3; each
R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R, a fluorescent moiety, a radioisotope or a therapeutic
agent; each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent; each R.sub.7 is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or
heterocycloaryl, optionally substituted with R.sub.5, or part of a
cyclic structure with a D residue; each R.sub.8 is independently
--H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,
optionally substituted with R.sub.5, or part of a cyclic structure
with an E residue; each v is independently an integer; each w is
independently an integer from 3-1000; u is an integer from 1-10;
each x, y and z is independently an integer from 0-10; and each n
is independently an integer from 1-5.
[0195] In some embodiments, each v and w is independently integers
between 1-30. In some embodiments, w is an integer from 3-1000, for
example 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, or 3-10. In some
embodiments, the sum of x+y+z is 3 or 6. In some embodiments, the
sum of x+y+z is 3. In other embodiments, the sum of x+y+z is 6.
[0196] In some embodiments, peptidomimetic macrocycles are also
provided of the formula:
##STR00015##
wherein: each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-Hiss-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.sub.10-
-X.sub.11-Ser.sub.12 (SEQ ID NO: 8), where each X is an amino acid;
each D and E is independently an amino acid; each R.sub.1 and
R.sub.2 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,
unsubstituted or substituted with halo-; or at least one of R.sub.1
and R.sub.2 forms a macrocycle-forming linker L' connected to the
alpha position of one of said D or E amino acids; each L or L' is
independently a macrocycle-forming linker; each R.sub.5 is
independently halogen, alkyl, --OR.sub.6, --N(R.sub.6).sub.2,
--SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6, --CO.sub.2R.sub.6, a
fluorescent moiety, a radioisotope or a therapeutic agent; each
R.sub.6 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a
radioisotope or a therapeutic agent; each R.sub.7 is independently
--H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,
optionally substituted with R.sub.5, or part of a cyclic structure
with a D residue; each R.sub.8 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,
optionally substituted with R.sub.5, or part of a cyclic structure
with an E residue; v is an integer from 1-1000, for example 1-500,
1-200, 1-100, 1-50, 1-30, 1-20 or 1-10; and w is an integer from
3-1000, for example 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, or
3-10.
[0197] In some embodiments, each v and w is independently an
integer between 1-30. In some embodiments, w is an integer from
3-1000, for example 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, or 3-10.
In some embodiments, the sum of x+y+z is 3 or 6. In some
embodiments, the sum of x+y+z is 3. In other embodiments, the sum
of x+y+z is 6.
[0198] In some embodiments of any of the Formulas described herein,
at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino acid as the
amino acid at the corresponding position of the sequence
Phe.sub.3-X.sub.4-Hiss-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.sub.10-
-X.sub.11-Ser.sub.12 (SEQ ID NO: 8). In other embodiments, at least
four of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8,
Xaa.sub.9, and Xaa.sub.10 are the same amino acid as the amino acid
at the corresponding position of the sequence
Phe.sub.3-X.sub.4-Hiss-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.sub.10-
-X.sub.11-Ser.sub.12 (SEQ ID NO: 8). In other embodiments, at least
five of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8,
Xaa.sub.9, and Xaa.sub.10 are the same amino acid as the amino acid
at the corresponding position of the sequence
Phe.sub.3-X.sub.4-Hiss-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.sub.10-
-X.sub.11-Ser.sub.12 (SEQ ID NO: 8). In other embodiments, at least
six of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8,
Xaa.sub.9, and Xaa.sub.10 are the same amino acid as the amino acid
at the corresponding position of the sequence
Phe.sub.3-X.sub.4-Hiss-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.sub.10-
-X.sub.11-Ser.sub.12 (SEQ ID NO: 8). In other embodiments, at least
seven of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8,
Xaa.sub.9, and Xaa.sub.10 are the same amino acid as the amino acid
at the corresponding position of the sequence
Phe.sub.3-X.sub.4-Hiss-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.sub.10-
-X.sub.11-Ser.sub.12 (SEQ ID NO: 8).
[0199] In some embodiments, a peptidomimetic macrocycle has the
Formula:
##STR00016##
wherein: each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 9), where each X
is an amino acid; each D is independently an amino acid; each E is
independently an amino acid, for example an amino acid selected
from Ala (alanine), D-Ala (D-alanine), Aib (.alpha.-aminoisobutyric
acid), Sar (N-methyl glycine), and Ser (serine); each R.sub.1 and
R.sub.2 are independently --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,
unsubstituted or substituted with halo-; or at least one of R.sub.1
and R.sub.2 forms a macrocycle-forming linker L' connected to the
alpha position of one of said D or E amino acids; each L or L' is
independently a macrocycle-forming linker; each R.sub.5 is
independently halogen, alkyl, --OR.sub.6, --N(R.sub.6).sub.2,
--SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6, --CO.sub.2R.sub.6, a
fluorescent moiety, a radioisotope or a therapeutic agent; each
R.sub.6 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a
radioisotope or a therapeutic agent; each R.sub.7 is independently
--H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,
optionally substituted with R.sub.5, or part of a cyclic structure
with a D residue; each R.sub.8 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,
optionally substituted with R.sub.5, or part of a cyclic structure
with an E residue; v is an integer from 1-1000, for example 1-500,
1-200, 1-100, 1-50, 1-30, 1-20, or 1-10; w is an integer from
3-1000, for example 3-500, 3-200, 3-100, 3-50, 3-30, 3-20, or 3-10;
and.
[0200] In some embodiments of the above Formula, at least three of
Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8, Xaa.sub.9,
and Xaa.sub.10 are the same amino acid as the amino acid at the
corresponding position of the sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp-Ala.sub.8-Gln.sub.9-Leu.sub.10/-
Cba.sub.10-X.sub.1-Ala.sub.2 (SEQ ID NO: 9). In other embodiments
of the above Formula, at least four of Xaa.sub.3, Xaa.sub.5,
Xaa.sub.6, Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the
same amino acid as the amino acid at the corresponding position of
the sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 9). In other
embodiments of the above Formula, at least five of Xaa.sub.3,
Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and
Xaa.sub.10 are the same amino acid as the amino acid at the
corresponding position of the sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.1-Ala.sub.2 (SEQ ID NO: 9). In other
embodiments of the above Formula, at least six of Xaa.sub.3,
Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and
Xaa.sub.10 are the same amino acid as the amino acid at the
corresponding position of the sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 9). In other
embodiments of the above Formula, at least seven of Xaa.sub.3,
Xaa.sub.5, Xaa.sub.6, Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and
Xaa.sub.10 are the same amino acid as the amino acid at the
corresponding position of the sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 9).
[0201] In some embodiments, w is an integer from 3-10, for example
3-6, 3-8, 6-8, or 6-10. In some embodiments, w is 3. In other
embodiments, w is 6. In some embodiments, v is an integer from
1-10, for example 2-5. In some embodiments, v is 2.
[0202] In some embodiments, a peptidomimetic macrocycle of Formula
(I) has Formula (Ic):
##STR00017##
wherein: each A, C, D, and E is independently a natural or
non-natural amino acid; each B is independently a natural or
non-natural amino acid, amino acid analog,
##STR00018##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or [--NH-L.sub.3-];
each L is independently a macrocycle-forming linker; each L' is
independently alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, each
being optionally substituted with R.sub.5, or a bond, or together
with R.sub.1 and the atom to which both R.sub.1 and L' are bound
forms a ring; each L'' is independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
arylene, or heteroarylene, each being optionally substituted with
R.sub.5, or a bond, or together with R.sub.2 and the atom to which
both R.sub.2 and L'' are bound forms a ring; each R.sub.1 is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or
substituted with halo-, or together with L' and the atom to which
both R, and L' are bound forms a ring; each R.sub.2 is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or
substituted with halo-, or together with L'' and the atom to which
both R.sub.2 and L'' are bound forms a ring; R.sub.3 is hydrogen,
alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, cycloalkylalkyl, aryl, or heteroaryl, optionally
substituted with R.sub.5; each L.sub.3 is independently alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, heteroarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5; each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene; each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3; each n is an integer from 1-5; each R.sub.5 is
independently halogen, alkyl, --OR.sub.6, --N(R.sub.6).sub.2,
--SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6, --CO.sub.2R.sub.6, a
fluorescent moiety, a radioisotope or a therapeutic agent; each
R.sub.6 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a
radioisotope or a therapeutic agent; each R.sub.7 is independently
--H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally
substituted with R.sub.5, or part of a cyclic structure with a D
residue; each R.sub.8 is independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, aryl, or heteroaryl, optionally substituted with
R.sub.5, or part of a cyclic structure with an E residue; each v
and w is independently an integer from 1-1000, for example 1-500,
1-200, 1-100, 1-50, 1-40, 1-25, 1-20, 1-15, or 1-10; and each u, x,
y and z is independently an integer from 0-10.
[0203] In some embodiments, the peptidomimetic macrocycles have the
Formula (I):
##STR00019##
wherein: each A, C, D, and E is independently a natural or
non-natural amino acid; each B is independently a natural or
non-natural amino acid, amino acid analog,
##STR00020##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or [--NH-L.sub.3-];
each R.sub.1 and R.sub.2 is independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; each
R.sub.3 is independently hydrogen, alkyl, alkenyl, alkynyl,
arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, aryl, or heteroaryl, optionally substituted with
R.sub.5; each L is independently a macrocycle-forming linker of the
formula
##STR00021##
each L.sub.1, L.sub.2 and L.sub.3 is independently alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, heteroarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5; each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene; each K is independently O, S, SO, SO.sub.2, CO,
CO.sub.2, or CONR.sub.3; each R.sub.5 is independently halogen,
alkyl, --OR.sub.6, --N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6,
--SO.sub.2R.sub.6, --CO.sub.2R.sub.6, a fluorescent moiety, a
radioisotope or a therapeutic agent; each R.sub.6 is independently
--H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl,
heterocycloalkyl, a fluorescent moiety, a radioisotope or a
therapeutic agent; each R.sub.7 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally
substituted with R.sub.5, or part of a cyclic structure with a D
residue; each R.sub.8 is independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, aryl, or heteroaryl, optionally substituted with
R.sub.5, or part of a cyclic structure with an E residue; each v
and w is independently an integer from 1-1000; each u, x, y and z
is independently integers from 0-10; and n is an integer from
1-5.
[0204] In an embodiment of any of the Formulas described herein, of
the macrocycle-forming linker (L) has a formula -L.sub.1-L.sub.2-,
wherein
each L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5; each R.sub.4 is independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
arylene, or heteroarylene; each K is independently O, S, SO,
SO.sub.2, CO, CO.sub.2, or CONR.sub.3; each R.sub.3 is
independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R; and n
is an integer from 1-5.
[0205] In some embodiments in the Formulas described herein, L (or
L') is a macrocycle-forming linker of the formula
##STR00022##
[0206] Exemplary embodiments of such macrocycle-forming linkers L
are shown below.
##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027##
##STR00028## ##STR00029## ##STR00030## ##STR00031##
[0207] In an embodiment of any of the Formulas described herein,
L.sub.1 and L.sub.2, either alone or in combination, form a
triazole or a thioether.
[0208] In an embodiment of any of the Formulas described herein,
L.sub.1 and L.sub.2, either alone or in combination, do not form a
triazole or a thioether.
[0209] In one example, at least one of R.sub.1 and R.sub.2 is
alkyl, unsubstituted or substituted with halo-. In another example,
each R, and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some embodiments, at least one of R, and
R.sub.2 is methyl. In other embodiments, R.sub.1 and R.sub.2 are
methyl.
[0210] In some embodiments, x+y+z is at least 3. In other
embodiments, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some
embodiments, the sum of x+y+z is 3 or 6. In some embodiments, the
sum of x+y+z is 3. In other embodiments, the sum of x+y+z is 6.
Each occurrence of A, B, C, D or E in a macrocycle or macrocycle
precursor is independently selected. For example, a sequence
represented by the formula [A].sub.x, when x is 3, encompasses
embodiments where the amino acids are not identical, e.g.
Gln-Asp-Ala as well as embodiments where the amino acids are
identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or
z in the indicated ranges. Similarly, when u is greater than 1,
each compound can encompass peptidomimetic macrocycles which are
the same or different. For example, a compound can comprise
peptidomimetic macrocycles comprising different linker lengths or
chemical compositions.
[0211] In some embodiments, the peptidomimetic macrocycle comprises
a secondary structure which is an .alpha.-helix and R.sub.5 is --H,
allowing intrahelical hydrogen bonding. In some embodiments, at
least one of A, B, C, D or E is an .alpha.,.alpha.-disubstituted
amino acid. In one example, B is an .alpha.,.alpha.-disubstituted
amino acid. For instance, at least one of A, B, C, D or E is
2-aminoisobutyric acid. In other embodiments, at least one of A, B,
C, D or E is
##STR00032##
[0212] In other embodiments, the length of the macrocycle-forming
linker L as measured from a first C.alpha. to a second C.alpha. is
selected to stabilize a desired secondary peptide structure, such
as an .alpha.-helix formed by residues of the peptidomimetic
macrocycle including, but not necessarily limited to, those between
the first C.alpha. to a second C.alpha..
[0213] In one embodiment, the peptidomimetic macrocycle of Formula
(I) is:
##STR00033##
wherein each R.sub.1 and R.sub.2 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,
heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with
halo-.
[0214] In related embodiments, the peptidomimetic macrocycle of
Formula (I) is:
##STR00034##
wherein each R.sub.1' and R.sub.2' is independently an amino
acid.
[0215] In other embodiments, the peptidomimetic macrocycle of
Formula (I) is a compound of any of the formulas shown below:
##STR00035## ##STR00036##
wherein "AA" represents any natural or non-natural amino acid side
chain and "" is [D].sub.v, [E].sub.w as defined above, and n is an
integer between 0 and 20, 50, 100, 200, 300, 400 or 500. In some
embodiments, n is 0. In other embodiments, n is less than 50.
[0216] Exemplary embodiments of the macrocycle-forming linker L are
shown below.
##STR00037##
[0217] In other embodiments, D and/or E in the compound of Formula
I are further modified in order to facilitate cellular uptake. In
some embodiments, lipidating or PEGylating a peptidomimetic
macrocycle facilitates cellular uptake, increases bioavailability,
increases blood circulation, alters pharmacokinetics, decreases
immunogenicity and/or decreases the needed frequency of
administration.
[0218] In other embodiments, at least one of [D] and [E] in the
compound of Formula I represents a moiety comprising an additional
macrocycle-forming linker such that the peptidomimetic macrocycle
comprises at least two macrocycle-forming linkers. In a specific
embodiment, a peptidomimetic macrocycle comprises two
macrocycle-forming linkers. In an embodiment, u is 2.
[0219] In some embodiments, any of the macrocycle-forming linkers
described herein can be used in any combination with any of the
sequences shown in Table 3, Table 3a, Table 3b, or Table 3c and
also with any of the R-substituents indicated herein.
[0220] In some embodiments, the peptidomimetic macrocycle comprises
at least one .alpha.-helix motif. For example, A, B and/or C in the
compound of Formula I include one or more .alpha.-helices. As a
general matter, .alpha.-helices include between 3 and 4 amino acid
residues per turn. In some embodiments, the .alpha.-helix of the
peptidomimetic macrocycle includes 1 to 5 turns and, therefore, 3
to 20 amino acid residues. In specific embodiments, the
.alpha.-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5
turns. In some embodiments, the macrocycle-forming linker
stabilizes an .alpha.-helix motif included within the
peptidomimetic macrocycle. Thus, in some embodiments, the length of
the macrocycle-forming linker L from a first C.alpha. to a second
C.alpha. is selected to increase the stability of an .alpha.-helix.
In some embodiments, the macrocycle-forming linker spans from 1
turn to 5 turns of the .alpha.-helix. In some embodiments, the
macrocycle-forming linker spans approximately 1 turn, 2 turns, 3
turns, 4 turns, or 5 turns of the .alpha.-helix. In some
embodiments, the length of the macrocycle-forming linker is
approximately 5 .ANG. to 9 .ANG. per turn of the .alpha.-helix, or
approximately 6 .ANG. to 8 .ANG. per turn of the .alpha.-helix.
Where the macrocycle-forming linker spans approximately 1 turn of
an .alpha.-helix, the length is equal to approximately 5
carbon-carbon bonds to 13 carbon-carbon bonds, approximately 7
carbon-carbon bonds to 11 carbon-carbon bonds, or approximately 9
carbon-carbon bonds. Where the macrocycle-forming linker spans
approximately 2 turns of an .alpha.-helix, the length is equal to
approximately 8 carbon-carbon bonds to 16 carbon-carbon bonds,
approximately 10 carbon-carbon bonds to 14 carbon-carbon bonds, or
approximately 12 carbon-carbon bonds. Where the macrocycle-forming
linker spans approximately 3 turns of an .alpha.-helix, the length
is equal to approximately 14 carbon-carbon bonds to 22
carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20
carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where
the macrocycle-forming linker spans approximately 4 turns of an
.alpha.-helix, the length is equal to approximately 20
carbon-carbon bonds to 28 carbon-carbon bonds, approximately 22
carbon-carbon bonds to 26 carbon-carbon bonds, or approximately 24
carbon-carbon bonds. Where the macrocycle-forming linker spans
approximately 5 turns of an .alpha.-helix, the length is equal to
approximately 26 carbon-carbon bonds to 34 carbon-carbon bonds,
approximately 28 carbon-carbon bonds to 32 carbon-carbon bonds, or
approximately 30 carbon-carbon bonds. Where the macrocycle-forming
linker spans approximately 1 turn of an .alpha.-helix, the linkage
contains approximately 4 atoms to 12 atoms, approximately 6 atoms
to 10 atoms, or approximately 8 atoms. Where the macrocycle-forming
linker spans approximately 2 turns of the .alpha.-helix, the
linkage contains approximately 7 atoms to 15 atoms, approximately 9
atoms to 13 atoms, or approximately 11 atoms. Where the
macrocycle-forming linker spans approximately 3 turns of the
.alpha.-helix, the linkage contains approximately 13 atoms to 21
atoms, approximately 15 atoms to 19 atoms, or approximately 17
atoms. Where the macrocycle-forming linker spans approximately 4
turns of the .alpha.-helix, the linkage contains approximately 19
atoms to 27 atoms, approximately 21 atoms to 25 atoms, or
approximately 23 atoms. Where the macrocycle-forming linker spans
approximately 5 turns of the .alpha.-helix, the linkage contains
approximately 25 atoms to 33 atoms, approximately 27 atoms to 31
atoms, or approximately 29 atoms. Where the macrocycle-forming
linker spans approximately 1 turn of the .alpha.-helix, the
resulting macrocycle forms a ring containing approximately 17
members to 25 members, approximately 19 members to 23 members, or
approximately 21 members. Where the macrocycle-forming linker spans
approximately 2 turns of the .alpha.-helix, the resulting
macrocycle forms a ring containing approximately 29 members to 37
members, approximately 31 members to 35 members, or approximately
33 members. Where the macrocycle-forming linker spans approximately
3 turns of the .alpha.-helix, the resulting macrocycle forms a ring
containing approximately 44 members to 52 members, approximately 46
members to 50 members, or approximately 48 members. Where the
macrocycle-forming linker spans approximately 4 turns of the
.alpha.-helix, the resulting macrocycle forms a ring containing
approximately 59 members to 67 members, approximately 61 members to
65 members, or approximately 63 members. Where the
macrocycle-forming linker spans approximately 5 turns of the
.alpha.-helix, the resulting macrocycle forms a ring containing
approximately 74 members to 82 members, approximately 76 members to
80 members, or approximately 78 members.
[0221] In other embodiments, provided are peptidomimetic
macrocycles of Formula (IV) or (IVa):
##STR00038##
wherein: each A, C, D, and E is independently a natural or
non-natural amino acid, and the terminal D and E independently
optionally include a capping group; each B is a natural or
non-natural amino acid, amino acid analog,
##STR00039##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or [--NH-L.sub.3-];
each R, and R.sub.2 are independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R, and R.sub.2 forms a macrocycle-forming linker L'
connected to the alpha position of one of said D or E amino acids;
each R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5;
each L is a macrocycle-forming linker of the formula
-L.sub.1-L.sub.2-; each L.sub.1, L.sub.2 and L.sub.3 are
independently alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, cycloarylene,
heterocycloarylene, or [--R.sub.4--K--R.sub.4--].sub.n, each being
optionally substituted with R.sub.5; each R.sub.4 is alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene; each K is O, S, SO,
SO.sub.2, CO, CO.sub.2, or CONR.sub.3; each R.sub.5 is
independently halogen, alkyl, --OR, --N(R.sub.6).sub.2, --SR.sub.6,
--SOR.sub.6, --SO.sub.2R.sub.6, --CO.sub.2R.sub.6, a fluorescent
moiety, a radioisotope or a therapeutic agent; each R.sub.6 is
independently --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a
radioisotope or a therapeutic agent; each R.sub.7 is --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,
cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,
optionally substituted with R.sub.5; each v and w are independently
integers from 1-1000; u is an integer from 1-10; each x, y and z
are independently integers from 0-10; and each n is an integer from
1-5.
[0222] In one example, L.sub.1 and L.sub.2, either alone or in
combination, do not form a triazole or a thioether.
[0223] In one example, at least one of R.sub.1 and R.sub.2 is
alkyl, unsubstituted or substituted with halo-. In another example,
both R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some embodiments, at least one of
R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and
R.sub.2 are methyl.
[0224] In some embodiments, x+y+z is at least 1. In other
embodiments, x+y+z is at least 2. In other embodiments, x+y+z is 1,
2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence of A, B, C, D or E in
a macrocycle or macrocycle precursor is independently selected. For
example, a sequence represented by the formula [A].sub.x, when x is
3, encompasses embodiments where the amino acids are not identical,
e.g. Gln-Asp-Ala as well as embodiments where the amino acids are
identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or
z in the indicated ranges.
[0225] In some embodiments, the peptidomimetic macrocycle comprises
a secondary structure which is an .alpha.-helix and R.sub.8 is --H,
allowing intrahelical hydrogen bonding. In some embodiments, at
least one of A, B, C, D or E is an .alpha.,.alpha.-disubstituted
amino acid. In one example, B is an .alpha.,.alpha.-disubstituted
amino acid. For instance, at least one of A, B, C, D or E is
2-aminoisobutyric acid. In other embodiments, at least one of A, B,
C, D or E is
##STR00040##
[0226] In other embodiments, the length of the macrocycle-forming
linker L as measured from a first C.alpha. to a second C.alpha. is
selected to stabilize a desired secondary peptide structure, such
as an .alpha.-helix formed by residues of the peptidomimetic
macrocycle including, but not necessarily limited to, those between
the first C.alpha. to a second C.alpha..
[0227] Exemplary embodiments of the macrocycle-forming linker
-L.sub.1-L.sub.2-are shown below.
##STR00041##
[0228] Unless otherwise stated, any compounds (including
peptidomimetic macrocycles, macrocycle precursors, and other
compositions) are also meant to encompass compounds which differ
only in the presence of one or more isotopically enriched atoms.
For example, compounds having the described structures except for
the replacement of a hydrogen by a deuterium or tritium, or the
replacement of a carbon by .sup.13C- or .sup.14C-enriched carbon
are within the scope of this disclosure.
[0229] In some embodiments, the compounds disclosed herein can
contain unnatural proportions of atomic isotopes at one or more of
atoms that constitute such compounds. For example, the compounds
can be radiolabeled with radioactive isotopes, such as for example
tritium (.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C).
In other embodiments, one or more carbon atoms is replaced with a
silicon atom. All isotopic variations of the compounds disclosed
herein, whether radioactive or not, are contemplated herein.
[0230] The circulating half-life of the peptidomimetic macrocycles
in human blood can be about 1-24 h. For example the circulating
half-life of the peptidomimetic macrocycles in human blood can be
about 2-24 h, 4-24 h, 6-24 h, 8-24 h, 10-24 h, 12-24 h, 14-24 h,
16-24 h, 18-24 h, 20-24 h, 22-24 h, 1-20 h, 4-20 h, 6-20 h, 8-20 h,
10-20 h, 12-20 h, 14-20 h, 16-20 h, 18-20 h, 1-16 h, 4-16 h, 6-16
h, 8-16 h, 10-16 h, 12-16 h, 14-16 h, 1-12 h, 4-12 h, 6-12 h, 8-12
h, 10-12 h, 1-8 h, 4-8 h, 6-8 h, or 1-4 h. In some examples, the
circulating half-life of the peptidomimetic macrocycles in human
blood can be bout 1-12 h, for example about 1 h, 2 h, 3 h, 4 h, 5
h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, or 12 h. In some examples, the
circulating half-life of the peptidomimetic macrocycles in human
blood is about 2 h. In some examples, the circulating half-life of
the peptidomimetic macrocycles in human blood is about 4 h. In some
examples, the circulating half-life of the peptidomimetic
macrocycles in human blood is about 6 h. In some examples, the
circulating half-life of the peptidomimetic macrocycles in human
blood is about 8 h. In some examples, the circulating half-life of
the peptidomimetic macrocycles in human blood is about 10 h.
[0231] The half-life of the peptidomimetic macrocycles in
biological tissue can be about 1-24 h. For example the circulating
half-life of the peptidomimetic macrocycles in human blood can be
about 1-24 h, 5-24 h, 10-24 h, 15-24 h, 20-24 h, 1-22 h, 5-22 h,
10-22 h, 15-22 h, 20-22 h, 1-20 h, 5-20 h, 15-20 h, 1-18 h, 5-18 h,
10-18 h, 15-18 h, 1-16 h, 5-16 h, 10-16 h, 15-16 h, 1-14 h, 5-14 h,
10-14 h, 1-12 h, 5-12 h, 10-12 h, 1-10 h, 5-10 h, 1-8 h, 5-8 h, 1-6
h, 5-6 h, or 1-4 h. In some examples, the circulating half-life of
the peptidomimetic macrocycles in human blood can be bout 5-20 h,
for example about 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h,
14 h, 15 h, 16 h, 17 h, 18 h, 19 h or 20 h. In some examples, the
circulating half-life of the peptidomimetic macrocycles in human
blood is about 2 h. In some examples, the circulating half-life of
the peptidomimetic macrocycles in human blood is about 4 h. In some
examples, the circulating half-life of the peptidomimetic
macrocycles in human blood is about 6 h. In some examples, the
circulating half-life of the peptidomimetic macrocycles in human
blood is about 8 h. In some examples, the circulating half-life of
the peptidomimetic macrocycles in human blood is about 10 h.
[0232] The circulating half-life of the peptidomimetic macrocycles
in human blood can be greater than, equal to, or less than the
half-life of the peptidomimetic macrocycles in biological tissue.
In some examples, the circulating half-life of the peptidomimetic
macrocycles in human blood can be greater than the half-life of the
peptidomimetic macrocycles in biological tissue. In some examples,
the circulating half-life of the peptidomimetic macrocycles in
human blood can be equal to the half-life of the peptidomimetic
macrocycles in biological tissue. In some examples, the half-life
of the peptidomimetic macrocycles in biological tissue is greater
than the circulating half-life of the peptidomimetic macrocycles in
human blood. This can facilitate administration of the
peptidomimetic macrocycles at a lower dose and/or at lower
frequency. In some embodiments, the half-life of the peptidomimetic
macrocycles in biological tissue is at least 1 h, 2 h, 3 h, 4 h, 5
h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, or 12 h greater than the than
the circulating half-life of the peptidomimetic macrocycles in
human blood. In some examples, the circulating half-life of the
peptidomimetic macrocycles in human blood is about 4 h and the
half-life of the in biological tissue is about 10 h. In some
examples, the circulating half-life of the peptidomimetic
macrocycles in human blood is about 6 h and the half-life of the in
biological tissue is about 10 h.
Preparation of Peptidomimetic Macrocycles
[0233] Peptidomimetic macrocycles can be prepared by any of a
variety of methods known in the art. For example, any of the
residues indicated by "$" or "$r8" in Table 3, Table 3a, Table 3b,
or Table 3c can be substituted with a residue capable of forming a
crosslinker with a second residue in the same molecule or a
precursor of such a residue.
[0234] Various methods to effect formation of peptidomimetic
macrocycles are known in the art. For example, the preparation of
peptidomimetic macrocycles of Formula I is described in
Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000);
Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005);
Walensky et al., Science 305:1466-1470 (2004); U.S. Pat. No.
7,192,713 and PCT application WO 2008/121767. The
.alpha.,.alpha.-disubstituted amino acids and amino acid precursors
disclosed in the cited references can be employed in synthesis of
the peptidomimetic macrocycle precursor polypeptides. For example,
the "S5-olefin amino acid" is (S)-.alpha.-(2'-pentenyl) alanine and
the "R8 olefin amino acid" is (R)-.alpha.-(2'-octenyl) alanine.
Following incorporation of such amino acids into precursor
polypeptides, the terminal olefins are reacted with a metathesis
catalyst, leading to the formation of the peptidomimetic
macrocycle. In various embodiments, the following amino acids can
be employed in the synthesis of the peptidomimetic macrocycle:
##STR00042## ##STR00043##
[0235] In other embodiments, the peptidomimetic macrocycles are of
Formula IV or IVa. Methods for the preparation of such macrocycles
are described, for example, in U.S. Pat. No. 7,202,332.
[0236] Additional methods of forming peptidomimetic macrocycles
which are envisioned as suitable include those disclosed by
Mustapa, M. Firouz Mohd et al., J. Org. Chem (2003), 68, pp.
8193-8198; Yang, Bin et al. Bioorg Med. Chem. Lett. (2004), 14, pp.
1403-1406; U.S. Pat. Nos. 5,364,851; 5,446,128; 5,824,483;
6,713,280; and 7,202,332. In such embodiments, amino acid
precursors are used containing an additional substituent R-- at the
alpha position. Such amino acids are incorporated into the
macrocycle precursor at the desired positions, which can be at the
positions where the crosslinker is substituted or, alternatively,
elsewhere in the sequence of the macrocycle precursor. Cyclization
of the precursor is then effected according to the indicated
method.
[0237] The peptidomimetic macrocycles described herein can be at
least 1% pure, at least 2% pure, at least 3% pure, at least 4%
pure, at least 5% pure, at least 6% pure, at least 7% pure, at
least 8% pure, at least 9% pure, at least 10% pure, at least 11%
pure, at least 12% pure, at least 13% pure, at least 14% pure, at
least 15% pure, at least 16% pure, at least 17% pure, at least 18%
pure, at least 19% pure, at least 20% pure, at least 21% pure, at
least 22% pure, at least 23% pure, at least 24% pure, at least 25%
pure, at least 26% pure, at least 27% pure, at least 28% pure, at
least 29% pure, at least 30% pure, at least 31% pure, at least 32%
pure, at least 33% pure, at least 34% pure, at least 35% pure, at
least 36% pure, at least 37% pure, at least 38% pure, at least 39%
pure, at least 40% pure, at least 41% pure, at least 42% pure, at
least 43% pure, at least 44% pure, at least 45% pure, at least 46%
pure, at least 47% pure, at least 48% pure, at least 49% pure, at
least 50% pure, at least 51% pure, at least 52% pure, at least 53%
pure, at least 54% pure, at least 55% pure, at least 56% pure, at
least 57% pure, at least 58% pure, at least 59% pure, at least 60%
pure, at least 61% pure, at least 62% pure, at least 63% pure, at
least 64% pure, at least 65% pure, at least 66% pure, at least 67%
pure, at least 68% pure, at least 69% pure, at least 70% pure, at
least 71% pure, at least 72% pure, at least 73% pure, at least 74%
pure, at least 75% pure, at least 76% pure, at least 77% pure, at
least 78% pure, at least 79% pure, at least 80% pure, at least 81%
pure, at least 82% pure, at least 83% pure, at least 84% pure, at
least 85% pure, at least 86% pure, at least 87% pure, at least 88%
pure, at least 89% pure, at least 90% pure, at least 91% pure, at
least 92% pure, at least 93% pure, at least 94% pure, at least 95%
pure, at least 96% pure, at least 97% pure, at least 98% pure, at
least 99% pure, at least 99.1% pure, at least 99.2% pure, at least
99.3% pure, at least 99.4% pure, at least 99.5% pure, at least
99.6% pure, at least 99.7% pure, at least 99.8% pure, or at least
99.9% pure on a chemical, optical, isomeric, enantiomeric, or
diastereomeric basis. Purity can be assessed, for example, by HPLC,
MS, LC/MS, melting point, or NMR.
[0238] Two or more peptides/peptidomimetic macrocycles can share a
degree of homology. A pair of peptides/peptidomimetic macrocycles
can have, for example, up to about 20% pairwise homology, up to
about 25% pairwise homology, up to about 30% pairwise homology, up
to about 35% pairwise homology, up to about 40% pairwise homology,
up to about 45% pairwise homology, up to about 50% pairwise
homology, up to about 55% pairwise homology, up to about 60%
pairwise homology, up to about 65% pairwise homology, up to about
70% pairwise homology, up to about 75% pairwise homology, up to
about 80% pairwise homology, up to about 85% pairwise homology, up
to about 90% pairwise homology, up to about 95% pairwise homology,
up to about 96% pairwise homology, up to about 97% pairwise
homology, up to about 98% pairwise homology, up to about 99%
pairwise homology, up to about 99.5% pairwise homology, or up to
about 99.9% pairwise homology. A pair of peptides can have, for
example, at least about 20% pairwise homology, at least about 25%
pairwise homology, at least about 30% pairwise homology, at least
about 35% pairwise homology, at least about 40% pairwise homology,
at least about 45% pairwise homology, at least about 50% pairwise
homology, at least about 55% pairwise homology, at least about 60%
pairwise homology, at least about 65% pairwise homology, at least
about 70% pairwise homology, at least about 75% pairwise homology,
at least about 80% pairwise homology, at least about 85% pairwise
homology, at least about 90% pairwise homology, at least about 95%
pairwise homology, at least about 96% pairwise homology, at least
about 97% pairwise homology, at least about 98% pairwise homology,
at least about 99% pairwise homology, at least about 99.5% pairwise
homology, at least about 99.9% pairwise homology.
[0239] Various methods and software programs can be used to
determine the homology between two or more peptides, such as NCBI
BLAST, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, or
another suitable method or algorithm.
Assays
[0240] The properties of peptidomimetic macrocycles are assayed,
for example, by using the methods described below. In some
embodiments, a peptidomimetic macrocycle has improved biological
properties relative to a corresponding polypeptide lacking the
substituents described herein.
Assay to Determine .alpha.-Helicity
[0241] In solution, the secondary structure of polypeptides with
.alpha.-helical domains will reach a dynamic equilibrium between
random coil structures and .alpha.-helical structures, often
expressed as a "percent helicity". Thus, for example, alpha-helical
domains are predominantly random coils in solution, with
.alpha.-helical content usually under 25%. Peptidomimetic
macrocycles with optimized linkers, on the other hand, possess, for
example, an alpha-helicity that is at least two-fold greater than
that of a corresponding uncrosslinked polypeptide. In some
embodiments, macrocycles will possess an alpha-helicity of greater
than 50%. To assay the helicity of peptidomimetic macrocycles, the
compounds are dissolved in an aqueous solution (e.g. 50 mM
potassium phosphate solution at pH 7, or distilled H.sub.2O, to
concentrations of 25-50 .mu.M). Circular dichroism (CD) spectra are
obtained on a spectropolarimeter (e.g., Jasco J-710) using standard
measurement parameters (e.g. temperature, 20.degree. C.;
wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec;
accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length,
0.1 cm). The .alpha.-helical content of each peptide is calculated
by dividing the mean residue ellipticity (e.g. [.PHI.]222obs) by
the reported value for a model helical decapeptide (Yang et al.
(1986), Methods Enzymol. 130:208)).
Assay to Determine Melting Temperature (Tm)
[0242] A peptidomimetic macrocycle comprising a secondary structure
such as an .alpha.-helix exhibits, for example, a higher melting
temperature than a corresponding uncrosslinked polypeptide.
Typically peptidomimetic macrocycles exhibit Tm of >60.degree.
C. representing a highly stable structure in aqueous solutions. To
assay the effect of macrocycle formation on melting temperature,
peptidomimetic macrocycles or unmodified peptides are dissolved in
distilled H.sub.2O (e.g. at a final concentration of 50 .mu.M) and
the Tm is determined by measuring the change in ellipticity over a
temperature range (e.g. 4 to 95.degree. C.) on a spectropolarimeter
(e.g., Jasco J-710) using standard parameters (e.g. wavelength 222
nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10;
response, 1 sec; bandwidth, 1 nm; temperature increase rate:
1.degree. C./min; path length, 0.1 cm).
Protease Resistance Assay
[0243] The amide bond of the peptide backbone is susceptible to
hydrolysis by proteases, thereby rendering peptidic compounds
vulnerable to rapid degradation in vivo. Peptide helix formation,
however, typically buries the amide backbone and therefore can
shield it from proteolytic cleavage. The peptidomimetic macrocycles
can be subjected to in vitro trypsin proteolysis to assess for any
change in degradation rate compared to a corresponding
uncrosslinked polypeptide. For example, the peptidomimetic
macrocycle and a corresponding uncrosslinked polypeptide are
incubated with trypsin agarose and the reactions quenched at
various time points by centrifugation and subsequent HPLC injection
to quantitate the residual substrate by ultraviolet absorption at
280 nm. Briefly, the peptidomimetic macrocycle and peptidomimetic
precursor (5 mcg) are incubated with trypsin agarose (Pierce) (S/E
.about.125) for 0, 10, 20, 90, and 180 minutes. Reactions are
quenched by tabletop centrifugation at high speed; remaining
substrate in the isolated supernatant is quantified by HPLC-based
peak detection at 280 nm. The proteolytic reaction displays first
order kinetics and the rate constant, k, is determined from a plot
of In[S] versus time (k=-1.times.slope).
Ex Vivo Stability Assay
[0244] Peptidomimetic macrocycles with optimized linkers possess,
for example, an ex vivo half-life that is at least two-fold greater
than that of a corresponding uncrosslinked polypeptide, and possess
an ex vivo half-life of 12 hours or more. For ex vivo serum
stability studies, a variety of assays can be used. For example, a
peptidomimetic macrocycle and a corresponding uncrosslinked
polypeptide (2 mcg) are incubated with fresh mouse, rat and/or
human serum (2 mL) at 37.degree. C. for 0, 1, 2, 4, 8, and 24
hours. To determine the level of intact compound, the following
procedure can be used: The samples are extracted by transferring
100 .mu.l of serato 2 ml centrifuge tubes followed by the addition
of 10 .mu.L of 50% formic acid and 500 .mu.L acetonitrile and
centrifugation at 14,000 RPM for 10 min at 4.+-.2.degree. C. The
supernatants are then transferred to fresh 2 ml tubes and
evaporated on Turbovap under N.sub.2<10 psi, 37.degree. C. The
samples are reconstituted in 100 .mu.L of 50:50 acetonitrile:water
and submitted to LC-MS/MS analysis.
In vitro Binding Assays
[0245] To assess the binding and affinity of peptidomimetic
macrocycles and peptidomimetic precursors to acceptor proteins, a
fluorescence polarization assay (FPA) is used, for example. The FPA
technique measures the molecular orientation and mobility using
polarized light and fluorescent tracer. When excited with polarized
light, fluorescent tracers (e.g., FITC) attached to molecules with
high apparent molecular weights (e.g. FITC-labeled peptides bound
to a large protein) emit higher levels of polarized fluorescence
due to their slower rates of rotation as compared to fluorescent
tracers attached to smaller molecules (e.g. FITC-labeled peptides
that are free in solution).
[0246] For example, fluoresceinated peptidomimetic macrocycles (25
nM) are incubated with the acceptor protein (25-1000 nM) in binding
buffer (140 mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room
temperature. Binding activity is measured, for example, by
fluorescence polarization on a luminescence spectrophotometer (e.g.
Perkin-Elmer LS50B). Kd values can be determined by nonlinear
regression analysis using, for example, Graphpad Prism software
(GraphPad Software, Inc., San Diego, Calif.). A peptidomimetic
macrocycle shows, In some embodiments, similar or lower Kd than a
corresponding uncrosslinked polypeptide.
In Vitro Displacement Assays to Characterize Antagonists of
Peptide-Protein Interactions
[0247] To assess the binding and affinity of compounds that
antagonize the interaction between a peptide and an acceptor
protein, a fluorescence polarization assay (FPA) utilizing a
fluoresceinated peptidomimetic macrocycle derived from a
peptidomimetic precursor sequence is used, for example. The FPA
technique measures the molecular orientation and mobility using
polarized light and fluorescent tracer. When excited with polarized
light, fluorescent tracers (e.g., FITC) attached to molecules with
high apparent molecular weights (e.g. FITC-labeled peptides bound
to a large protein) emit higher levels of polarized fluorescence
due to their slower rates of rotation as compared to fluorescent
tracers attached to smaller molecules (e.g. FITC-labeled peptides
that are free in solution). A compound that antagonizes the
interaction between the fluoresceinated peptidomimetic macrocycle
and an acceptor protein will be detected in a competitive binding
FPA experiment.
[0248] For example, putative antagonist compounds (1 nM to 1 mM)
and a fluoresceinated peptidomimetic macrocycle (25 nM) are
incubated with the acceptor protein (50 nM) in binding buffer (140
mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room
temperature. Antagonist binding activity is measured, for example,
by fluorescence polarization on a luminescence spectrophotometer
(e.g. Perkin-Elmer LS50B). Kd values can be determined by nonlinear
regression analysis using, for example, Graphpad Prism software
(GraphPad Software, Inc., San Diego, Calif.).
[0249] Any class of molecule, such as small organic molecules,
peptides, oligonucleotides or proteins can be examined as putative
antagonists in this assay.
Assay for Protein-Ligand Binding by Affinity Selection-Mass
Spectrometry
[0250] To assess the binding and affinity of test compounds for
proteins, an affinity-selection mass spectrometry assay is used,
for example. Protein-ligand binding experiments are conducted
according to the following representative procedure outlined for a
system-wide control experiment using 1 .mu.M peptidomimetic
macrocycle plus 5 .mu.M hMDM2. A 1 .mu.L DMSO aliquot of a 40 .mu.M
stock solution of peptidomimetic macrocycle is dissolved in 19
.mu.L of PBS (Phosphate-buffered saline: 50 mM, pH 7.5 Phosphate
buffer containing 150 mM NaCl). The resulting solution is mixed by
repeated pipetting and clarified by centrifugation at 10 000 g for
10 min. To a 4 .mu.L aliquot of the resulting supernatant is added
4 .mu.L of 10 M hMDM2 in PBS. Each 8.0 .mu.L experimental sample
thus contains 40 pmol (1.5 .mu.g) of protein at 5.0 M concentration
in PBS plus 1 .mu.M peptidomimetic macrocycle and 2.5% DMSO.
Duplicate samples thus prepared for each concentration point are
incubated for 60 min at room temperature, and then chilled to
4.degree. C. prior to size-exclusion chromatography-LC-MS analysis
of 5.0 .mu.L injections. Samples containing a target protein,
protein-ligand complexes, and unbound compounds are injected onto
an SEC column, where the complexes are separated from non-binding
component by a rapid SEC step. The SEC column eluate is monitored
using UV detectors to confirm that the early-eluting protein
fraction, which elutes in the void volume of the SEC column, is
well resolved from unbound components that are retained on the
column. After the peak containing the protein and protein-ligand
complexes elutes from the primary UV detector, it enters a sample
loop where it is excised from the flow stream of the SEC stage and
transferred directly to the LC-MS via a valving mechanism. The
(M+3H).sup.3+ ion of the peptidomimetic macrocycle is observed by
ESI-MS at the expected m/z, confirming the detection of the
protein-ligand complex.
Assay for Protein-Ligand Kd Titration Experiments
[0251] To assess the binding and affinity of test compounds for
proteins, a protein-ligand Kd titration experiment is performed,
for example. Protein-ligand K.sub.d titrations experiments are
conducted as follows: 2 .mu.L DMSO aliquots of a serially diluted
stock solution of titrant peptidomimetic macrocycle (5, 2.5, . . .
, 0.098 mM) are prepared then dissolved in 38 .mu.L of PBS. The
resulting solutions are mixed by repeated pipetting and clarified
by centrifugation at 10 000 g for 10 min. To 4.0 .mu.L aliquots of
the resulting supernatants is added 4.0 .mu.L of 10 M hMDM2 in PBS.
Each 8.0 .mu.L experimental sample thus contains 40 pmol (1.5
.mu.g) of protein at 5.0 .mu.M concentration in PBS, varying
concentrations (125, 62.5, . . . , 0.24 .mu.M) of the titrant
peptide, and 2.5% DMSO. Duplicate samples thus prepared for each
concentration point are incubated at room temperature for 30 min,
then chilled to 4.degree. C. prior to SEC-LC-MS analysis of 2.0
.mu.L injections. The (M+H).sup.1+, (M+2H).sup.2+, (M+3H).sup.3+,
and/or (M+Na).sup.1+ ion is observed by ESI-MS; extracted ion
chromatograms are quantified, then fit to equations to derive the
binding affinity K.sub.d as described in "A General Technique to
Rank Protein-Ligand Binding Affinities and Determine Allosteric vs.
Direct Binding Site Competition in Compound Mixtures." Annis, D.
A.; Nazef, N.; Chuang, C. C.; Scott, M. P.; Nash, H. M. J Am. Chem.
Soc. 2004, 126, 15495-15503; also in "ALIS: An Affinity
Selection-Mass Spectrometry System for the Discovery and
Characterization of Protein-Ligand Interactions" D. A. Annis, C.-C.
Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry.
Edited by Wanner K, Hofner G: Wiley-VCH; 2007:121-184. Mannhold R,
Kubinyi H, Folkers G (Series Editors): Methods and Principles in
Medicinal Chemistry.
Assay for Competitive Binding Experiments by Affinity
Selection-Mass Spectrometry
[0252] To determine the ability of test compounds to bind
competitively to proteins, an affinity selection mass spectrometry
assay is performed, for example. A mixture of ligands at 40 .mu.M
per component is prepared by combining 2 .mu.L aliquots of 400
.mu.M stocks of each of the three compounds with 14 .mu.L of DMSO.
Then, 1 .mu.L aliquots of this 40 .mu.M per component mixture are
combined with 1 .mu.L DMSO aliquots of a serially diluted stock
solution of titrant peptidomimetic macrocycle (10, 5, 2.5, . . . ,
0.078 mM). These 2 L samples are dissolved in 38 .mu.L of PBS. The
resulting solutions were mixed by repeated pipetting and clarified
by centrifugation at 10 000 g for 10 min. To 4.0 .mu.L aliquots of
the resulting supernatants is added 4.0 .mu.L of 10 .mu.M hMDM2
protein in PBS. Each 8.0 .mu.L experimental sample thus contains 40
pmol (1.5 .mu.g) of protein at 5.0 .mu.M concentration in PBS plus
0.5 .mu.M ligand, 2.5% DMSO, and varying concentrations (125, 62.5,
. . . , 0.98 .mu.M) of the titrant peptidomimetic macrocycle.
Duplicate samples thus prepared for each concentration point are
incubated at room temperature for 60 min, then chilled to 4.degree.
C. prior to SEC-LC-MS analysis of 2.0 .mu.L injections. Additional
details on these and other methods are provided in "A General
Technique to Rank Protein-Ligand Binding Affinities and Determine
Allosteric vs. Direct Binding Site Competition in Compound
Mixtures." Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.;
Nash, H. M. J Am. Chem. Soc. 2004, 126, 15495-15503; also in "ALIS:
An Affinity Selection-Mass Spectrometry System for the Discovery
and Characterization of Protein-Ligand Interactions" D. A. Annis,
C.-C. Chuang, and N. Nazef. In Mass Spectrometry in Medicinal
Chemistry. Edited by Wanner K, Hofner G: Wiley-VCH; 2007:121-184.
Mannhold R, Kubinyi H, Folkers G (Series Editors): Methods and
Principles in Medicinal Chemistry.
Binding Assays in Intact Cells
[0253] It is possible to measure binding of peptides or
peptidomimetic macrocycles to their natural acceptors in intact
cells by immunoprecipitation experiments. For example, intact cells
are incubated with fluoresceinated (FITC-labeled) compounds for 4
hrs in the absence of serum, followed by serum replacement and
further incubation that ranges from 4-18 hrs. Cells are then
pelleted and incubated in lysis buffer (50 mM Tris [pH 7.6], 150 mM
NaCl, 1% CHAPS and protease inhibitor cocktail) for 10 minutes at
4.degree. C. Extracts are centrifuged at 14,000 rpm for 15 minutes
and supernatants collected and incubated with 10 .mu.l goat
anti-FITC antibody for 2 hrs, rotating at 4.degree. C. followed by
further 2 hrs incubation at 4.degree. C. with protein A/G Sepharose
(50 .mu.l of 50% bead slurry). After quick centrifugation, the
pellets are washed in lysis buffer containing increasing salt
concentration (e.g., 150, 300, 500 mM). The beads are then
re-equilibrated at 150 mM NaCl before addition of SDS-containing
sample buffer and boiling. After centrifugation, the supernatants
are optionally electrophoresed using 4%-12% gradient Bis-Tris gels
followed by transfer into Immobilon-P membranes. After blocking,
blots are optionally incubated with an antibody that detects FITC
and also with one or more antibodies that detect proteins that bind
to the peptidomimetic macrocycle.
Cellular Penetrability Assays
[0254] A peptidomimetic macrocycle is, for example, more cell
penetrable compared to a corresponding uncrosslinked macrocycle.
Peptidomimetic macrocycles with optimized linkers possess, for
example, cell penetrability that is at least two-fold greater than
a corresponding uncrosslinked macrocycle, and often 20% or more of
the applied peptidomimetic macrocycle will be observed to have
penetrated the cell after 4 hours. To measure the cell
penetrability of peptidomimetic macrocycles and corresponding
uncrosslinked macrocycle, intact cells are incubated with
fluorescently-labeled (e.g. fluoresceinated) peptidomimetic
macrocycles or corresponding uncrosslinked macrocycle (10 .mu.M)
for 4 hrs in serum free media at 37.degree. C., washed twice with
media and incubated with trypsin (0.25%) for 10 min at 37.degree.
C. The cells are washed again and resuspended in PBS. Cellular
fluorescence is analyzed, for example, by using either a
FACSCalibur flow cytometer or Cellomics' KineticScan.RTM. HCS
Reader.
Cellular Efficacy Assays
[0255] The efficacy of certain peptidomimetic macrocycles is
determined, for example, in cell-based killing assays using a
variety of tumorigenic and non-tumorigenic cell lines and primary
cells derived from human or mouse cell populations. Cell viability
is monitored, for example, over 24-96 hrs of incubation with
peptidomimetic macrocycles (0.5 to 50 .mu.M) to identify those that
kill at EC.sub.50<10 .mu.M. Several standard assays that measure
cell viability are commercially available and are optionally used
to assess the efficacy of the peptidomimetic macrocycles. In
addition, assays that measure Annexin V and caspase activation are
optionally used to assess whether the peptidomimetic macrocycles
kill cells by activating the apoptotic machinery. For example, the
Cell Titer-glo assay is used which determines cell viability as a
function of intracellular ATP concentration.
In Vivo Stability Assay
[0256] To investigate the in vivo stability of the peptidomimetic
macrocycles, the compounds are, for example, administered to mice
and/or rats by IV, IP, PO or inhalation routes at concentrations
ranging from 0.1 to 50 mg/kg and blood specimens withdrawn at 0',
5', 15', 30', 1 hr, 4 hrs, 8 hrs and 24 hours post-injection.
Levels of intact compound in 25 .mu.L of fresh serum are then
measured by LC-MS/MS as above.
In Vivo Efficacy in Animal Models
[0257] To determine the anti-oncogenic activity of peptidomimetic
macrocycles in vivo, the compounds are, for example, given alone
(IP, IV, PO, by inhalation or nasal routes) or in combination with
sub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide,
doxorubicin, etoposide). In one example, 5.times.10.sup.6 RS4;11
cells (established from the bone marrow of a patient with acute
lymphoblastic leukemia) that stably express luciferase are injected
by tail vein in NOD-SCID mice 3 hrs after they have been subjected
to total body irradiation. If left untreated, this form of leukemia
is fatal in 3 weeks in this model. The leukemia is readily
monitored, for example, by injecting the mice with D-luciferin (60
mg/kg) and imaging the anesthetized animals (e.g., Xenogen In Vivo
Imaging System, Caliper Life Sciences, Hopkinton, Mass.). Total
body bioluminescence is quantified by integration of photonic flux
(photons/sec) by Living Image Software (Caliper Life Sciences,
Hopkinton, Mass.). Peptidomimetic macrocycles alone or in
combination with sub-optimal doses of relevant chemotherapeutics
agents are, for example, administered to leukemic mice (10 days
after injection/day 1 of experiment, in bioluminescence range of
14-16) by tail vein or IP routes at doses ranging from 0.1 mg/kg to
50 mg/kg for 7 to 21 days. Optionally, the mice are imaged
throughout the experiment every other day and survival monitored
daily for the duration of the experiment. Expired mice are
optionally subjected to necropsy at the end of the experiment.
Another animal model is implantation into NOD-SCID mice of DoHH2, a
cell line derived from human follicular lymphoma, that stably
expresses luciferase. These in vivo tests optionally generate
preliminary pharmacokinetic, pharmacodynamic and toxicology
data.
Clinical Trials
[0258] To determine the suitability of the peptidomimetic
macrocycles for treatment of humans, clinical trials are performed.
For example, patients diagnosed with solid tumor and in need of
treatment can be selected and separated in treatment and one or
more control groups, wherein the treatment group is administered a
peptidomimetic macrocycle, while the control groups receive a
placebo or a known anti-cancer drug. The treatment safety and
efficacy of the peptidomimetic macrocycles can thus be evaluated by
performing comparisons of the patient groups with respect to
factors such as survival and quality-of-life. In this example, the
patient group treated with a peptidomimetic macrocycle can show
improved long-term survival compared to a patient control group
treated with a placebo.
Formulation and Administration
Mode of Administration
[0259] An effective amount of a peptidomimetic macrocycles or a
pharmaceutically acceptable salt thereof of the disclosure can be
administered in a pharmaceutical composition, as either single or
multiple doses by an accepted mode of administration. In some
embodiments, a pharmaceutical composition comprising a
therapeutically effective amount of a peptidomimetic macrocycle or
a therapeutically equivalent amount of a pharmaceutically
acceptable salt thereof, of the disclosure are administered
parenterally, for example, by subcutaneous, intramuscular,
intrathecal, intravenous or epidural injection. For example, a
pharmaceutical composition comprising a therapeutically effective
amount of a peptidomimetic macrocycle or a therapeutically
equivalent amount of a pharmaceutically acceptable salt thereof is
administered intravenously, intraarterially, subcutaneously or by
infusion. In some examples, a pharmaceutical composition comprising
a therapeutically effective amount of a peptidomimetic macrocycle
or a therapeutically equivalent amount of a pharmaceutically
acceptable salt thereof is administered intravenously. In some
examples, a pharmaceutical composition comprising a therapeutically
effective amount of a peptidomimetic macrocycle or a
therapeutically equivalent amount of a pharmaceutically acceptable
salt thereof is administered intraarterially.
[0260] Regardless of the route of administration selected, the
peptidomimetic macrocycles of the present disclosure, and/or the
pharmaceutical compositions of the present disclosure, are
formulated into pharmaceutically-acceptable dosage forms by
conventional methods known to those of skill in the art. The
peptidomimetic macrocycles according to the disclosure can be
formulated for administration in any convenient way for use in
human or veterinary medicine, by analogy with other
pharmaceuticals.
[0261] In one aspect, the disclosure provides pharmaceutical
composition comprising a therapeutically-effective amount of one or
more of the peptidomimetic macrocycles described above, formulated
together with one or more pharmaceutically acceptable carriers
(additives) and/or diluents. In one embodiment, one or more of the
peptidomimetic macrocycles described herein are formulated for
parenteral administration for parenteral administration, one or
more peptidomimetic macrocycles disclosed herein can be formulated
as aqueous or nonaqueous solutions, dispersions, suspensions or
emulsions or sterile powders which can be reconstituted into
sterile injectable solutions or dispersions just prior to use. Such
a pharmaceutical composition can comprise sugars, alcohols,
antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic with the blood of the intended recipient or
suspending or thickening agents, A pharmaceutical composition can
also contain adjuvants such as preservatives, wetting agents,
emulsifying agents and dispersing agents. Prevention of the action
of microorganisms upon the subject compounds can be ensured by the
inclusion of various antibacterial and antifungal agents, for
example, paraben, chlorobutanol, phenol sorbic acid, and the like.
It can also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into a pharmaceutical
composition. In addition, prolonged absorption of an injectable
pharmaceutical composition can be brought about by the inclusion of
agents which delay absorption such as aluminum monostearate and
gelatin. If desired a pharmaceutical composition can be diluted
prior to use with, for example, an isotonic saline solution or a
dextrose solution. In some examples, the peptidomimetic macrocycle
is formulated as an aqueous solution and is administered
intravenously.
Amount and Frequency of Administration
[0262] Dosing can be determined using techniques known to one
skilled in the art. The selected dosage level can depend upon a
variety of factors including the activity of the particular
peptidomimetic macrocycle employed, the route of administration,
the time of administration, the rate of excretion or metabolism of
the particular peptidomimetic macrocycle being employed, the
duration of the treatment, other drugs, compounds and/or materials
used in combination with the particular peptidomimetic macrocycle
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts. The dosage values can also vary with the
severity of the condition to be alleviated. For any particular
subject, specific dosage regimens can be adjusted over time
according to the individual need and the professional judgment of
the person administering or supervising the administration of the
compositions.
[0263] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the disclosure
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved.
[0264] In some embodiments, a suitable daily dose of a
peptidomimetic macrocycle of the disclosure can be that amount of
the peptidomimetic macrocycle which is the lowest dose effective to
produce a therapeutic effect. Such an effective dose will generally
depend upon the factors described above. The precise time of
administration and amount of any particular peptidomimetic
macrocycle that will yield the most effective treatment in a given
patient will depend upon the activity, pharmacokinetics, and
bioavailability of a particular peptidomimetic macrocycle,
physiological condition of the patient (including age, sex, disease
type and stage, general physical condition, responsiveness to a
given dosage and type of medication), route of administration, and
the like.
[0265] Dosage can be based on the amount of the peptidomimetic
macrocycle per kg body weight of the patient. Other amounts are
known to those of skill in the art and readily determined.
Alternatively, the dosage of the subject disclosure can be
determined by reference to the plasma concentrations of the
peptidomimetic macrocycle. For example, the maximum plasma
concentration (Cmax) and the area under the plasma
concentration-time curve from time 0 to infinity (AUC) can be
used.
[0266] In some embodiment, the subject is a human subject and the
amount of the peptidomimetic macrocycle administered is 0.01-100 mg
per kilogram body weight of the human subject. For example, in
various examples, the amount of the peptidomimetic macrocycle
administered is about 0.01-50 mg/kg, about 0.01-20 mg/kg, about
0.01-10 mg/kg, about 0.1-100 mg/kg, about 0.1-50 mg/kg, about
0.1-20 mg/kg, about 0.1-10 mg/kg, about 0.5-100 mg/kg, about 0.5-50
mg/kg, about 0.5-20 mg/kg, about 0.5-10 mg/kg, about 1-100 mg/kg,
about 1-50 mg/kg, about 1-20 mg/kg, about 1-10 mg/kg body weight of
the human subject. In one embodiment, about 0.5 mg-10 mg of the
peptidomimetic macrocycle per kilogram body weight of the human
subject is administered. In some examples the amount of the
peptidomimetic macrocycle administered is about 0.16 mg, 0.32 mg,
0.64 mg, 1.28 mg, 3.56 mg, 7.12 mg, 14.24, or 20 mg per kilogram
body weight of the human subject. In some examples the amount of
the peptidomimetic macrocycle administered is about 0.16 mg, 0.32
mg, 0.64 mg, 1.28 mg, 3.56 mg, 7.12 mg, or 14.24 mg per kilogram
body weight of the human subject. In some examples the amount of
the peptidomimetic macrocycle administered is 0.16 mg per kilogram
body weight of the human subject. In some examples the amount of
the peptidomimetic macrocycle administered is 0.32 mg per kilogram
body weight of the human subject. In some examples the amount of
the peptidomimetic macrocycle administered is 0.64 mg per kilogram
body weight of the human subject. In some examples the amount of
the peptidomimetic macrocycle administered is 1.28 mg per kilogram
body weight of the human subject. In some examples the amount of
the peptidomimetic macrocycle administered is 3.56 mg per kilogram
body weight of the human subject. In some examples the amount of
the peptidomimetic macrocycle administered is 7.12 mg per kilogram
body weight of the human subject. In some examples the amount of
the peptidomimetic macrocycle administered is 14.24 mg per kilogram
body weight of the human subject.
[0267] In some embodiments about 0.5-20 mg or 0.5-10 mg of the
peptidomimetic macrocycle per kilogram body weight of the human
subject is administered two times a week. For example about 0.5-1.0
mg, 0.5-5.0 mg, 0.5-10.0 mg, 0.5-15 mg, or 1-5 mg, 1-10 mg, 1-15
mg, 1-20 mg, 5-10 mg, 1-15 mg, 5-20 mg, 10-15 mg, 10-20 mg, 15-20
mg of the peptidomimetic macrocycle per kilogram body weight of the
human subject is administrated about twice a week. In some examples
about 1.0 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2.0 mg, 2.25 mg, 2.5 mg,
2.75 mg, 3.0 mg, 3.25 mg, 3.5 mg, 3.75 mg, 4.0 mg, 4.25 mg, 4.5 mg,
4.75 mg, 5.0 mg, 5.25 mg, 5.5 mg, 5.75 mg, 6.0 mg, 6.25 mg, 6.5 mg,
6.75 mg, 7.0 mg, 7.25 mg, 7.5 mg, 7.75 mg, 8.0 mg, 8.25 mg, 8.5 mg,
8.75 mg, 9.0 mg, 9.25 mg, 9.5 mg, 9.75 mg, 10.0 mg, 10.25 mg, 10.5
mg, 10.75 mg, 11.0 mg, 11.25 mg, 11.5 mg, 11.75 mg, 12.0 mg, 12.25
mg, 12.5 mg, 12.75 mg, 13.0 mg, 13.25 mg, 13.5 mg, 13.75 mg, 14.0
mg, 14.25 mg, 14.5 mg, 14.75 mg, 15.0 mg, 15.25 mg, 15.5 mg, 15.75
mg, 16.0 mg, 16.5 mg, 17.0 mg, 17.5 mg, 18.0 mg, 18.5 mg, 19.0 mg,
19.5 mg, or 20.0 mg of the peptidomimetic macrocycle per kilogram
body weight of the human subject is administered two times a week.
In some examples, the amount of the peptidomimetic macrocycle
administered is about 1.25 mg, 2.5 mg, 5.0 mg, 10.0, or 20 mg per
kilogram body weight of the human subject and the peptidomimetic
macrocycle is administered two times a week. In some examples, the
amount of the peptidomimetic macrocycle administered is about 1.25
mg, 2.5 mg, 5.0 mg or 10.0 mg per kilogram body weight of the human
subject and the peptidomimetic macrocycle is administered two times
a week.
[0268] In some embodiments about 0.5-20 mg or 0.5-10 mg of the
peptidomimetic macrocycle per kilogram body weight of the human
subject is administered once a week. For example about 0.5-1.0 mg,
0.5-5.0 mg, 0.5-10.0 mg, 0.5-15 mg, or 1-5 mg, 1-10 mg, 1-15 mg,
1-20 mg, 5-10 mg, 1-15 mg, 5-20 mg, 10-15 mg, 10-20 mg, 15-20 mg of
the peptidomimetic macrocycle per kilogram body weight of the human
subject is administrated once a week. In some examples about 1.0
mg, 1.25 mg, 1.5 mg, 1.75 mg, 2.0 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3.0
mg, 3.25 mg, 3.5 mg, 3.75 mg, 4.0 mg, 4.25 mg, 4.5 mg, 4.75 mg, 5.0
mg, 5.25 mg, 5.5 mg, 5.75 mg, 6.0 mg, 6.25 mg, 6.5 mg, 6.75 mg, 7.0
mg, 7.25 mg, 7.5 mg, 7.75 mg, 8.0 mg, 8.25 mg, 8.5 mg, 8.75 mg, 9.0
mg, 9.25 mg, 9.5 mg, 9.75 mg, 10.0 mg, 10.25 mg, 10.5 mg, 10.75 mg,
11.0 mg, 11.25 mg, 11.5 mg, 11.75 mg, 12.0 mg, 12.25 mg, 12.5 mg,
12.75 mg, 13.0 mg, 13.25 mg, 13.5 mg, 13.75 mg, 14.0 mg, 14.25 mg,
14.5 mg, 14.75 mg, 15.0 mg, 15.25 mg, 15.5 mg, 15.75 mg, 16.0 mg,
16.5 mg, 17.0 mg, 17.5 mg, 18.0 mg, 18.5 mg, 19.0 mg, 19.5 mg, or
20.0 mg of the peptidomimetic macrocycle per kilogram body weight
of the human subject is administered once a week. In some examples,
the amount of the peptidomimetic macrocycle administered is about
1.25 mg, 2.5 mg, 5.0 mg 10.0 mg, or 20.0 mg per kilogram body
weight of the human subject and the peptidomimetic macrocycle is
administered once a week. In some examples, the amount of the
peptidomimetic macrocycle administered is about 1.25 mg, 2.5 mg,
5.0 mg or 10.0 mg per kilogram body weight of the human subject and
the peptidomimetic macrocycle is administered once a week
[0269] In some embodiments about 0.5-20 mg or 0.5-10 mg of the
peptidomimetic macrocycle per kilogram body weight of the human
subject is administered 3, 4, 5, 6 or 7 times a week. For example
about 0.5-1.0 mg, 0.5-5.0 mg, 0.5-10.0 mg, 0.5-15 mg, or 1-5 mg,
1-10 mg, 1-15 mg, 1-20 mg, 5-10 mg, 1-15 mg, 5-20 mg, 10-15 mg,
10-20 mg, 15-20 mg of the peptidomimetic macrocycle per kilogram
body weight of the human subject is administrated 3, 4, 5, 6, or 7
times a week. In some examples about 1.0 mg, 1.25 mg, 1.5 mg, 1.75
mg, 2.0 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3.0 mg, 3.25 mg, 3.5 mg, 3.75
mg, 4.0 mg, 4.25 mg, 4.5 mg, 4.75 mg, 5.0 mg, 5.25 mg, 5.5 mg, 5.75
mg, 6.0 mg, 6.25 mg, 6.5 mg, 6.75 mg, 7.0 mg, 7.25 mg, 7.5 mg, 7.75
mg, 8.0 mg, 8.25 mg, 8.5 mg, 8.75 mg, 9.0 mg, 9.25 mg, 9.5 mg, 9.75
mg, 10.0 mg, 10.25 mg, 10.5 mg, 10.75 mg, 11.0 mg, 11.25 mg, 11.5
mg, 11.75 mg, 12.0 mg, 12.25 mg, 12.5 mg, 12.75 mg, 13.0 mg, 13.25
mg, 13.5 mg, 13.75 mg, 14.0 mg, 14.25 mg, 14.5 mg, 14.75 mg, 15.0
mg, 15.25 mg, 15.5 mg, 15.75 mg, 16.0 mg, 16.5 mg, 17.0 mg, 17.5
mg, 18.0 mg, 18.5 mg, 19.0 mg, 19.5 mg, or 20.0 mg of the
peptidomimetic macrocycle per kilogram body weight of the human
subject is administered 3, 4, 5, 6, or 7 times a week. In some
examples, the amount of the peptidomimetic macrocycle administered
is about 1.25 mg, 2.5 mg, 5.0 mg 10.0 mg, or 20.0 mg per kilogram
body weight of the human subject and the peptidomimetic macrocycle
is administered 3, 4, 5, 6, or 7 times a week. In some examples,
the amount of the peptidomimetic macrocycle administered is about
1.25 mg, 2.5 mg, 5.0 mg or 10.0 mg per kilogram body weight of the
human subject and the peptidomimetic macrocycle is administered 3,
4, 5, 6, or 7 times a week.
[0270] In some embodiments about 0.5-20 mg or 0.5-10 mg of the
peptidomimetic macrocycle per kilogram body weight of the human
subject is administered once every 2, 3, or 4 weeks. For example
about 0.5-1.0 mg, 0.5-5.0 mg, 0.5-10.0 mg, 0.5-15 mg, or 1-5 mg,
1-10 mg, 1-15 mg, 1-20 mg, 5-10 mg, 1-15 mg, 5-20 mg, 10-15 mg,
10-20 mg, 15-20 mg of the peptidomimetic macrocycle per kilogram
body weight of the human subject is administrated 3, 4, 5, 6, or 7
once every 2 or 3 week. In some examples about 1.0 mg, 1.25 mg, 1.5
mg, 1.75 mg, 2.0 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3.0 mg, 3.25 mg, 3.5
mg, 3.75 mg, 4.0 mg, 4.25 mg, 4.5 mg, 4.75 mg, 5.0 mg, 5.25 mg, 5.5
mg, 5.75 mg, 6.0 mg, 6.25 mg, 6.5 mg, 6.75 mg, 7.0 mg, 7.25 mg, 7.5
mg, 7.75 mg, 8.0 mg, 8.25 mg, 8.5 mg, 8.75 mg, 9.0 mg, 9.25 mg, 9.5
mg, 9.75 mg, 10.0 mg, 10.25 mg, 10.5 mg, 10.75 mg, 11.0 mg, 11.25
mg, 11.5 mg, 11.75 mg, 12.0 mg, 12.25 mg, 12.5 mg, 12.75 mg, 13.0
mg, 13.25 mg, 13.5 mg, 13.75 mg, 14.0 mg, 14.25 mg, 14.5 mg, 14.75
mg, 15.0 mg, 15.25 mg, 15.5 mg, 15.75 mg, 16.0 mg, 16.5 mg, 17.0
mg, 17.5 mg, 18.0 mg, 18.5 mg, 19.0 mg, 19.5 mg, or 20.0 mg of the
peptidomimetic macrocycle per kilogram body weight of the human
subject is administered once every 2 or 3 weeks. In some examples,
the amount of the peptidomimetic macrocycle administered is about
1.25 mg, 2.5 mg, 5.0 mg 10.0 mg, or 20.0 mg per kilogram body
weight of the human subject and the peptidomimetic macrocycle is
administered once every 2 weeks. In some examples, the amount of
the peptidomimetic macrocycle administered is about 1.25 mg, 2.5
mg, 5.0 mg or 10.0 mg per kilogram body weight of the human subject
and the peptidomimetic macrocycle is administered once every 2
weeks. In some examples, the amount of the peptidomimetic
macrocycle administered is about 1.25 mg, 2.5 mg, 5.0 mg 10.0 mg,
or 20.0 mg per kilogram body weight of the human subject and the
peptidomimetic macrocycle is administered once every 3 weeks. In
some examples, the amount of the peptidomimetic macrocycle
administered is about 1.25 mg, 2.5 mg, 5.0 mg or 10.0 mg per
kilogram body weight of the human subject and the peptidomimetic
macrocycle is administered once every 3 weeks.
[0271] In some embodiments, the peptidomimetic macrocycle is
administered gradually over a period of time. A desired amount of
peptidomimetic macrocycle can be administered gradually over a
period of from about 0.1 h-24 h. In some cases a desired amount of
peptidomimetic macrocycle is administered gradually over a period
of 0.1 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5
h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17
h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or 24 h. In some examples, a
desired amount of peptidomimetic macrocycle is administered
gradually over a period of 0.25-12 h, for example over a period of
0.25-1 h, 0.25-2 h, 0.25-3 h, 0.25-4 h, 0.25-6 h, 0.25-8 h, 0.25-10
h. In some examples, a desired amount of peptidomimetic macrocycle
is administered gradually over a period of 0.25-2 h. In some
examples, a desired amount of peptidomimetic macrocycle is
administered gradually over a period of 0.25-1 h. In some examples,
a desired amount of peptidomimetic macrocycle is administered
gradually over a period of 0.25 h, 0.3 h, 0.4 h, 0.5 h, 0.6 h, 0.7
h, 0.8 h, 0.9 h, 1.0 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h,
1.7 h, 1.8 h, 1.9 h, or 2.0 h. In some examples, a desired amount
of peptidomimetic macrocycle is administered gradually over a
period of 1 h. In some examples, a desired amount of peptidomimetic
macrocycle is administered gradually over a period of 2 h.
[0272] Administration of the peptidomimetic macrocycles can
continue as long as necessary to treat a solid tumor in a subject
in need thereof. In some embodiments, one or more peptidomimetic
macrocycle of the disclosure is administered for more than 1 day, 1
week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7
months, 8 months, 9 months, 10 months, 11 months, 12 months, 13
months, 14 months, 15 months, 16 months, 17 months, 18 months, 19
months, 20 months, 21 months, 22 months, 23 months, or 24 months.
In some embodiments, one or more peptidomimetic macrocycle of the
disclosure is administered for less than 1 week, 1 month, 2 months,
3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9
months, 10 months, 11 months, 12 months, 13 months, 14 months, 15
months, 16 months, 17 months, 18 months, 19 months, 20 months, 21
months, 22 months, 23 months, or 24 months.
[0273] In some embodiments, the peptidomimetic macrocycle is
administered on day 1, 8, 15 and 28 of a 28 day cycle. In some
embodiments, the peptidomimetic macrocycle is administered on day
1, 8, 15 and 28 of a 28 day cycle and administration is continued
for two cycles. In some embodiments, the peptidomimetic macrocycle
is administered on day 1, 8, 15 and 28 of a 28 day cycle and
administration is continued for three cycles. In some embodiments,
the peptidomimetic macrocycle is administered on day 1, 8, 15 and
28 of a 28 day cycle and administration is continued for 4, 5, 6,
7, 8, 9, 10, or more cycles.
[0274] In some embodiments, one or more peptidomimetic macrocycle
of the disclosure is administered chronically on an ongoing basis.
In some embodiments administration of one or more peptidomimetic
macrocycle of the disclosure is continued until documentation of
disease progression, unacceptable toxicity, or patient or physician
decision to discontinue administration.
Method and Uses
[0275] In one aspect, the disclosure provides a method of treating
solid tumor in a subject, the method comprising administering to
the subject a pharmaceutical composition comprising a
therapeutically effective amount of a peptidomimetic macrocycle or
a therapeutically equivalent amount of a pharmaceutically
acceptable salt thereof, wherein the peptidomimetic macrocycle
binds to MDM2 and/or MDMX proteins. In some embodiments, the
peptidomimetic macrocycle can disrupt the interaction between p53
and MDM2 and MDMX. In some embodiments, treatment according to the
method disclosed herein can result in re-activation of the p53
pathway, decreased tumor cell proliferation, increased p53 protein,
increased p21, and/or increased apoptosis in the human subject.
[0276] In one aspect, the disclosure provides a method of treating
solid tumor, that lacks a p53 deactivating mutation, in a subject
the method comprising administering to the subject a pharmaceutical
composition comprising a therapeutically effective amount of a
peptidomimetic macrocycle or a therapeutically equivalent amount of
a pharmaceutically acceptable salt thereof, wherein the
peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins. In
some embodiments, the solid tumor is determined to lack a p53
mutation prior to treatment In some embodiments, the peptidomimetic
macrocycle can disrupt the interaction between p53 and MDM2 and
MDMX. The method further can comprise confirming the lack of the
p53 deactivating mutation in the subject prior to the
administration of the peptidomimetic macrocycle. In some
embodiments, treatment according to the method disclosed herein can
result in re-activation of the p53 pathway, decreased tumor cell
proliferation, increased p53 protein, increased p21, and/or
increased apoptosis in the human subject.
[0277] In one aspect, the disclosure provides a method of treating
solid tumor in a subject expressing wild type p53, the method
comprising administering to the subject a pharmaceutical
composition comprising a therapeutically effective amount of a
peptidomimetic macrocycle or a therapeutically equivalent amount of
a pharmaceutically acceptable salt thereof, wherein the
peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins. In
some embodiments, the peptidomimetic macrocycle can disrupt the
interaction between p53 and MDM2 and MDMX. The method further can
comprise confirming the wild type p53 status of the subject prior
to the administration of the peptidomimetic macrocycle. In some
embodiments, treatment according to the method disclosed herein can
result in re-activation of the p53 pathway, decreased tumor cell
proliferation, increased p53 protein, increased p21, and/or
increased apoptosis in the human subject.
[0278] In some embodiments, the methods for treating solid tumor
provided herein inhibit, reduce, diminish, arrest, or stabilize a
tumor associated with the solid tumor. In other embodiments, the
methods for treating solid tumor provided herein inhibit, reduce,
diminish, arrest, or stabilize the blood flow, metabolism, or edema
in a tumor associated with the solid tumor or one or more symptoms
thereof. In some examples, the methods for treating solid tumor
provided herein cause the regression of a tumor, tumor blood flow,
tumor metabolism, or peritumor edema, and/or one or more symptoms
associated with the solid tumor. In other examples, the methods for
treating solid tumor provided herein maintain the size of the tumor
so that it does not increase, or so that it increases by less than
the increase of a tumor after administration of a standard therapy
as measured by conventional methods available to one of skill in
the art, such as ultrasound, CT Scan, MRI, dynamic
contrast-enhanced MRI, or PET Scan. In some examples, the methods
for treating solid tumor provided herein decrease tumor size. In
some examples, the methods for treating solid tumor provided herein
reduce the formation of a tumor. In some examples, the methods for
treating solid tumor provided herein eradicate, remove, or control
primary, regional and/or metastatic tumors associated with the
solid tumor. In some examples, the methods for treating solid tumor
provided herein decrease the number or size of metastases
associated with the solid tumor. In some examples, the methods for
treating solid tumor provided herein reduce the tumor volume or
tumor size (e.g., diameter) in a subject by an amount in the range
of about 5-10%, 5-20%, 10-20%, 15-20%, 10-30%, 20-30%, 20-40%,
30-40%, 10-50%, 20-50%, 30-50%, 40-50%, 10-60%, 20-60%, 30-60%,
40-60%, 50-60%, 10-70%, 20-70%, 30-70%, 40-70%, 50-70%, 60-70%,
10-80%, 20-80%, 30-80%, 40-80%, 50-80%, 60-80%, 70- 80%, 10-90%,
20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 80-90%, 10-100%,
20%-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%,
90-100%, 95-100%, or any range in between, relative to tumor size
(e.g., volume or diameter) in a subject prior to administration of
the peptidomimetic macrocycles as assessed by methods well known in
the art, e.g., CT Scan, MRI, DCE-MRI, or PET Scan. In certain
embodiments, the methods herein reduce the tumor size (e.g., volume
or diameter) in a subject by at least about 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, 99%, or
100%, relative to tumor volume or tumor size (e.g., diameter) prior
to administration of the peptidomimetic macrocycle as assessed by
methods well known in the art, e.g., CT Scan, MRI, DCE-MRI, or PET
Scan.
[0279] In some embodiments, the methods provided herein reduce the
tumor perfusion in a subject by an amount in the range of about
5-10%, 5-20%, 10-20%, 15-20%, 10-30%, 20-30%, 20-40%, 30-40%,
10-50%, 20-50%, 30-50%, 40-50%, 10-60%, 20-60%, 30-60%, 40-60%,
50-60%, 10-70%, 20-70%, 30-70%, 40-70%, 50-70%, 60-70%, 10-80%,
20-80%, 30-80%, 40-80%, 50-80%, 60-80%, 70-80%, 10-90%, 20-90%, 30-
90%, 40-90%, 50-90%, 60-90%, 70-90%, 80-90%, 10-100%, 20%-100%,
30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%,
95-100%, or any range in between, relative to tumor perfusion prior
to administration of the peptidomimetic macrocycle, as assessed by
methods well known in the art, e.g., MRI, DCE-MRI, or PET Scan. In
certain embodiments, the methods provided herein reduce the tumor
perfusion in a subject by at least about 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, 99%, or
100%, relative to tumor perfusion prior to administration of the
peptidomimetic macrocycle as assessed by methods well known in the
art, e.g., MRI, DCE-MRI, or PET Scan.
[0280] In some embodiments, the methods provided herein inhibit or
decrease tumor metabolism in a subject in the range of about 5-10%,
5-20%, 10-20%, 15-20%, 10-30%, 20-30%, 20-40%, 30-40%, 10-50%,
20-50%, 30-50%, 40-50%, 10-60%, 20-60%, 30-60%, 40-60%, 50-60%,
10-70%, 20-70%, 30-70%, 40-70%, 50-70%, 60-70%, 10-80%, 20-80%,
30-80%, 40-80%, 50-80%, 60-80%, 70-80%, 10-90%, 20-90%, 30- 90%,
40-90%, 50-90%, 60-90%, 70-90%, 80-90%, 10-100%, 20%-100%, 30-100%,
40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, or
any range in between, relative to tumor metabolism prior to
administration of the peptidomimetic macrocycle, as assessed by
methods well known in the art. In certain embodiments, the methods
provided herein inhibit or decrease tumor metabolism in a subject
as assessed by methods well known in the art, e.g., PET scanning.
In specific embodiments, the methods provided herein inhibit or
decrease tumor metabolism in a subject by at least about 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%,
90%, 95%, or 100%, relative to tumor metabolism prior to
administration of the peptidomimetic macrocycle, as assessed by
methods well known in the art.
[0281] In other aspect, the disclosure provides a method for
increasing the survival time of a subject with solid tumor
determined to lack a p53 deactivating mutation and/or with solid
tumor expressing wild type p53, the method comprising administering
to the subject a pharmaceutical composition comprising a
therapeutically effective amount of a peptidomimetic macrocycle or
a therapeutically equivalent amount of a pharmaceutically
acceptable salt thereof, wherein the peptidomimetic macrocycle
binds to MDM2 and/or MDMX proteins. In some examples, the survival
time of the subject is at least 30 days longer than the expected
survival time of the subject if the subject was not treated
according to the methods provided herein. In some examples, the
survival time of the subject is at 1 month-about 5 years longer
than the expected survival time of the subject if the subject was
not treated according to the methods provided herein. For example,
the survival time of the subject is at least 3 months, at least 6
months, at least 9 months, at least 12 months, at least 15 months,
at least 18 months, at least 21 months, or at least 24 months
longer than the expected survival time of the subject if the
subject was not treated according to the methods disclosed herein
disclosure.
[0282] In one aspect, the disclosure provides a method to access
presence, absence or amount of the biomarker biomarkers in a
subject suffering with solid tumor, the method comprising. In some
examples, the biomarkers include patient biomarkers for example the
p53 status of the subject and the MDM2 and MDMX expression levels
in the subject.
[0283] The method of the disclosure can also optionally include
studying and/or evaluating the safety and/or tolerability of the
peptidomimetic macrocycles disclosed herein in the subject.
[0284] Also provided herein is a method to re-activate the p53
pathway in a subject with a solid tumor lacking a p53 deactivating
mutation and/or expressing wild type p53, the method comprising
administering to the subject a pharmaceutical composition
comprising a therapeutically effective amount of a peptidomimetic
macrocycle or a therapeutically equivalent amount of a
pharmaceutically acceptable salt thereof, wherein the
peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.
[0285] Also provided herein is a method to decrease tumor cell
proliferation in a human subject with a solid tumor lacking a p53
deactivating mutation and/or expressing wild type p53, the method
comprising administering to the subject a pharmaceutical
composition comprising a therapeutically effective amount of a
peptidomimetic macrocycle or a therapeutically equivalent amount of
a pharmaceutically acceptable salt thereof, wherein the
peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.
[0286] Also provided herein is a method to increased p53 protein in
a subject with a solid tumor lacking a p53 deactivating mutation
and/or expressing wild type p53, the method comprising
administering to the subject a pharmaceutical composition
comprising a therapeutically effective amount of a peptidomimetic
macrocycle or a therapeutically equivalent amount of a
pharmaceutically acceptable salt thereof, wherein the
peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.
[0287] Also provided herein is a method to increased p21 in a
subject with a solid tumor lacking a p53 deactivating mutation
and/or expressing wild type p53, the method comprising
administering to the subject a pharmaceutical composition
comprising a therapeutically effective amount of a peptidomimetic
macrocycle or a therapeutically equivalent amount of a
pharmaceutically acceptable salt thereof, wherein the
peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.
[0288] Also provided herein is a method to increased apoptosis in a
subject with a solid tumor lacking a p53 deactivating mutation
and/or expressing wild type p53, the method comprising
administering to the subject a pharmaceutical composition
comprising a therapeutically effective amount of a peptidomimetic
macrocycle or a therapeutically equivalent amount of a
pharmaceutically acceptable salt thereof, wherein the
peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins.
[0289] In some embodiments, the disclosure also provides a method
to determine the dose limiting toxicities (DLTs) and/or maximum
tolerated dose (MTD) of the peptidomimetic macrocycles disclosed
herein in subject with a solid tumor lacking a p53 deactivating
mutation and/or expressing wild type p53.
[0290] The methods of the disclosure can optionally include
pharmacokinetic analysis of the peptidomimetic macrocycles
disclosed herein. Accordingly, the methods can further comprise
collecting one or more biological sample from the subject at one or
more specific time point and analyzing the one or more biological
sample for levels of the peptidomimetic macrocycles and/or it
metabolites. The biological sample can be a blood sample from the
subject, for example blood sample from a human subject. The one or
more specific time point can include time points before, after
and/or during the administration of the peptidomimetic macrocycle
to the subject. In some embodiments one or more biological sample
includes biological samples collected before and after each
administration of the peptidomimetic macrocycle to the subject. In
some embodiments a biological sample for pharmacokinetic analysis
is collected before the first administration of the peptidomimetic
macrocycle to the subject and at one or more time points after each
administration of the peptidomimetic macrocycles to the subject.
The biological sample collected before the administration of the
peptidomimetic macrocycle to the subject can be done within 0-24
hour before the start of administration of the peptidomimetic
macrocycle to the subject. For example, the biological sample can
be collected within 24 h, 23 h, 22 h, 21 h, 20 h, 19 h, 18 h, 17 h,
16 h, 15 h, 14 h, 13 h, 12 h, 11 h, 10 h, 9 h, 8 h, 7 h, 6 h, 5 h,
4 h, 3 h, 2 h, 1 h, 30 min, 15 min, or immediately before the
administration of the peptidomimetic macrocycle to the subject. One
or more biological samples collected after the administration of
the peptidomimetic macrocycle to the subject can be collected from
0-about 72 h, for example after 0 min, 5 min, 10 min, 20 min, 30
min, 45 min, 60 min, 1.25 h, 1.5 h, 1.75 h, 2.0 h, 2.25 h, 2.5 h,
2.75 h, 3.0 h, 3.25 h, 3.5 h, 3.75 h, 4.0 h, 4.25 h, 4.5 h, 4.75 h,
5.0 h, 5.25 h, 5.5 h, 5.75 h, 6.0 h, 6.25 h, 6.5 h, 6.75 h, 7.0 h,
7.25 h, 7.5 h, 7.75 h, 8.0 h, 8.25 h, 8.5 h, 8.75 h, 9.0 h, 9.25 h,
9.5 h, 9.75 h, 10.0 h, 10.25 h, 10.5 h, 10.75 h, 11.0 h, 11.25 h,
11.5 h, 11.75 h, 12.0 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h,
48 h, 52 h, 56 h, 60 h, 64 h, 68 h, or 72 h after the
administration of the peptidomimetic macrocycle to the subject. In
some embodiments, the peptidomimetic macrocycle is administered on
day 1, day 8, day 15 of a 28 day cycle and biological sample is
collected before administration on day 1, after the administration
on day 1 (multiple biological samples can be collected, for example
after about 0 min, about 30 min, about 1 h, about 2 h, about 4 h,
about 8 h, about 24 h, and 48 hour after administration), before
administration on day 8, after administration on day 8 (multiple
biological samples can be collected, for example after about 0 min,
about 30 min, about 1 h, about 2 h, and about 4 h after
administration), before administration on day 15 and after
administration on day 15(multiple biological samples can be
collected, for example after about 0 min, about 30 min, about 1 h,
about 2 h, about 4 h, about 8 h, and about 24 h after
administration).
[0291] The method of the disclosure can optionally include
pharmacodynamic analysis of the peptidomimetic macrocycles
disclosed herein. Accordingly, the methods can comprise collecting
one or more biological samples from the subject at one or more
specific time points for pharmacodynamic analysis. Pharmacodynamic
analysis can include analyzing the levels of biomarkers including
MIC-1, p53, MDM2, MDMX, p21 and/or cases in the biological sample.
Detection of biomarkers in a biological sample can be performed by
any conventional method for detecting the type of biomarker, e.g.,
direct measurement, immunohistochemistry, immunoblotting,
immunoflourescense, immunoabsorbence, immunoprecipitations, protein
array, flourescence in situ hybridization, FACS analysis,
hybridization, in situ hybridization, Northern blots, Southern
blots, Western blots, ELISA, radioimmunoassay, gene array/chip,
PCR, RT-PCR, or cytogenetic analysis. The biological sample for
pharmacodynamic analysis can be a blood sample or a tumor specimen
from the subject, for example biological sample for pharmacodynamic
analysis can be a blood sample or a tumor specimen from the human
subject. The biological samples for pharmacodynamic analysis of the
peptidomimetic macrocycles can be collected any time before,
during, or after the administration of the peptidomimetic
macrocycle to the subject. In some embodiments a blood sample for
pharmacokinetic analysis is collected before the first
administration of the peptidomimetic macrocycle to the subject and
at one or more time points after each administration of the
peptidomimetic macrocycles to the subject. The blood sample
collected before the administration of the peptidomimetic
macrocycle to the subject can be done within 0-24 hour before the
start of administration of the peptidomimetic macrocycle to the
subject. For example, the biological sample can be collected within
24 h, 23 h, 22 h, 21 h, 20 h, 19 h, 18 h, 17 h, 16 h, 15 h, 14 h,
13 h, 12 h, 11 h, 10 h, 9 h, 8 h, 7 h, 6 h, 5 h, 4 h, 3 h, 2 h, 1
h, 30 min, 15 min, or immediately before the administration of the
peptidomimetic macrocycle to the subject. One or more blood samples
for pharmacodynamic analysis collected after the administration of
the peptidomimetic macrocycle to the subject can be collected from
0-about 72 h, for example after about 12 h, 24 h, 36 h or 48 h
after the administration of the peptidomimetic macrocycle to the
subject. In some embodiments, the peptidomimetic macrocycle is
administered on day 1, day 8, day 15 of a 28 day cycle and blood
samples for pharmacodynamic analysis are collected before
administration on day 1, after the administration on day 1
(multiple samples can be collected, for example after about 24 h
and 48 hour after administration), before administration on day 8,
after administration on day 8 (multiple samples can be collected,
for example with about 1 h administration), before administration
on day 15 and after administration on day 15(multiple samples can
be collected, for example with about 1 h and about 48 h after
administration), and day 22. Tumor specimens for pharmacodynamic
analysis can be collected at any time before, after or during the
administration of the peptidomimetic macrocycle to the subject. For
example the peptidomimetic macrocycle can be administered on day 1,
day 8, day 15 of a 28 day cycle and tumor samples for
pharmacodynamic analysis are collected before administration on day
1 and between day 14-day 18, for example of day 16.
[0292] The method of the disclosure can optionally include clinical
activity analysis of the peptidomimetic macrocycles disclosed
herein. Accordingly, the methods can comprise analyzing one or more
biological samples collected from the subject at one or more
specific time points. Any appropriate analytical procedure can be
used for the analysis of the biological samples. For example,
imaging techniques like radiographs, ultrasound, CT scan, PET scan,
MRI scan, chest x-ray, laparoscopy, complete blood count (CBC)
test, bone scanning and fecal occult blood test can be used.
Further analytical procedures that can be used include blood
chemistry analysis, chromosomal translocation analysis, needle
biopsy, tissue biopsy, fluorescence in situ hybridization,
laboratory biomarker analysis, immunohistochemistry staining
method, flow cytometry, or a combination thereof. The method can
further comprise tabulating and/or plotting results of the
analytical procedure.
Biological Samples
[0293] As used in the present application, "biological sample"
means any fluid or other material derived from the body of a normal
or diseased subject, such as blood, serum, plasma, lymph, urine,
saliva, tears, cerebrospinal fluid, milk, amniotic fluid, bile,
ascites fluid, pus, and the like. Also included within the meaning
of the term "biological sample" is an organ or tissue extract and
culture fluid in which any cells or tissue preparation from a
subject has been incubated. Biological samples also include tumor
samples or specimens. Tumor sample can be a tumor tissue sample.
Methods of obtaining tumor tissue samples are well known in the art
and can vary according to the type and location of a tumor and
preferences of the physician. In some embodiments, the tumor tissue
sample can obtained from surgically excised tissue. Tissue samples
and cellular samples can also be obtained without invasive surgery,
for example by punctuating the chest wall or the abdominal wall or
from masses of breast, thyroid or other sites with a fine needle
and withdrawing cellular material (fine needle aspiration
biopsy).
[0294] The biological samples obtained can be used in fresh,
frozen, or fixed (e.g., paraffin-embedded) form, depending on the
nature of the sample, the assay used, and the convenience of the
practitioner. Although fresh, frozen and fixed materials are
suitable for various RNA and protein assays, generally, fresh
tissues can be preferred for ex vivo measurements of activity.
[0295] Fixed tissue samples can also be employed. Tissue obtained
by biopsy is often fixed, usually by formalin, formaldehyde, or
gluteraldehyde, for example, or by alcohol immersion. Fixed
biological samples are often dehydrated and embedded in paraffin or
other solid supports, as is known in the art. See the reference
Plenat et al., 2001, Ann. Pathol. 21:29-47. Non-embedded, fixed
tissue, as well as fixed and embedded tissue, can be used in the
present methods. Solid supports for embedding fixed tissue can be
removed with organic solvents to enable subsequent rehydration of
preserved tissue.
[0296] In some cases, the assay includes a step of cell or tissue
culture. Culture methods are well known in the art. For example,
cells from a biopsy can be disaggregated using enzymes (such as
collagenase and hyaluronidase) and or physical disruption (e.g.,
repeated passage through a 25-gauge needle) to dissociate the
cells, collected by centrifugation, and resuspended in desired
buffer or culture medium for culture, immediate analysis, or
further processing.
Subject/Patient Population
[0297] In some embodiments, a subject treated for solid tumor in
accordance with the methods provided herein is a human, who has or
is diagnosed with a solid tumor. In other embodiments, a subject
treated for solid tumor in accordance with the methods provided
herein is a human, predisposed or susceptible to a solid tumor. In
some embodiments, a subject treated for solid tumor in accordance
with the methods provided herein is a human, at risk of developing
a solid tumor.
[0298] In some embodiments, a subject treated for solid tumor in
accordance with the methods provided herein is a human, who has or
is diagnosed with a solid tumor, determined to lack a p53
deactivating mutation and/or expressing wild type p53. In other
embodiments, a subject treated for solid tumor in accordance with
the methods provided herein is a human, predisposed or susceptible
to a solid tumor, determined to lack a p53 deactivating mutation
and/or expressing wild type p53. In some embodiments, a subject
treated for solid tumor in accordance with the methods provided
herein is a human, at risk of developing a solid tumor, determined
to lack a p53 deactivating mutation and/or expressing wild type
p53. A p53 deactivating mutation, as used herein is any mutation
that leads to loss of (or a decrease in) the in vitro apoptotic
activity of p53. Non limiting examples of p53 deactivating
mutations are included in Table 1a. Accordingly, in some
embodiments, a subject with a solid tumor in accordance with the
composition as provided herein is a human who has or is diagnosed
with a solid tumor that is determined to lack a p53 deactivation
mutation, such as those shown in Table 1a.
[0299] In some embodiments, the subject treated for solid tumor in
accordance with the methods provided herein is a human, who has or
is diagnosed with a solid tumor, determined to have a p53 gain of
function mutation. In other embodiments, a subject treated for
solid tumor in accordance with the methods provided herein is a
human, predisposed or susceptible to a solid tumor, determined to
have a p53 gain of function mutation. In some embodiments, a
subject treated for solid tumor in accordance with the methods
provided herein is a human, at risk of developing a solid tumor,
determined to have a p53 gain of function mutation. A p53 gain of
function mutation, as used herein is any mutation such that the
mutant p53 exerts oncogenic functions beyond their negative
domination over the wild-type p53 tumor suppressor functions. The
p53 gain of function mutant protein mat exhibit new activities that
can contribute actively to various stages of tumor progression and
to increased resistance to anticancer treatments. Non limiting
examples of p53 gain of function mutation mutations are included in
Table 1b. Accordingly, in some embodiments, a subject with a solid
tumor in accordance with the composition as provided herein is a
human who has or is diagnosed with a solid tumor that is determined
to have a p53 gain of function mutation, such as those shown in
Table 1b.
[0300] In some embodiments, the subject treated for solid tumor in
accordance with the methods provided herein is a human, who has or
is diagnosed with a solid tumor that is not p53 negative. In other
embodiments, a subject treated for solid tumor in accordance with
the methods provided herein is a human, predisposed or susceptible
to a solid tumor that is not p53 negative. In some embodiments, a
subject treated for solid tumor in accordance with the methods
provided herein is a human, at risk of developing a solid tumor
that is not p53 negative.
[0301] In some embodiments, the subject treated for solid tumor in
accordance with the methods provided herein is a human, who has or
is diagnosed with a solid tumor that expresses p53 with partial
loss of function mutation. In other embodiments, a subject treated
for solid tumor in accordance with the methods provided herein is a
human, predisposed or susceptible to a solid tumor that expresses
p53 with partial loss of function mutation. In some embodiments, a
subject treated for solid tumor in accordance with the methods
provided herein is a human, at risk of developing a solid tumor
that expresses p53 with partial loss of function mutation. As used
herein "a partial loss of p53 function" mutation means that the
mutant p53 exhibits some level of function of normal p53, but to a
lesser or slower extent. For example, a partial loss of p53
function can mean that the cells become arrested in cell division
to a lesser or slower extent.
[0302] In some embodiments, the subject treated for solid tumor in
accordance with the methods provided herein is a human, who has or
is diagnosed with a solid tumor that expresses p53 with a copy loss
mutation and a deactivating mutation. In other embodiments, a
subject treated for solid tumor in accordance with the methods
provided herein is a human, predisposed or susceptible to a solid
tumor that expresses p53 with a copy loss mutation and a
deactivating mutation. In some embodiments, a subject treated for
solid tumor in accordance with the methods provided herein is a
human, at risk of developing a solid tumor that expresses p53 with
a copy loss mutation and a deactivating mutation.
[0303] In some embodiments, the subject treated for solid tumor in
accordance with the methods provided herein is a human, who has or
is diagnosed with a solid tumor that expresses p53 with a copy loss
mutation. In other embodiments, a subject treated for solid tumor
in accordance with the methods provided herein is a human,
predisposed or susceptible to a solid tumor that expresses p53 with
a copy loss mutation. In some embodiments, a subject treated for
solid tumor in accordance with the methods provided herein is a
human, at risk of developing a solid tumor that expresses p53 with
a copy loss mutation.
[0304] In some embodiments, the subject treated for solid tumor in
accordance with the methods provided herein is a human, who has or
is diagnosed with a solid tumor that expresses p53 with one or more
silent mutations. In other embodiments, a subject treated for solid
tumor in accordance with the methods provided herein is a human,
predisposed or susceptible to a solid tumor that expresses p53 with
one or more silent mutations. In some embodiments, a subject
treated for solid tumor in accordance with the methods provided
herein is a human, at risk of developing a solid tumor that
expresses p53 with one or more silent mutations. Silent mutations
as used herein are mutations which cause no change in the encoded
p53 amino acid sequence.
[0305] In some embodiments, a subject treated for solid tumor in
accordance with the methods provided herein is a human, who has or
is diagnosed with a solid tumor, determined to lack a dominant p53
deactivating mutation. Dominant p53 deactivating mutation or
dominant negative mutation, as used herein, is a mutation wherein
the mutated p53 inhibits or disrupt the activity of the wild-type
p53 gene.
TABLE-US-00002 TABLE 1a Exemplary of p53 deactivating mutations
Mutation at position Amino acid change 62 E62_W91del 122 V122X 135
C135S 143 V143A 144 Q144P 146 W146X 157 V157F 158 R158H 163 Y163N
168 H168Y 173 V173L 175 R175H 175 R175P 175 R175Q 175 R175S 219
P219H 234 Y234C 234 Y234H 237 M237I 240 S240R 245 G245C 245 G245S
246 M246I 248 R248Q 248 R248W 249 R249S 272 V272M 273 R273H 274
V274F 279 G279E 280 R280K 281 D281H 282 R282W 306 R306P 308
P300_L308del 327 P300_Y327del 332 D324_I332del 337 R337C 344
L344P
TABLE-US-00003 TABLE 1b Exemplary p53 activating mutations AA
position AAchange 46 S46F 121 S121F 123 T123A 288 N288K
[0306] Table 1a and 1b refer to the sequence of the wild-type human
TP53 tumor protein p53 shown in FIG. 1. Amino acid changes are
reported as: the amino acid being substituted followed by the
position of the amino acid being substituted in the wild type p53
sequence, followed by the amino acid used for substitution. For
example L344P, indicates that the lysine (K) at the 344 position in
the wild type sequence is replaced by a proline (P).
[0307] In some embodiments, a subject treated for solid tumor in
accordance with the methods provided herein is a refractory
patient. In a certain embodiment, a refractory patient is a patient
refractory to a standard therapy (e.g., surgery, radiation,
anti-androgen therapy and/or drug therapy such as chemotherapy). In
certain embodiments, a patient with the solid tumor is refractory
to a therapy when the solid tumor has not significantly been
eradicated and/or the one or more symptoms have not been
significantly alleviated. The determination of whether a patient is
refractory can be made either in vivo or in vitro by any method
known in the art for assaying the effectiveness of a treatment of
solid tumor. In various embodiments, a patient with solid tumor is
refractory when one or more tumors associated with the solid tumor,
have not decreased or have increased. In various embodiments, a
patient with solid tumor is refractory when one or more tumors
metastasize and/or spread to another organ.
[0308] In some embodiments, a subject treated for solid tumor
accordance with the methods provided herein is a human that has
proven refractory to therapies other than treatment with the
peptidomimetic macrocycles of the disclosure, but is no longer on
these therapies. In certain embodiments, a subject treated for
solid tumor in accordance with the methods provided herein is a
human already receiving one or more conventional anti-cancer
therapies, such as surgery, drug therapy such as chemotherapy,
anti-androgen therapy or radiation. Among these patients are
refractory patients, patients who are too young for conventional
therapies, and patients with recurring tumors despite treatment
with existing therapies.
[0309] In some embodiments, the subject is a human who has had at
least one unsuccessful prior treatment and/or therapy of the solid
tumor.
Methods of Detecting Wild Type p53 and/or p53 Mutations
[0310] The tumor samples from a subject can be assayed in order to
determine the lack of a p53 deactivating mutation and/or expression
of wild type p53.
[0311] In order to detect the p53 wild-type gene and/or lack of p53
deactivation mutation in a tissue, it can be helpful to isolate the
tissue free from surrounding normal tissues. Means for enriching a
tissue preparation for tumor cells are known in the art. For
example, the tissue can be isolated from paraffin or cryostat
sections. Cancer cells can also be separated from normal cells by
flow cytometry. These as well as other techniques for separating
tumor from normal cells are well known in the art. If the tumor
tissue is highly contaminated with normal cells, detection of
mutations can be more difficult.
[0312] Detection of point mutations can be accomplished by
molecular cloning of the p53 allele (or alleles) present in the
tumor tissue and sequencing that allele(s) using techniques well
known in the art. Alternatively, the polymerase chain reaction can
be used to amplify p53 gene sequences directly from a genomic DNA
preparation from the tumor tissue. The DNA sequence of the
amplified sequences can then be determined. The polymerase chain
reaction itself is well known in the art. See e.g., Saiki et al.,
Science, Vol. 239, p. 487, 1988; U.S. Pat. Nos. 4,683,202; and
4,683,195.
[0313] Specific deletions of p53 genes can also be detected. For
example, restriction fragment length polymorphism (RFLP) probes for
the p53 gene or surrounding marker genes can be used to score loss
of a p53 allele. Other techniques for detecting deletions, as are
known in the art can be used.
[0314] Loss of wild-type p53 genes can also be detected on the
basis of the loss of a wild-type expression product of the p53
gene. Such expression products include both the mRNA as well as the
p53 protein product itself. Point mutations can be detected by
sequencing the mRNA directly or via molecular cloning of cDNA made
from the mRNA. The sequence of the cloned cDNA can be determined
using DNA sequencing techniques which are well known in the art.
The cDNA can also be sequenced via the polymerase chain reaction
(PCR).
[0315] Alternatively, mismatch detection can be used to detect
point mutations in the p53 gene or its mRNA product. The method can
involve the use of a labeled riboprobe which is complementary to
the human wild-type p53 gene. The riboprobe and either mRNA or DNA
isolated from the tumor tissue are annealed (hybridized) together
and subsequently digested with the enzyme RNase A which is able to
detect some mismatches in a duplex RNA structure. If a mismatch is
detected by RNase A, it cleaves at the site of the mismatch. Thus,
when the annealed RNA preparation is separated on an
electrophoretic gel matrix, if a mismatch has been detected and
cleaved by RNase A, an RNA product will be seen which is smaller
than the full-length duplex RNA for the riboprobe and the p53 mRNA
or DNA. The riboprobe need not be the full length of the p53 mRNA
or gene but can be a segment of either. If the riboprobe comprises
only a segment of the p53 mRNA or gene it will be desirable to use
a number of these probes to screen the whole mRNA sequence for
mismatches.
[0316] In similar fashion, DNA probes can be used to detect
mismatches, through enzymatic or chemical cleavage. See, e.g.,
Cotton et al., Proc. Natl. Acad. Sci. USA, vol. 85, 4397, 1988; and
Shenk et al., Proc. Natl. Acad. Sci. USA, vol. 72, p. 989, 1975.
Alternatively, mismatches can be detected by shifts in the
electrophoretic mobility of mismatched duplexes relative to matched
duplexes. See, e.g., Cariello, Human Genetics, vol. 42, p. 726,
1988. With either riboprobes or DNA probes, the cellular mRNA or
DNA which might contain a mutation can be amplified using PCR (see
below) before hybridization.
[0317] DNA sequences of the p53 gene from the tumor tissue which
have been amplified by use of polymerase chain reaction can also be
screened using allele-specific probes. These probes are nucleic
acid oligomers, each of which contains a region of the p53 gene
sequence harboring a known mutation. For example, one oligomer can
be about 30 nucleotides in length, corresponding to a portion of
the p53 gene sequence. At the position coding for the 175th codon
of p53 gene the oligomer encodes an alanine, rather than the
wild-type codon valine. By use of a battery of such allele-specific
probes, the PCR amplification products can be screened to identify
the presence of a previously identified mutation in the p53 gene.
Hybridization of allele-specific probes with amplified p53
sequences can be performed, for example, on a nylon filter.
Hybridization to a particular probe indicates the presence of the
same mutation in the tumor tissue as in the allele-specific
probe.
[0318] The identification of p53 gene structural changes in tumor
cells has been facilitated through the development and application
of a diverse series of high resolution, high throughput microarray
platforms. Essentially there are two types of array; those that
carry PCR products from cloned nucleic acids {e.g. cDNA, BACs,
cosmids) and those that use oligonucleotides. Each has advantages
and disadvantages but it is now possible to survey genome wide DNA
copy number abnormalities and expression levels to allow
correlations between losses, gains and amplifications in tumor
cells with genes that are over- and under-expressed in the same
samples. The gene expression arrays that provide estimates of mRNA
levels in tumors have given rise to exon-specific arrays that can
identify both gene expression levels, alternative splicing events
and mRNA processing alterations. Oligonucleotide arrays are also
being used to interrogate single nucleotide polymorphisms (SNPs)
throughout the genome for linkage and association studies and these
have been adapted to quantify copy number abnormalities and loss of
heterozygosity events. Ultimately DNA sequencing arrays will allow
resequencing of chromosome regions and whole genomes.
[0319] SNP-based arrays or other gene arrays or chips are also
contemplated to determine the presence of wild-type p53 allele and
the structure of mutations. A single nucleotide polymorphism (SNP),
a variation at a single site in DNA, is the most frequent type of
variation in the genome. For example, there are an estimated 5-10
million SNPs in the human genome. As SNPs are highly conserved
throughout evolution and within a population, the map of SNPs
serves as an excellent genotypic marker for research. An SNP array
is a useful tool to study the whole genome.
[0320] In addition, SNP array can be used for studying the Loss Of
Heterozygosity (LOH). LOH is a form of allelic imbalance that can
result from the complete loss of an allele or from an increase in
copy number of one allele relative to the other. While other
chip-based methods (e.g., comparative genomic hybridization can
detect only genomic gains or deletions), SNP array has the
additional advantage of detecting copy number neutral LOH due to
uniparental disomy (UPD). In UPD, one allele or whole chromosome
from one parent are missing leading to reduplication of the other
parental allele (uni-parental=from one parent, disomy=duplicated).
In a disease setting this occurrence can be pathologic when the
wild-type allele (e.g., from the mother) is missing and instead two
copies of the heterozygous allele (e.g., from the father) are
present. This usage of SNP array has a huge potential in cancer
diagnostics as LOH is a prominent characteristic of most human
cancers. Recent studies based on the SNP array technology have
shown that not only solid tumors (e.g. gastric cancer, liver cancer
etc) but also hematologic malignancies (ALL, MDS, CML etc) have a
high rate of LOH due to genomic deletions or UPD and genomic gains.
In the present disclosure, using high density SNP array to detect
LOH allows identification of pattern of allelic imbalance to
determine the presence of wild-type p53 allele (Lips et ah, 2005;
Lai et al, 2007).
[0321] Examples for current p53 gene sequence and single nucleotide
polymorphism arrays include p53 Gene Chip (Affymetrix, Santa Clara,
Calif.), Roche p53 Ampli-Chip (Roche Molecular Systems, Pleasanton,
Calif.), GeneChip Mapping arrays (Affymetrix, Santa Clara, Calif.),
SNP Array 6.0 (Affymetrix, Santa Clara, Calif.), BeadArrays
(Illumina, San Diego, Calif.), etc.
[0322] Mutations of wild-type p53 genes can also be detected on the
basis of the mutation of a wild-type expression product of the p53
gene. Such expression products include both the mRNA as well as the
p53 protein product itself. Point mutations can be detected by
sequencing the mRNA directly or via molecular cloning of cDNA made
from the mRNA. The sequence of the cloned cDNA can be determined
using DNA sequencing techniques which are well known in the art.
The cDNA can also be sequenced via the polymerase chain reaction
(PCR). A panel of monoclonal antibodies could be used in which each
of the epitopes involved in p53 functions are represented by a
monoclonal antibody. Loss or perturbation of binding of a
monoclonal antibody in the panel would indicate mutational
alteration of the p53 protein and thus of the p53 gene itself.
Mutant p53 genes or gene products can also be detected in body
samples, such as, serum, stool, or other body fluids, such as urine
and sputum. The same techniques discussed above for detection of
mutant p53 genes or gene products in tissues can be applied to
other body samples. 2. Assessment of p53 Protein Level
[0323] Loss of wild-type p53 genes can also be detected by
screening for loss of wild-type p53 protein function. Although all
of the functions which the p53 protein undoubtedly possesses have
yet to be elucidated, at least two specific functions are known.
Protein p53 binds to the SV40 large T antigen as well as to the
adenovirus E1B antigen. Loss of the ability of the p53 protein to
bind to either or both of these antigens indicates a mutational
alteration in the protein which reflects a mutational alteration of
the gene itself. Alternatively, a panel of monoclonal antibodies
could be used in which each of the epitopes involved in p53
functions are represented by a monoclonal antibody. Loss or
perturbation of binding of a monoclonal antibody in the panel would
indicate mutational alteration of the p53 protein and thus of the
p53 gene itself. Any means for detecting an altered p53 protein can
be used to detect loss of wild-type p53 genes.
[0324] Mutant p53 genes or gene products can also be detected in
body samples, such as, serum, stool, or other body fluids, such as
urine and sputum. The same techniques discussed above for detection
of mutant p53 genes or gene products in tissues can be applied to
other body samples.
[0325] Determination of the lack of p53 deactivating mutation
and/or expression of wild type p53 in the subject with solid tumor
can be performed any time before, during or after the
administration of the peptidomimetic macrocycles. In some
embodiments, the determination of the lack of a p53 deactivating
mutation and/or expression of wild type p53 is performed before the
first administration of the peptidomimetic macrocycle to the
subject, for example about 5 years-1 month, 4 years-1 month, 3
years-1 month, 2 years-1 month, 1 years-1 month, 5 years-1 week, 4
years-1 week, 3 years-1 month, 2 years-1 week, 1 year-1 week, 5
years-1 day, 4 years-1 day, 3 years-1 days, 2 years-1 day, 1 year-1
day, 15 months-1 month, 15 months-1 week, 15 months-1 day, 12
months-1 month, 12 months-1 week, 12 months-1 day, 6 months-1
month, 6 months-1 week, 6 months-1 day, 3 months-1 month, 3
months-1 week, or 3 months-1 day prior to the first administration
of the peptidomimetic macrocycle to the subject. In some examples,
the confirmation of the lack of the p53 deactivating mutation
and/or expression of wild type p53 is performed up to 6 years, 5
years, 4 years, 3 years, 24 months, 23 months, 22 months, 21
months, 20 months, 19 months, 18 months, 17 months, 16 months, 15
months, 14 months, 13 months, 12 months, 11 months, 10 months, 9
months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months,
2 months, 1 months, 4 weeks (28 days), 3 weeks (21 days), 2 weeks
(14 days), 1 week (7 days), 6 days, 5 days, 4 days, 3 days, 2 days
or 1 day before the first administration of the peptidomimetic
macrocycle to the subject. In some examples the confirmation of the
lack of the p53 deactivating mutation is performed within 1 month
of the first administration of the peptidomimetic macrocycle to the
subject. In some examples the confirmation of the lack of the p53
deactivating mutation is performed within 21 days of the first
administration of the peptidomimetic macrocycle to the subject.
Solid Tumors
[0326] Solid tumors that can be treated by the instant methods
include tumors and/or metastasis (wherever located) other than
lymphatic cancer, for example brain and other central nervous
system tumors (including but not limited to tumors of the meninges,
brain, spinal cord, cranial nerves and other parts of central
nervous system, e.g. glioblastomas or medulla blastomas); head
and/or neck cancer; breast tumors; circulatory system tumors
(including but not limited to heart, mediastinum and pleura, and
other intrathoracic organs, vascular tumors and tumor-associated
vascular tissue); excretory system tumors (including but not
limited to tumors of kidney, renal pelvis, ureter, bladder, other
and unspecified urinary organs); gastrointestinal tract tumors
(including but not limited to tumors of oesophagus, stomach, small
intestine, colon, colorectal, rectosigmoid junction, rectum, anus
and anal canal, tumors involving the liver and intrahepatic bile
ducts, gall bladder, other and unspecified parts of biliary tract,
pancreas, other and digestive organs); oral cavity tumors
(including but not limited to tumors of lip, tongue, gum, floor of
mouth, palate, and other parts of mouth, parotid gland, and other
parts of the salivary glands, tonsil, oropharynx, nasopharynx,
pyriform sinus, hypopharynx, and other sites in the lip, oral
cavity and pharynx); reproductive system tumors (including but not
limited to tumors of vulva, vagina, Cervix uteri, Corpus uteri,
uterus, ovary, and other sites associated with female genital
organs, placenta, penis, prostate, testis, and other sites
associated with male genital organs); respiratory tract tumors
(including but not limited to tumors of nasal cavity and middle
ear, accessory sinuses, larynx, trachea, bronchus and lung, e.g.
small cell lung cancer or non-small cell lung cancer); skeletal
system tumors (including but not limited to tumors of bone and
articular cartilage of limbs, bone articular cartilage and other
sites); skin tumors (including but not limited to malignant
melanoma of the skin, non-melanoma skin cancer, basal cell
carcinoma of skin, squamous cell carcinoma of skin, mesothelioma,
Kaposi's sarcoma); and tumors involving other tissues including
peripheral nerves and autonomic nervous system, connective and soft
tissue, retroperitoneum and peritoneum, eye and adnexa, thyroid,
adrenal gland and other endocrine glands and related structures,
secondary and unspecified malignant neoplasm of lymph nodes,
secondary malignant neoplasm of respiratory and digestive systems
and secondary malignant neoplasm of other sites.
[0327] In some examples, the solid tumor treated by the methods of
the instant disclosure is pancreatic cancer, bladder cancer, colon
cancer, liver cancer, colorectal cancer (colon cancer or rectal
cancer), breast cancer, prostate cancer, renal cancer,
hepatocellular cancer, lung cancer, ovarian cancer, cervical
cancer, gastric cancer, esophageal cancer, head and neck cancer,
melanoma, neuroendocrine cancers, CNS cancers, brain tumors, bone
cancer, skin cancer, ocular tumor, choriocarcinoma (tumor of the
placenta), sarcoma or soft tissue cancer.
[0328] In some examples, the solid tumor to be treated by the
methods of the instant disclosure is selected bladder cancer, bone
cancer, breast cancer, cervical cancer, CNS cancer, colon cancer,
ocular tumor, renal cancer, liver cancer, lung cancer, pancreatic
cancer, choriocarcinoma (tumor of the placenta), prostate cancer,
sarcoma, skin cancer, soft tissue cancer or gastric cancer.
[0329] In some examples, the solid tumor treated by the methods of
the instant disclosure is breast cancer. Non limiting examples of
breast cancer that can be treated by the instant methods include
ductal carcinoma in situ (DCIS or intraductal carcinoma), lobular
carcinoma in situ (LCIS), invasive (or infiltrating) ductal
carcinoma, invasive (or infiltrating) lobular carcinoma,
inflammatory breast cancer, triple-negative breast cancer, paget
disease of the nipple, phyllodes tumor (phylloides tumor or
cystosarcoma phyllodes), angiosarcoma, adenoid cystic (or
adenocystic) carcinoma, low-grade adenosquamous carcinoma,
medullary carcinoma, papillary carcinoma, tubular carcinoma,
metaplastic carcinoma, micropapillary carcinoma, and mixed
carcinoma.
[0330] In some examples, the solid tumor treated by the methods of
the instant disclosure is bone cancer. Non limiting examples of
bone cancer that can be treated by the instant methods include
osteosarcoma, chondrosarcoma, the Ewing Sarcoma Family of Tumors
(ESFTs).
[0331] In some examples, the solid tumor treated by the methods of
the instant disclosure is skin cancer. Non limiting examples of
skin cancer that can be treated by the instant methods include
melanoma, basal cell skin cancer, and squamous cell skin
cancer.
[0332] In some examples, the solid tumor treated by the methods of
the instant disclosure is ocular tumor. Non limiting examples of
ocular tumor that can be treated by the methods of the instant
disclosure include ocular tumor is choroidal nevus, choroidal
melanoma, choroidal metastasis, choroidal hemangioma, choroidal
osteoma, iris melanoma, uveal melanoma, intraocular lymphoma,
melanocytoma, metastasis retinal capillary hemangiomas, congenital
hypertrophy of the RPE, RPE adenoma or retinoblastoma.
[0333] In some embodiments solid tumors treated by the methods
disclosed herein exclude cancers that are known to be associated
with HPV (Human papillomavirus). The excluded group includes HPV
positive cervical cancer, HPV positive anal cancer, and HPV head
and neck cancers, such as oropharyngeal cancers.
[0334] The effectiveness and/or response of cancer treatment by the
methods disclosed herein can be determined by any method known in
art. The response can be a complete response, and which can be an
objective response, a clinical response, or a pathological response
to treatment. For example, the response can be determined based
upon the techniques for evaluating response to treatment of solid
tumors as described in Therese et al., New Guidelines to Evaluate
the Response to Treatment in Solid Tumors, J. of the National
Cancer Institute 92(3):205-207 (2000), which is hereby incorporated
by reference in its entirety. The response can be a duration of
survival (or probability of such duration) or progression-free
interval. The timing or duration of such events can be determined
from about the time of diagnosis, or from about the time treatment
is initiated or from about the time treatment is finished (like the
final administration of the peptidomimetic macrocycle).
Alternatively, the response can be based upon a reduction in tumor
size, tumor volume, or tumor metabolism, or based upon overall
tumor burden, or based upon levels of serum markers especially
where elevated in the disease state.
[0335] The response in individual patients can be characterized as
a complete response, a partial response, stable disease, and
progressive disease, as these terms are understood in the art. In
some embodiments, the response is complete response (CR). Complete
response, in some examples can be defined as disappearance of all
target lesions i.e. any pathological lymph nodes (whether target or
non-target) must have reduction in short axis to <10 mm. In
certain embodiments, the response is a partial response (PR).
Partial response can be defined to mean at least 30% decrease in
the sum of diameters of target lesions, taking as reference the
baseline sum diameters. In some embodiments, the response is
progressive disease (PD). Progressive disease can be defined as at
least a 20% increase in the sum of diameters of target lesions,
taking as reference the smallest sum on study (this includes the
baseline sum if that is the smallest) and an absolute increase of
at least 5 mm in the sum of diameters of target lesions. The
appearance of one or more new lesions can also be considered as
progression. In some embodiments, the disease can be stable disease
(SD). Stable disease can be characterized by neither sufficient
shrinkage to qualify for PR nor sufficient increase to qualify for
PD, taking as reference the smallest sum diameters while on study.
In certain embodiments, the response is a pathological complete
response. A pathological complete response, e.g., as determined by
a pathologist following examination of tissue removed at the time
of surgery or biopsy, generally refers to an absence of
histological evidence of invasive tumor cells in the surgical
specimen.
Combination Treatment
[0336] Also provided herein are combination therapies for the
treatment of a solid tumor which involve the administration of the
peptidomimetic macrocycles disclosed herein in combination with one
or more additional therapies to a subject with solid tumor
determined to lack a p53 deactivating mutation and/or express wild
type p53. In a specific embodiment, presented herein are
combination therapies for the treatment of solid tumor which
involve the administration of an effective amount of the
peptidomimetic macrocycles in combination with an effective amount
of another therapy to a subject with a solid tumor determined to
lack a p53 deactivating mutation and/or with a solid tumor
expressing wild type p53.
[0337] As used herein, the term "in combination," refers, in the
context of the administration of the peptidomimetic macrocycles, to
the administration of the peptidomimetic macrocycles prior to,
concurrently with, or subsequent to the administration of one or
more additional therapies (e.g., agents, surgery, or radiation) for
use in treating solid tumor. The use of the term "in combination"
does not restrict the order in which the peptidomimetic macrocycles
and one or more additional therapies are administered to a subject.
In specific embodiments, the interval of time between the
administration of the peptidomimetic macrocycles and the
administration of one or more additional therapies can be about 1-5
minutes, 1-30 minutes, 30 minutes to 60 minutes, 1 hour, 1-2 hours,
2-6 hours, 2-12 hours, 12-24 hours, 1-2 days, 2 days, 3 days, 4
days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20
weeks, 26 weeks, 52 weeks, 11-15 weeks, 15-20 weeks, 20-30 weeks,
30-40 weeks, 40-50 weeks, 1 month, 2 months, 3 months, 4 months 5
months, 6 months, 7 months, 8 months, 9 months, 10 months, 11
months, 12 months, 1 year, 2 years, or any period of time in
between. In certain embodiments, the peptidomimetic macrocycles and
one or more additional therapies are administered less than 1 day,
1 week, 2 weeks, 3 weeks, 4 weeks, one month, 2 months, 3 months, 6
months, 1 year, 2 years, or 5 years apart.
[0338] In some embodiments, the combination therapies provided
herein involve administering of the peptidomimetic macrocycles 1-2
times a week, once every week, once every 2 weeks, once every 3
weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks,
once every 7 weeks or once every 8 weeks and administering one or
more additional therapies once a week, once every 2 weeks, once
every 3 weeks, once every 4 weeks, once every month, once every 2
months (e.g., approximately 8 weeks), once every 3 months (e.g.,
approximately 12 weeks), or once every 4 months (e.g.,
approximately 16 weeks). In certain embodiments, the peptidomimetic
macrocycles and one or more additional therapies are cyclically
administered to a subject. Cycling therapy involves the
administration of the peptidomimetic macrocycles compounds for a
period of time, followed by the administration of one or more
additional therapies for a period of time, and repeating this
sequential administration. In certain embodiments, cycling therapy
can also include a period of rest where the peptidomimetic
macrocycles or the additional therapy is not administered for a
period of time (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 7
days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 10 weeks, 20
weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7
months, 8 months, 9 months, 10 months, 11 months, 12 months, 2
years, or 3 years). In an embodiment, the number of cycles
administered is from 1 to 12 cycles, from 2 to 10 cycles, or from 2
to 8 cycles.
[0339] In some embodiments, the methods for treating solid tumor
provided herein comprise administering the peptidomimetic
macrocycles as a single agent for a period of time prior to
administering the peptidomimetic macrocycles in combination with an
additional therapy. In certain embodiments, the methods for
treating cancer provided herein comprise administering an
additional therapy alone for a period of time prior to
administering the peptidomimetic macrocycles in combination with
the additional therapy.
[0340] In some embodiments, the administration of the
peptidomimetic macrocycles and one or more additional therapies in
accordance with the methods presented herein have an additive
effect relative the administration of the peptidomimetic
macrocycles or said one or more additional therapies alone. In some
embodiments, the administration of the peptidomimetic macrocycles
and one or more additional therapies in accordance with the methods
presented herein have a synergistic effect relative to the
administration of the peptidomimetic macrocycles or said one or
more additional therapies alone.
[0341] As used herein, the term "synergistic," refers to the effect
of the administration of the peptidomimetic macrocycles in
combination with one or more additional therapies (e.g., agents),
which combination is more effective than the additive effects of
any two or more single therapies (e.g., agents). In a specific
embodiment, a synergistic effect of a combination therapy permits
the use of lower dosages (e.g., sub-optimal doses) of the
peptidomimetic macrocycles or an additional therapy and/or less
frequent administration of the peptidomimetic macrocycles or an
additional therapy to a subject. In certain embodiments, the
ability to utilize lower dosages of the peptidomimetic macrocycles
or of an additional therapy and/or to administer the peptidomimetic
macrocycles or said additional therapy less frequently reduces the
toxicity associated with the administration of the peptidomimetic
macrocycles or of said additional therapy, respectively, to a
subject without reducing the efficacy of the peptidomimetic
macrocycles or of said additional therapy, respectively, in the
treatment of solid tumor. In some embodiments, a synergistic effect
results in improved efficacy of the peptidomimetic macrocycles and
each of said additional therapies in treating cancer. In some
embodiments, a synergistic effect of a combination of the
peptidomimetic macrocycles and one or more additional therapies
avoids or reduces adverse or unwanted side effects associated with
the use of any single therapy.
[0342] The combination of the peptidomimetic macrocycles and one or
more additional therapies can be administered to a subject in the
same pharmaceutical composition. Alternatively, the peptidomimetic
macrocycles and one or more additional therapies can be
administered concurrently to a subject in separate pharmaceutical
compositions. The peptidomimetic macrocycles and one or more
additional therapies can be administered sequentially to a subject
in separate pharmaceutical compositions. The peptidomimetic
macrocycles compounds and one or more additional therapies can also
be administered to a subject by the same or different routes of
administration.
[0343] The combination therapies provided herein involve
administering to a subject to in need thereof the peptidomimetic
macrocycles in combination with conventional, or known, therapies
for treating cancer. Other therapies for cancer or a condition
associated therewith are aimed at controlling or relieving one or
more symptoms. Accordingly, in some embodiments, the combination
therapies provided herein involve administering to a subject to in
need thereof a pain reliever, or other therapies aimed at
alleviating or controlling one or more symptoms associated with or
a condition associated therewith.
[0344] Non-limiting specific examples of anti-cancer agents that
can be used in combination with the peptidomimetic macrocycles
include: a hormonal agent (e.g., aromatase inhibitor, selective
estrogen receptor modulator (SERM), and estrogen receptor
antagonist), chemotherapeutic agent (e.g., microtubule disassembly
blocker, antimetabolite, topoisomerase inhibitor, and DNA
crosslinker or damaging agent), anti-antigenic agent (e.g., VEGF
antagonist, receptor antagonist, integrin antagonist, vascular
targeting agent (VTA)/vascular disrupting agent (VDA)), radiation
therapy, and conventional surgery.
[0345] Non-limiting examples of hormonal agents that can be used in
combination with the peptidomimetic macrocycles include aromatase
inhibitors, SERMs, and estrogen receptor antagonists. Hormonal
agents that are aromatase inhibitors can be steroidal or no
steroidal. Non-limiting examples of no steroidal hormonal agents
include letrozole, anastrozole, aminoglutethimide, fadrozole, and
vorozole. Non-limiting examples of steroidal hormonal agents
include aromasin (exemestane), formestane, and testolactone.
Non-limiting examples of hormonal agents that are SERMs include
tamoxifen (branded/marketed as Nolvadex.RTM.), afimoxifene,
arzoxifene, bazedoxifene, clomifene, femarelle, lasofoxifene,
ormeloxifene, raloxifene, and toremifene. Non-limiting examples of
hormonal agents that are estrogen receptor antagonists include
fulvestrant. Other hormonal agents include but are not limited to
abiraterone and lonaprisan.
[0346] Non-limiting examples of chemotherapeutic agents that can be
used in combination with of peptidomimetic macrocycles include
microtubule disassembly blocker, antimetabolite, topoisomerase
inhibitor, and DNA crosslinker or damaging agent. Chemotherapeutic
agents that are microtubule disassembly blockers include, but are
not limited to, taxanes (e.g., paclitaxel (branded/marketed as
TAXOL.RTM.), docetaxel, abraxane, larotaxel, ortataxel, and
tesetaxel); epothilones (e.g., ixabepilone); and vinca alkaloids
(e.g., vinorelbine, vinblastine, vindesine, and vincristine
(branded/marketed as ONCOVIN.RTM.)).
[0347] Chemotherapeutic agents that are antimetabolites include,
but are not limited to, folate anitmetabolites (e.g., methotrexate,
aminopterin, pemetrexed, raltitrexed); purine antimetabolites
(e.g., cladribine, clofarabine, fludarabine, mercaptopurine,
pentostatin, thioguanine); pyrimidine antimetabolites (e.g.,
5-fluorouracil, capcitabine, gemcitabine (GEMZAR.RTM.), cytarabine,
decitabine, floxuridine, tegafur); and deoxyribonucleotide
antimetabolites (e.g., hydroxyurea).
[0348] Chemotherapeutic agents that are topoisomerase inhibitors
include, but are not limited to, class I (camptotheca)
topoisomerase inhibitors (e.g., topotecan (branded/marketed as
HYCAMTIN.RTM.) irinotecan, rubitecan, and belotecan); class II
(podophyllum) topoisomerase inhibitors (e.g., etoposide or VP-16,
and teniposide); anthracyclines (e.g., doxorubicin, epirubicin,
Doxil, aclarubicin, amrubicin, daunorubicin, idarubicin,
pirarubicin, valrubicin, and zorubicin); and anthracenediones
(e.g., mitoxantrone, and pixantrone).
[0349] Chemotherapeutic agents that are DNA crosslinkers (or DNA
damaging agents) include, but are not limited to, alkylating agents
(e.g., cyclophosphamide, mechlorethamine, ifosfamide
(branded/marketed as IFEX.RTM.), trofosfamide, chlorambucil,
melphalan, prednimustine, bendamustine, uramustine, estramustine,
carmustine (branded/marketed as BiCNU.RTM.), lomustine, semustine,
fotemustine, nimustine, ranimustine, streptozocin, busulfan,
mannosulfan, treosulfan, carboquone,
N,N'N'-triethylenethiophosphoramide, triaziquone,
triethylenemelamine); alkylating-like agents (e.g., carboplatin
(branded/marketed as PARAPLATIN.RTM.), cisplatin, oxaliplatin,
nedaplatin, triplatin tetranitrate, satraplatin, picoplatin);
nonclassical DNA crosslinkers (e.g., procarbazine, dacarbazine,
temozolomide (branded/marketed as TEMODAR.RTM.), altretamine,
mitobronitol); and intercalating agents (e.g., actinomycin,
bleomycin, mitomycin, and plicamycin).
[0350] Non-limiting examples of other therapies that can be
administered to a subject in combination with the peptidomimetic
macrocycles include: (1) a statin such as lovastatin (e.g.,
branded/marketed as MEVACOR.RTM.); (2) an mTOR inhibitor such as
sirolimus which is also known as Rapamycin (e.g., branded/marketed
as RAPAMUNE.RTM.), temsirolimus (e.g., branded/marketed as
TORISEL.RTM.), evorolimus (e.g., branded/marketed as
AFINITOR.RTM.), and deforolimus; (3) a famesyltransferase inhibitor
agent such as tipifarnib; (4) an antifibrotic agent such as
pirfenidone; (5) a pegylated interferon such as PEG-interferon
alfa-2b; (6) a CNS stimulant such as methylphenidate
(branded/marketed as RITALIN.RTM.); (7) a HER-2 antagonist such as
anti-HER-2 antibody (e.g., trastuzumab) and kinase inhibitor (e.g.,
lapatinib); (8) an IGF-1 antagonist such as an anti-IGF-1 antibody
(e.g., AVE1642 and IMC-A11) or an IGF-1 kinase inhibitor; (9)
EGFR/HER-1 antagonist such as an anti-EGFR antibody (e.g.,
cetuximab, panitumamab) or EGFR kinase inhibitor (e.g., erlotinib;
gefitinib); (10) SRC antagonist such as bosutinib; (11) cyclin
dependent kinase (CDK) inhibitor such as seliciclib; (12) Janus
kinase 2 inhibitor such as lestaurtinib; (13) proteasome inhibitor
such as bortezomib; (14) phosphodiesterase inhibitor such as
anagrelide; (15) inosine monophosphate dehydrogenase inhibitor such
as tiazofurine; (16) lipoxygenase inhibitor such as masoprocol;
(17) endothelin antagonist; (18) retinoid receptor antagonist such
as tretinoin or alitretinoin; (19) immune modulator such as
lenalidomide, pomalidomide, or thalidomide; (20) kinase (e.g.,
tyrosine kinase) inhibitor such as imatinib, dasatinib, erlotinib,
nilotinib, gefitinib, sorafenib, sunitinib, lapatinib, or TG100801;
(21) non-steroidal anti-inflammatory agent such as celecoxib
(branded/marketed as CELEBREX.RTM.); (22) human granulocyte
colony-stimulating factor (G-CSF) such as filgrastim
(branded/marketed as NEUPOGEN.RTM.); (23) folinic acid or
leucovorin calcium; (24) integrin antagonist such as an integrin
.alpha.5.beta.1-antagonist (e.g., JSM6427); (25) nuclear factor
kappa beta (NF-.kappa..beta.) antagonist such as OT-551, which is
also an anti-oxidant. (26) hedgehog inhibitor such as CUR61414,
cyclopamine, GDC-0449, and anti-hedgehog antibody; (27) histone
deacetylase (HDAC) inhibitor such as SAHA (also known as vorinostat
(branded/marketed as ZOLINZA)), PCI-24781, SB939, CHR-3996,
CRA-024781, ITF2357, JNJ-26481585, or PCI-24781; (28) retinoid such
as isotretinoin (e.g., branded/marketed as ACCUTANE.RTM.); (29)
hepatocyte growth factor/scatter factor (HGF/SF) antagonist such as
HGF/SF monoclonal antibody (e.g., AMG 102); (30) synthetic chemical
such as antineoplaston; (31) anti-diabetic such as rosaiglitazone
(e.g., branded/marketed as AVANDIA.RTM.); (32) antimalarial and
amebicidal drug such as chloroquine (e.g., branded/marketed as
ARALEN.RTM.); (33) synthetic bradykinin such as RMP-7; (34)
platelet-derived growth factor receptor inhibitor such as SU-101;
(35) receptor tyrosine kinase inhibitorsof Flk-1/KDR/VEGFR2, FGFR1
and PDGFR beta such as SU5416 and SU6668; (36) anti-inflammatory
agent such as sulfasalazine (e.g., branded/marketed as
AZULFIDINE.RTM.); and (37) TGF-beta antisense therapy.
[0351] In some embodiments a peptidomimetic macrocycles disclosed
herein can inhibit one or more transporter enzymes (e.g., OATP1B1,
OATP1B3, BSEP) at concentrations that can be clinically relevant.
Therefore such a peptidomimetic macrocycles disclosed herein can
interact with medications that are predominantly cleared by
hepatobiliary transporters. In particular, methotrexate and statins
(e.g., atorvastatin, fluvastatin lovastatin, pitavastatin
pravastatin, rosuvastatin and simvastatin) may not be dosed within
48 h, 36 h, 24 h, or 12 h ((for example within 24 h) of the
administration of such a peptidomimetic macrocycle. Exemplary
medications that can be affected by co-administration of such a
peptidomimetic macrocycles are listed below. In various embodiments
one or more of the medications selected from Table 2 may not be
dosed within 48 h, 36 h, 24 h, or 12 h (for example within 24 h) of
the administration of such a peptidomimetic macrocycle.
TABLE-US-00004 TABLE 2 Exemplary medications that can be affected
by co-administration with a peptidomimetic macrocycles disclosed
herein. Medication Therapeutic Area Irinotecan Oncology Bosentan
Pulmonary artery hypertension Caspofungin Antifungal Methotrexate
Oncology & rheumatology Repaglinide Diabetes mellitus
Atorvastatin Hypercholesterolemia Cerivastatin Hypercholesterolemia
Fluvastatin Hypercholesterolemia Lovastatin Hypercholesterolemia
Pitavastatin Hypercholesterolemia Pravastatin Hypercholesterolemia
Rosuvastatin Hypercholesterolemia Simvastatin
Hypercholesterolemia
EXAMPLES
Example 1: Peptidomimetic Macrocycles
[0352] Peptidomimetic macrocycles were synthesized, purified and
analyzed as previously described and as described below
(Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000);
Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005);
Walensky et al., Science 305:1466-1470 (2004); and U.S. Pat. No.
7,192,713). Peptidomimetic macrocycles were designed by replacing
two or more naturally occurring amino acids with the corresponding
synthetic amino acids. Substitutions were made at i and i+4, and i
and i+7 positions. Peptide synthesis was performed either manually
or on an automated peptide synthesizer (Applied Biosystems, model
433A), using solid phase conditions, rink amide AM resin
(Novabiochem), and Fmoc main-chain protecting group chemistry. For
the coupling of natural Fmoc-protected amino acids (Novabiochem),
10 equivalents of amino acid and a 1:1:2 molar ratio of coupling
reagents HBTU/HOBt (Novabiochem)/DIEA were employed. Non-natural
amino acids (4 equiv) were coupled with a 1:1:2 molar ratio of HATU
(Applied Biosystems)/HOBt/DIEA. The N-termini of the synthetic
peptides were acetylated, while the C-termini were amidated.
[0353] Purification of cross-linked compounds was achieved by high
performance liquid chromatography (HPLC) (Varian ProStar) on a
reverse phase C18 column (Varian) to yield the pure compounds.
Chemical composition of the pure products was confirmed by LC/MS
mass spectrometry (Micromass LCT interfaced with Agilent 1100 HPLC
system) and amino acid analysis (Applied Biosystems, model
420A).
[0354] The following protocol was used in the synthesis of
dialkyne-crosslinked peptidomimetic macrocycles, including SP662,
SP663 and SP664. Fully protected resin-bound peptides were
synthesized on a PEG-PS resin (loading 0.45 mmol/g) on a 0.2 mmol
scale. Deprotection of the temporary Fmoc group was achieved by
3.times.10 min treatments of the resin bound peptide with 20% (v/v)
piperidine in DMF. After washing with NMP (3.times.),
dichloromethane (3.times.) and NMP (3.times.), coupling of each
successive amino acid was achieved with 1.times.60 min incubation
with the appropriate preactivated Fmoc-amino acid derivative. All
protected amino acids (0.4 mmol) were dissolved in NMP and
activated with HCTU (0.4 mmol) and DIEA (0.8 mmol) prior to
transfer of the coupling solution to the deprotected resin-bound
peptide. After coupling was completed, the resin was washed in
preparation for the next deprotection/coupling cycle. Acetylation
of the amino terminus was carried out in the presence of acetic
anhydride/DIEA in NMP. The LC-MS analysis of a cleaved and
deprotected sample obtained from an aliquot of the fully assembled
resin-bound peptide was accomplished in order to verifying the
completion of each coupling. In a typical example, tetrahydrofuran
(4 ml) and triethylamine (2 ml) were added to the peptide resin
(0.2 mmol) in a 40 ml glass vial and shaken for 10 minutes.
Pd(PPh.sub.3).sub.2Cl.sub.2 (0.014 g, 0.02 mmol) and copper iodide
(0.008 g, 0.04 mmol) were then added and the resulting reaction
mixture was mechanically shaken 16 hours while open to atmosphere.
The diyne-cyclized resin-bound peptides were deprotected and
cleaved from the solid support by treatment with TFA/H.sub.2O/TIS
(95/5/5 v/v) for 2.5 h at room temperature. After filtration of the
resin the TFA solution was precipitated in cold diethyl ether and
centrifuged to yield the desired product as a solid. The crude
product was purified by preparative HPLC.
[0355] The following protocol was used in the synthesis of single
alkyne-crosslinked peptidomimetic macrocycles, including SP665.
Fully protected resin-bound peptides were synthesized on a Rink
amide MBHA resin (loading 0.62 mmol/g) on a 0.1 mmol scale.
Deprotection of the temporary Fmoc group was achieved by 2.times.20
min treatments of the resin bound peptide with 25% (v/v) piperidine
in NMP. After extensive flow washing with NMP and dichloromethane,
coupling of each successive amino acid was achieved with 1.times.60
min incubation with the appropriate preactivated Fmoc-amino acid
derivative. All protected amino acids (1 mmol) were dissolved in
NMP and activated with HCTU (1 mmol) and DIEA (1 mmol) prior to
transfer of the coupling solution to the deprotected resin-bound
peptide. After coupling was completed, the resin was extensively
flow washed in preparation for the next deprotection/coupling
cycle. Acetylation of the amino terminus was carried out in the
presence of acetic anhydride/DIEA in NMP/NMM. The LC-MS analysis of
a cleaved and deprotected sample obtained from an aliquot of the
fully assembled resin-bound peptide was accomplished in order to
verifying the completion of each coupling. In a typical example,
the peptide resin (0.1 mmol) was washed with DCM. Resin was loaded
into a microwave vial. The vessel was evacuated and purged with
nitrogen. Molybdenumhexacarbonyl (0.01 eq, Sigma Aldrich 199959)
was added. Anhydrous chlorobenzene was added to the reaction
vessel. Then 2-fluorophenol (1 eq, Sigma Aldrich F 12804) was
added. The reaction was then loaded into the microwave and held at
130.degree. C. for 10 minutes. Reaction may need to be pushed a
subsequent time for completion. The alkyne metathesized resin-bound
peptides were deprotected and cleaved from the solid support by
treatment with TFA/H.sub.2O/TIS (94/3/3 v/v) for 3 h at room
temperature. After filtration of the resin the TFA solution was
precipitated in cold diethyl ether and centrifuged to yield the
desired product as a solid. The crude product was purified by
preparative HPLC.
[0356] Table 3 shows a list of peptidomimetic macrocycles
prepared.
TABLE-US-00005 TABLE 3 SEQ ID Exact Found Calc Calc Calc Sequence
NO: Isomer Mass Mass (M + 1)/1 (M + 2)/2 (M + 3)/3 1
Ac-F$r8AYWEAc3cL$AAA-NH.sub.2 10 1456.78 729.44 1457.79 729.4 486.6
2 Ac-F$r8AYWEAc3cL$AAibA-NH.sub.2 11 1470.79 736.4 1471.8 736.4
491.27 3 Ac-LTF$r8AYWAQL$SANle-NH.sub.2 12 1715.97 859.02 1716.98
858.99 573 4 Ac-LTF$r8AYWAQL$SAL-NH.sub.2 13 1715.97 859.02 1716.98
858.99 573 5 Ac-LTF$r8AYWAQL$SAM-NH.sub.2 14 1733.92 868.48 1734.93
867.97 578.98 6 Ac-LTF$r8AYWAQL$SAhL-NH.sub.2 15 1729.98 865.98
1730.99 866 577.67 7 Ac-LTF$r8AYWAQL$SAF-NH.sub.2 16 1749.95 876.36
1750.96 875.98 584.32 8 Ac-LTF$r8AYWAQL$SAI-NH.sub.2 17 1715.97
859.02 1716.98 858.99 573 9 Ac-LTF$r8AYWAQL$SAChg-NH.sub.2 18
1741.98 871.98 1742.99 872 581.67 10 Ac-LTF$r8AYWAQL$SAAib-NH.sub.2
19 1687.93 845.36 1688.94 844.97 563.65 11
Ac-LTF$r8AYWAQL$SAA-NH.sub.2 20 1673.92 838.01 1674.93 837.97
558.98 12 Ac-LTF$r8AYWA$L$S$Nle-NH.sub.2 21 1767.04 884.77 1768.05
884.53 590.02 13 Ac-LTF$r8AYWA$L$S$A-NH.sub.2 22 1724.99 864.23
1726 863.5 576 14 Ac-F$r8AYWEAc3cL$AANle-NH.sub.2 23 1498.82 750.46
1499.83 750.42 500.61 15 Ac-F$r8AYWEAc3cL$AAL-NH.sub.2 24 1498.82
750.46 1499.83 750.42 500.61 16 Ac-F$r8AYWEAc3cL$AAM-NH.sub.2 25
1516.78 759.41 1517.79 759.4 506.6 17
Ac-F$r8AYWEAc3cL$AAhL-NH.sub.2 26 1512.84 757.49 1513.85 757.43
505.29 18 Ac-F$r8AYWEAc3cL$AAF-NH.sub.2 27 1532.81 767.48 1533.82
767.41 511.94 19 Ac-F$r8AYWEAc3cL$AAI-NH.sub.2 28 1498.82 750.39
1499.83 750.42 500.61 20 Ac-F$r8AYWEAc3cL$AAChg-NH.sub.2 29 1524.84
763.48 1525.85 763.43 509.29 21 Ac-F$r8AYWEAc3cL$AACha-NH.sub.2 30
1538.85 770.44 1539.86 770.43 513.96 22
Ac-F$r8AYWEAc3cL$AAAib-NH.sub.2 31 1470.79 736.84 1471.8 736.4
491.27 23 Ac-LTF$r8AYWAQL$AAAibV-NH.sub.2 32 1771.01 885.81 1772.02
886.51 591.34 24 Ac-LTF$r8AYWAQL$AAAibV-NH.sub.2 33 iso2 1771.01
886.26 1772.02 886.51 591.34 25 Ac-LTF$r8AYWAQL$SAibAA-NH.sub.2 34
1758.97 879.89 1759.98 880.49 587.33 26
Ac-LTF$r8AYWAQL$SAibAA-NH.sub.2 35 iso2 1758.97 880.34 1759.98
880.49 587.33 27 Ac-HLTF$r8HHWHQL$AANleNle-NH.sub.2 36 2056.15
1028.86 2057.16 1029.08 686.39 28 Ac-DLTF$r8HHWHQL$RRLV-NH.sub.2 37
2190.23 731.15 2191.24 1096.12 731.08 29
Ac-HHTF$r8HHWHQL$AAML-NH.sub.2 38 2098.08 700.43 2099.09 1050.05
700.37 30 Ac-F$r8HHWHQL$RRDCha-NH.sub.2 39 1917.06 959.96 1918.07
959.54 640.03 31 Ac-F$r8HHWHQL$HRFV-NH.sub.2 40 1876.02 938.65
1877.03 939.02 626.35 32 Ac-HLTF$r8HHWHQL$AAhLA-NH.sub.2 41 2028.12
677.2 2029.13 1015.07 677.05 33 Ac-DLTF$r8HHWHQL$RRChgl-NH.sub.2 42
2230.26 1115.89 2231.27 1116.14 744.43 34
Ac-DLTF$r8HHWHQL$RRChgl-NH.sub.2 43 iso2 2230.26 1115.96 2231.27
1116.14 744.43 35 Ac-HHTF$r8HHWHQL$AAChav-NH.sub.2 44 2106.14
1053.95 2107.15 1054.08 703.05 36 Ac-F$r8HHWHQL$RRDa-NH.sub.2 45
1834.99 918.3 1836 918.5 612.67 37 Ac-F$r8HHWHQL$HRAibG-NH.sub.2 46
1771.95 886.77 1772.96 886.98 591.66 38
Ac-F$r8AYWAQL$HHNleL-NH.sub.2 47 1730.97 866.57 1731.98 866.49 578
39 Ac-F$r8AYWSAL$HQANle-NH.sub.2 48 1638.89 820.54 1639.9 820.45
547.3 40 Ac-F$r8AYWVQL$QHChgl-NH.sub.2 49 1776.01 889.44 1777.02
889.01 593.01 41 Ac-F$r8AYWTAL$QQNlev-NH.sub.2 50 1671.94 836.97
1672.95 836.98 558.32 42 Ac-F$r8AYWYQL$HAibAa-NH.sub.2 51 1686.89
844.52 1687.9 844.45 563.3 43 Ac-LTF$r8AYWAQL$HHLa-NH.sub.2 52
1903.05 952.27 1904.06 952.53 635.36 44
Ac-LTF$r8AYWAQL$HHLa-NH.sub.2 53 iso2 1903.05 952.27 1904.06 952.53
635.36 45 Ac-LTF$r8AYWAQL$HQNlev-NH.sub.2 54 1922.08 962.48 1923.09
962.05 641.7 46 Ac-LTF$r8AYWAQL$HQNlev-NH.sub.2 55 iso2 1922.08
962.4 1923.09 962.05 641.7 47 Ac-LTF$r8AYWAQL$QQMl-NH.sub.2 56
1945.05 973.95 1946.06 973.53 649.36 48
Ac-LTF$r8AYWAQL$QQMl-NH.sub.2 57 iso2 1945.05 973.88 1946.06 973.53
649.36 49 Ac-LTF$r8AYWAQL$HAibhLV-NH.sub.2 58 1893.09 948.31 1894.1
947.55 632.04 50 Ac-LTF$r8AYWAQL$AHFA-NH.sub.2 59 1871.01 937.4
1872.02 936.51 624.68 51 Ac-HLTF$r8HHWHQL$AANlel-NH.sub.2 60
2056.15 1028.79 2057.16 1029.08 686.39 52
Ac-DLTF$r8HHWHQL$RRLa-NH.sub.2 61 2162.2 721.82 2163.21 1082.11
721.74 53 Ac-HHTF$r8HHWHQL$AAMv-NH.sub.2 62 2084.07 1042.92 2085.08
1043.04 695.7 54 Ac-F$r8HHWHQL$RRDA-NH.sub.2 63 1834.99 612.74 1836
918.5 612.67 55 Ac-F$r8HHWHQL$HRFCha-NH.sub.2 64 1930.06 966.47
1931.07 966.04 644.36 56 Ac-F$r8AYWEAL$AA-NHAm 65 1443.82 1445.71
1444.83 722.92 482.28 57 Ac-F$r8AYWEAL$AA-NHiAm 66 1443.82 723.13
1444.83 722.92 482.28 58 Ac-F$r8AYWEAL$AA-NHnPr3Ph 67 1491.82 747.3
1492.83 746.92 498.28 59 Ac-F$r8AYWEAL$AA-NHnBu33Me 68 1457.83
1458.94 1458.84 729.92 486.95 60 Ac-F$r8AYWEAL$AA-NHnPr 69 1415.79
709.28 1416.8 708.9 472.94 61 Ac-F$r8AYWEAL$AA-NHnEt2Ch 70 1483.85
1485.77 1484.86 742.93 495.62 62 Ac-F$r8AYWEAL$AA-NHnEt2Cp 71
1469.83 1470.78 1470.84 735.92 490.95 63 Ac-F$r8AYWEAL$AA-NHHex 72
1457.83 730.19 1458.84 729.92 486.95 64
Ac-LTF$r8AYWAQL$AAIA-NH.sub.2 73 1771.01 885.81 1772.02 886.51
591.34 65 Ac-LTF$r8AYWAQL$AAIA-NH.sub.2 74 iso2 1771.01 866.8
1772.02 886.51 591.34 66 Ac-LTF$r8AYWAAL$AAMA-NH.sub.2 75 1731.94
867.08 1732.95 866.98 578.32 67 Ac-LTF$r8AYWAAL$AAMA-NH.sub.2 76
iso2 1731.94 867.28 1732.95 866.98 578.32 68
Ac-LTF$r8AYWAQL$AANleA-NH.sub.2 77 1771.01 867.1 1772.02 886.51
591.34 69 Ac-LTF$r8AYWAQL$AANleA-NH.sub.2 78 iso2 1771.01 886.89
1772.02 886.51 591.34 70 Ac-LTF$r8AYWAQL$AAIa-NH.sub.2 79 1771.01
886.8 1772.02 886.51 591.34 71 Ac-LTF$r8AYWAQL$AAIa-NH.sub.2 80
iso2 1771.01 887.09 1772.02 886.51 591.34 72
Ac-LTF$r8AYWAAL$AAMa-NH.sub.2 81 1731.94 867.17 1732.95 866.98
578.32 73 Ac-LTF$r8AYWAAL$AAMa-NH.sub.2 82 iso2 1731.94 867.37
1732.95 866.98 578.32 74 Ac-LTF$r8AYWAQL$AANlea-NH.sub.2 83 1771.01
887.08 1772.02 886.51 591.34 75 Ac-LTF$r8AYWAQL$AANlea-NH.sub.2 84
iso2 1771.01 887.08 1772.02 886.51 591.34 76
Ac-LTF$r8AYWAAL$AAIv-NH.sub.2 85 1742.02 872.37 1743.03 872.02
581.68 77 Ac-LTF$r8AYWAAL$AAIv-NH.sub.2 86 iso2 1742.02 872.74
1743.03 872.02 581.68 78 Ac-LTF$r8AYWAQL$AAMv-NH.sub.2 87 1817
910.02 1818.01 909.51 606.67 79 Ac-LTF$r8AYWAAL$AANlev-NH.sub.2 88
1742.02 872.37 1743.03 872.02 581.68 80
Ac-LTF$r8AYWAAL$AANlev-NH.sub.2 89 iso2 1742.02 872.28 1743.03
872.02 581.68 81 Ac-LTF$r8AYWAQL$AAIl-NH.sub.2 90 1813.05 907.81
1814.06 907.53 605.36 82 Ac-LTF$r8AYWAQL$AAIl-NH.sub.2 91 iso2
1813.05 907.81 1814.06 907.53 605.36 83
Ac-LTF$r8AYWAAL$AAMl-NH.sub.2 92 1773.99 887.37 1775 888 592.34 84
Ac-LTF$r8AYWAQL$AANlel-NH.sub.2 93 1813.05 907.61 1814.06 907.53
605.36 85 Ac-LTF$r8AYWAQL$AANlel-NH.sub.2 94 iso2 1813.05 907.71
1814.06 907.53 605.36 86 Ac-F$r8AYWEAL$AAMA-NH.sub.2 95 1575.82
789.02 1576.83 788.92 526.28 87 Ac-F$r8AYWEAL$AANleA-NH.sub.2 96
1557.86 780.14 1558.87 779.94 520.29 88 Ac-F$r8AYWEAL$AAIa-NH.sub.2
97 1557.86 780.33 1558.87 779.94 520.29 89
Ac-F$r8AYWEAL$AAMa-NH.sub.2 98 1575.82 789.3 1576.83 788.92 526.28
90 Ac-F$r8AYWEAL$AANlea-NH.sub.2 99 1557.86 779.4 1558.87 779.94
520.29 91 Ac-F$r8AYWEAL$AAIv-NH.sub.2 100 1585.89 794.29 1586.9
793.95 529.64 92 Ac-F$r8AYWEAL$AAMv-NH.sub.2 101 1603.85 803.08
1604.86 802.93 535.62 93 Ac-F$r8AYWEAL$AANlev-NH.sub.2 102 1585.89
793.46 1586.9 793.95 529.64 94 Ac-F$r8AYWEAL$AAIl-NH.sub.2 103
1599.91 800.49 1600.92 800.96 534.31 95 Ac-F$r8AYWEAL$AAMl-NH.sub.2
104 1617.86 809.44 1618.87 809.94 540.29 96
Ac-F$r8AYWEAL$AANlel-NH.sub.2 105 1599.91 801.7 1600.92 800.96
534.31 97 Ac-F$r8AYWEAL$AANlel-NH.sub.2 106 iso2 1599.91 801.42
1600.92 800.96 534.31 98 Ac-LTF$r8AY6clWAQL$SAA-NH.sub.2 107
1707.88 855.72 1708.89 854.95 570.3 99
Ac-LTF$r8AY6clWAQL$SAA-NH.sub.2 108 iso2 1707.88 855.35 1708.89
854.95 570.3 100 Ac-WTF$r8FYWSQL$AVAa-NH.sub.2 109 1922.01 962.21
1923.02 962.01 641.68 101 Ac-WTF$r8FYWSQL$AVAa-NH.sub.2 110 iso2
1922.01 962.49 1923.02 962.01 641.68 102
Ac-WTF$r8VYWSQL$AVA-NH.sub.2 111 1802.98 902.72 1803.99 902.5 602
103 Ac-WTF$r8VYWSQL$AVA-NH.sub.2 112 iso2 1802.98 903 1803.99 902.5
602 104 Ac-WTF$r8FYWSQL$SAAa-NH.sub.2 113 1909.98 956.47 1910.99
956 637.67 105 Ac-WTF$r8FYWSQL$SAAa-NH.sub.2 114 iso2 1909.98
956.47 1910.99 956 637.67 106 Ac-WTF$r8VYWSQL$AVAaa-NH.sub.2 115
1945.05 974.15 1946.06 973.53 649.36 107
Ac-WTF$r8VYWSQL$AVAaa-NH.sub.2 116 iso2 1945.05 973.78 1946.06
973.53 649.36 108 Ac-LTF$r8AYWAQL$AVG-NH.sub.2 117 1671.94 837.52
1672.95 836.98 558.32 109 Ac-LTF$r8AYWAQL$AVG-NH.sub.2 118 iso2
1671.94 837.21 1672.95 836.98 558.32 110
Ac-LTF$r8AYWAQL$AVQ-NH.sub.2 119 1742.98 872.74 1743.99 872.5 582
111 Ac-LTF$r8AYWAQL$AVQ-NH.sub.2 120 iso2 1742.98 872.74 1743.99
872.5 582 112 Ac-LTF$r8AYWAQL$SAa-NH.sub.2 121 1673.92 838.23
1674.93 837.97 558.98 113 Ac-LTF$r8AYWAQL$SAa-NH.sub.2 122 iso2
1673.92 838.32 1674.93 837.97 558.98 114
Ac-LTF$r8AYWAQhL$SAA-NH.sub.2 123 1687.93 844.37 1688.94 844.97
563.65 115 Ac-LTF$r8AYWAQhL$SAA-NH.sub.2 124 iso2 1687.93 844.81
1688.94 844.97 563.65 116 Ac-LTF$r8AYWEQLStSA$-NH.sub.2 125 1826
905.27 1827.01 914.01 609.67 117 Ac-LTF$r8AYWAQL$SLA-NH.sub.2 126
1715.97 858.48 1716.98 858.99 573 118 Ac-LTF$r8AYWAQL$SLA-NH.sub.2
127 iso2 1715.97 858.87 1716.98 858.99 573 119
Ac-LTF$r8AYWAQL$SWA-NH.sub.2 128 1788.96 895.21 1789.97 895.49
597.33 120 Ac-LTF$r8AYWAQL$SWA-NH.sub.2 129 iso2 1788.96 895.28
1789.97 895.49 597.33 121 Ac-LTF$r8AYWAQL$SVS-NH.sub.2 130 1717.94
859.84 1718.95 859.98 573.65 122 Ac-LTF$r8AYWAQL$SAS-NH.sub.2 131
1689.91 845.85 1690.92 845.96 564.31 123
Ac-LTF$r8AYWAQL$SVG-NH.sub.2 132 1687.93 844.81 1688.94 844.97
563.65 124 Ac-ETF$r8VYWAQL$SAa-NH.sub.2 133 1717.91 859.76 1718.92
859.96 573.64 125 Ac-ETF$r8VYWAQL$SAA-NH.sub.2 134 1717.91 859.84
1718.92 859.96 573.64 126 Ac-ETF$r8VYWAQL$SVA-NH.sub.2 135 1745.94
873.82 1746.95 873.98 582.99 127 Ac-ETF$r8VYWAQL$SLA-NH.sub.2 136
1759.96 880.85 1760.97 880.99 587.66 128
Ac-ETF$r8VYWAQL$SWA-NH.sub.2 137 1832.95 917.34 1833.96 917.48
611.99 129 Ac-ETF$r8KYWAQL$SWA-NH.sub.2 138 1861.98 931.92 1862.99
932 621.67 130 Ac-ETF$r8VYWAQL$SVS-NH.sub.2 139 1761.93 881.89
1762.94 881.97 588.32 131 Ac-ETF$r8VYWAQL$SAS-NH.sub.2 140 1733.9
867.83 1734.91 867.96 578.97 132 Ac-ETF$r8VYWAQL$SVG-NH.sub.2 141
1731.92 866.87 1732.93 866.97 578.31 133
Ac-LTF$r8VYWAQL$SSa-NH.sub.2 142 1717.94 859.47 1718.95 859.98
573.65 134 Ac-ETF$r8VYWAQL$SSa-NH.sub.2 143 1733.9 867.83 1734.91
867.96 578.97 135 Ac-LTF$r8VYWAQL$SNa-NH.sub.2 144 1744.96 873.38
1745.97 873.49 582.66 136 Ac-ETF$r8VYWAQL$SNa-NH.sub.2 145 1760.91
881.3 1761.92 881.46 587.98 137 Ac-LTF$r8VYWAQL$SAa-NH.sub.2 146
1701.95 851.84 1702.96 851.98 568.32 138
Ac-LTF$r8VYWAQL$SVA-NH.sub.2 147 1729.98 865.53 1730.99 866 577.67
139 Ac-LTF$r8VYWAQL$SVA-NH.sub.2 148 iso2 1729.98 865.9 1730.99 866
577.67 140 Ac-LTF$r8VYWAQL$SWA-NH.sub.2 149 1816.99 909.42 1818
909.5 606.67 141 Ac-LTF$r8VYWAQL$SVS-NH.sub.2 150 1745.98 873.9
1746.99 874 583 142 Ac-LTF$r8VYWAQL$SVS-NH.sub.2 151 iso2 1745.98
873.9 1746.99 874 583 143 Ac-LTF$r8VYWAQL$SAS-NH.sub.2 152 1717.94
859.84 1718.95 859.98 573.65 144 Ac-LTF$r8VYWAQL$SAS-NH.sub.2 153
iso2 1717.94 859.91 1718.95 859.98 573.65 145
Ac-LTF$r8VYWAQL$SVG-NH.sub.2 154 1715.97 858.87 1716.98 858.99 573
146 Ac-LTF$r8VYWAQL$SVG-NH.sub.2 155 iso2 1715.97 858.87 1716.98
858.99 573 147 Ac-LTF$r8EYWAQCha$SAA-NH.sub.2 156 1771.96 886.85
1772.97 886.99 591.66 148 Ac-LTF$r8EYWAQCha$SAA-NH.sub.2 157 iso2
1771.96 886.85 1772.97 886.99 591.66 149
Ac-LTF$r8EYWAQCpg$SAA-NH.sub.2 158 1743.92 872.86 1744.93 872.97
582.31 150 Ac-LTF$r8EYWAQCpg$SAA-NH.sub.2 159 iso2 1743.92 872.86
1744.93 872.97 582.31 151 Ac-LTF$r8EYWAQF$SAA-NH.sub.2 160 1765.91
883.44 1766.92 883.96 589.64 152 Ac-LTF$r8EYWAQF$SAA-NH.sub.2 161
iso2 1765.91 883.89 1766.92 883.96 589.64 153
Ac-LTF$r8EYWAQCba$SAA-NH.sub.2 162 1743.92 872.42 1744.93 872.97
582.31 154 Ac-LTF$r8EYWAQCba$SAA-NH.sub.2 163 iso2 1743.92 873.39
1744.93 872.97 582.31 155 Ac-LTF3Cl$r8EYWAQL$SAA-NH.sub.2 164
1765.89 883.89 1766.9 883.95 589.64 156
Ac-LTF3Cl$r8EYWAQL$SAA-NH.sub.2 165 iso2 1765.89 883.96 1766.9
883.95 589.64 157 Ac-LTF34F2$r8EYWAQL$SAA-NH.sub.2 166 1767.91
884.48 1768.92 884.96 590.31 158 Ac-LTF34F2$r8EYWAQL$SAA-NH.sub.2
167 iso2 1767.91 884.48 1768.92 884.96 590.31
159 Ac-LTF34F2$r8EYWAQhL$SAA-NH.sub.2 168 1781.92 891.44 1782.93
891.97 594.98 160 Ac-LTF34F2$r8EYWAQhL$SAA-NH.sub.2 169 iso2
1781.92 891.88 1782.93 891.97 594.98 161
Ac-ETF$r8EYWAQL$SAA-NH.sub.2 170 1747.88 874.34 1748.89 874.95
583.63 162 Ac-LTF$r8AYWVQL$SAA-NH.sub.2 171 1701.95 851.4 1702.96
851.98 568.32 163 Ac-LTF$r8AHWAQL$SAA-NH.sub.2 172 1647.91 824.83
1648.92 824.96 550.31 164 Ac-LTF$r8AEWAQL$SAA-NH.sub.2 173 1639.9
820.39 1640.91 820.96 547.64 165 Ac-LTF$r8ASWAQL$SAA-NH.sub.2 174
1597.89 799.38 1598.9 799.95 533.64 166
Ac-LTF$r8AEWAQL$SAA-NH.sub.2 175 iso2 1639.9 820.39 1640.91 820.96
547.64 167 Ac-LTF$r8ASWAQL$SAA-NH.sub.2 176 iso2 1597.89 800.31
1598.9 799.95 533.64 168 Ac-LTF$r8AF4coohWAQL$SAA-NH.sub.2 177
1701.91 851.4 1702.92 851.96 568.31 169
Ac-LTF$r8AF4coohWAQL$SAA-NH.sub.2 178 iso2 1701.91 851.4 1702.92
851.96 568.31 170 Ac-LTF$r8AHWAQL$AAIa-NH.sub.2 179 1745 874.13
1746.01 873.51 582.67 171 Ac-ITF$r8FYWAQL$AAIa-NH.sub.2 180 1847.04
923.92 1848.05 924.53 616.69 172 Ac-ITF$r8EHWAQL$AAIa-NH.sub.2 181
1803.01 903.17 1804.02 902.51 602.01 173
Ac-ITF$r8EHWAQL$AAIa-NH.sub.2 182 iso2 1803.01 903.17 1804.02
902.51 602.01 174 Ac-ETF$r8EHWAQL$AAIa-NH.sub.2 183 1818.97 910.76
1819.98 910.49 607.33 175 Ac-ETF$r8EHWAQL$AAIa-NH.sub.2 184 iso2
1818.97 910.85 1819.98 910.49 607.33 176
Ac-LTF$r8AHWVQL$AAIa-NH.sub.2 185 1773.03 888.09 1774.04 887.52
592.02 177 Ac-ITF$r8FYWVQL$AAIa-NH.sub.2 186 1875.07 939.16 1876.08
938.54 626.03 178 Ac-ITF$r8EYWVQL$AAIa-NH.sub.2 187 1857.04 929.83
1858.05 929.53 620.02 179 Ac-ITF$r8EHWVQL$AAIa-NH.sub.2 188 1831.04
916.86 1832.05 916.53 611.35 180 Ac-LTF$r8AEWAQL$AAIa-NH.sub.2 189
1736.99 869.87 1738 869.5 580 181
Ac-LTF$r8AF4coohWAQL$AAIa-NH.sub.2 190 1799 900.17 1800.01 900.51
600.67 182 Ac-LTF$r8AF4coohWAQL$AAIa-NH.sub.2 191 iso2 1799 900.24
1800.01 900.51 600.67 183 Ac-LTF$r8AHWAQL$AHFA-NH.sub.2 192 1845.01
923.89 1846.02 923.51 616.01 184 Ac-ITF$r8FYWAQL$AHFA-NH.sub.2 193
1947.05 975.05 1948.06 974.53 650.02 185
Ac-ITF$r8FYWAQL$AHFA-NH.sub.2 194 iso2 1947.05 976.07 1948.06
974.53 650.02 186 Ac-ITF$r8FHWAQL$AEFA-NH.sub.2 195 1913.02 958.12
1914.03 957.52 638.68 187 Ac-ITF$r8FHWAQL$AEFA-NH.sub.2 196 iso2
1913.02 957.86 1914.03 957.52 638.68 188
Ac-ITF$r8EHWAQL$AHFA-NH.sub.2 197 1903.01 952.94 1904.02 952.51
635.34 189 Ac-ITF$r8EHWAQL$AHFA-NH.sub.2 198 iso2 1903.01 953.87
1904.02 952.51 635.34 190 Ac-LTF$r8AHWVQL$AHFA-NH.sub.2 199 1873.04
937.86 1874.05 937.53 625.35 191 Ac-ITF$r8FYWVQL$AHFA-NH.sub.2 200
1975.08 988.83 1976.09 988.55 659.37 192
Ac-ITF$r8EYWVQL$AHFA-NH.sub.2 201 1957.05 979.35 1958.06 979.53
653.36 193 Ac-ITF$r8EHWVQL$AHFA-NH.sub.2 202 1931.05 967 1932.06
966.53 644.69 194 Ac-ITF$r8EHWVQL$AHFA-NH.sub.2 203 iso2 1931.05
967.93 1932.06 966.53 644.69 195 Ac-ETF$r8EYWAAL$SAA-NH.sub.2 204
1690.86 845.85 1691.87 846.44 564.63 196
Ac-LTF$r8AYWVAL$SAA-NH.sub.2 205 1644.93 824.08 1645.94 823.47
549.32 197 Ac-LTF$r8AHWAAL$SAA-NH.sub.2 206 1590.89 796.88 1591.9
796.45 531.3 198 Ac-LTF$r8AEWAAL$SAA-NH.sub.2 207 1582.88 791.9
1583.89 792.45 528.63 199 Ac-LTF$r8AEWAAL$SAA-NH.sub.2 208 iso2
1582.88 791.9 1583.89 792.45 528.63 200
Ac-LTF$r8ASWAAL$SAA-NH.sub.2 209 1540.87 770.74 1541.88 771.44
514.63 201 Ac-LTF$r8ASWAAL$SAA-NH.sub.2 210 iso2 1540.87 770.88
1541.88 771.44 514.63 202 Ac-LTF$r8AYWAAL$AAIa-NH.sub.2 211 1713.99
857.39 1715 858 572.34 203 Ac-LTF$r8AYWAAL$AAIa-NH.sub.2 212 iso2
1713.99 857.84 1715 858 572.34 204 Ac-LTF$r8AYWAAL$AHFA-NH.sub.2
213 1813.99 907.86 1815 908 605.67 205
Ac-LTF$r8EHWAQL$AHIa-NH.sub.2 214 1869.03 936.1 1870.04 935.52
624.02 206 Ac-LTF$r8EHWAQL$AHIa-NH.sub.2 215 iso2 1869.03 937.03
1870.04 935.52 624.02 207 Ac-LTF$r8AHWAQL$AHIa-NH.sub.2 216 1811.03
906.87 1812.04 906.52 604.68 208 Ac-LTF$r8EYWAQL$AHIa-NH.sub.2 217
1895.04 949.15 1896.05 948.53 632.69 209
Ac-LTF$r8AYWAQL$AAFa-NH.sub.2 218 1804.99 903.2 1806 903.5 602.67
210 Ac-LTF$r8AYWAQL$AAFa-NH.sub.2 219 iso2 1804.99 903.28 1806
903.5 602.67 211 Ac-LTF$r8AYWAQL$AAWa-NH.sub.2 220 1844 922.81
1845.01 923.01 615.67 212 Ac-LTF$r8AYWAQL$AAVa-NH.sub.2 221 1756.99
878.86 1758 879.5 586.67 213 Ac-LTF$r8AYWAQL$AAVa-NH.sub.2 222 iso2
1756.99 879.3 1758 879.5 586.67 214 Ac-LTF$r8AYWAQL$AALa-NH.sub.2
223 1771.01 886.26 1772.02 886.51 591.34 215
Ac-LTF$r8AYWAQL$AALa-NH.sub.2 224 iso2 1771.01 886.33 1772.02
886.51 591.34 216 Ac-LTF$r8EYWAQL$AAIa-NH.sub.2 225 1829.01 914.89
1830.02 915.51 610.68 217 Ac-LTF$r8EYWAQL$AAIa-NH.sub.2 226 iso2
1829.01 915.34 1830.02 915.51 610.68 218
Ac-LTF$r8EYWAQL$AAFa-NH.sub.2 227 1863 932.87 1864.01 932.51 622.01
219 Ac-LTF$r8EYWAQL$AAFa-NH.sub.2 228 iso2 1863 932.87 1864.01
932.51 622.01 220 Ac-LTF$r8EYWAQL$AAVa-NH.sub.2 229 1815 908.23
1816.01 908.51 606.01 221 Ac-LTF$r8EYWAQL$AAVa-NH.sub.2 230 iso2
1815 908.31 1816.01 908.51 606.01 222 Ac-LTF$r8EHWAQL$AAIa-NH.sub.2
231 1803.01 903.17 1804.02 902.51 602.01 223
Ac-LTF$r8EHWAQL$AAIa-NH.sub.2 232 iso2 1803.01 902.8 1804.02 902.51
602.01 224 Ac-LTF$r8EHWAQL$AAWa-NH.sub.2 233 1876 939.34 1877.01
939.01 626.34 225 Ac-LTF$r8EHWAQL$AAWa-NH.sub.2 234 iso2 1876
939.62 1877.01 939.01 626.34 226 Ac-LTF$r8EHWAQL$AALa-NH.sub.2 235
1803.01 902.8 1804.02 902.51 602.01 227
Ac-LTF$r8EHWAQL$AALa-NH.sub.2 236 iso2 1803.01 902.9 1804.02 902.51
602.01 228 Ac-ETF$r8EHWVQL$AALa-NH.sub.2 237 1847 924.82 1848.01
924.51 616.67 229 Ac-LTF$r8AYWAQL$AAAa-NH.sub.2 238 1728.96 865.89
1729.97 865.49 577.33 230 Ac-LTF$r8AYWAQL$AAAa-NH.sub.2 239 iso2
1728.96 865.89 1729.97 865.49 577.33 231
Ac-LTF$r8AYWAQL$AAAibA-NH.sub.2 240 1742.98 872.83 1743.99 872.5
582 232 Ac-LTF$r8AYWAQL$AAAibA-NH.sub.2 241 iso2 1742.98 872.92
1743.99 872.5 582 233 Ac-LTF$r8AYWAQL$AAAAa-NH.sub.2 242 1800
901.42 1801.01 901.01 601.01 234 Ac-LTF$r5AYWAQL$s8AAIa-NH.sub.2
243 1771.01 887.17 1772.02 886.51 591.34 235
Ac-LTF$r5AYWAQL$s8SAA-NH.sub.2 244 1673.92 838.33 1674.93 837.97
558.98 236 Ac-LTF$r8AYWAQCba$AANleA-NH.sub.2 245 1783.01 892.64
1784.02 892.51 595.34 237 Ac-ETF$r8AYWAQCba$AANleA-NH.sub.2 246
1798.97 900.59 1799.98 900.49 600.66 238
Ac-LTF$r8EYWAQCba$AANleA-NH.sub.2 247 1841.01 922.05 1842.02 921.51
614.68 239 Ac-LTF$r8AYWAQCba$AWNleA-NH.sub.2 248 1898.05 950.46
1899.06 950.03 633.69 240 Ac-ETF$r8AYWAQCba$AWNleA-NH.sub.2 249
1914.01 958.11 1915.02 958.01 639.01 241
Ac-LTF$r8EYWAQCba$AWNleA-NH.sub.2 250 1956.06 950.62 1957.07 979.04
653.03 242 Ac-LTF$r8EYWAQCba$SAFA-NH.sub.2 251 1890.99 946.55 1892
946.5 631.34 243 Ac-LTF34F2$r8EYWAQCba$SANleA-NH.sub.2 252 1892.99
947.57 1894 947.5 632 244 Ac-LTF$r8EF4coohWAQCba$SANleA-NH.sub.2
253 1885 943.59 1886.01 943.51 629.34 245
Ac-LTF$r8EYWSQCba$SANleA-NH.sub.2 254 1873 937.58 1874.01 937.51
625.34 246 Ac-LTF$r8EYWWQCba$SANleA-NH.sub.2 255 1972.05 987.61
1973.06 987.03 658.36 247 Ac-LTF$r8EYWAQCba$AAIa-NH.sub.2 256
1841.01 922.05 1842.02 921.51 614.68 248
Ac-LTF34F2$r8EYWAQCba$AAIa-NH.sub.2 257 1876.99 939.99 1878 939.5
626.67 249 Ac-LTF$r8EF4coohWAQCba$AAIa-NH.sub.2 258 1869.01 935.64
1870.02 935.51 624.01 250 Pam-ETF$r8EYWAQCba$SAA-NH.sub.2 259
1956.1 979.57 1957.11 979.06 653.04 251
Ac-LThF$r8EFWAQCba$SAA-NH.sub.2 260 1741.94 872.11 1742.95 871.98
581.65 252 Ac-LTA$r8EYWAQCba$SAA-NH.sub.2 261 1667.89 835.4 1668.9
834.95 556.97 253 Ac-LTF$r8EYAAQCba$SAA-NH.sub.2 262 1628.88 815.61
1629.89 815.45 543.97 254 Ac-LTF$r8EY2NalAQCba$SAA-NH.sub.2 263
1754.93 879.04 1755.94 878.47 585.98 255
Ac-LTF$r8AYWAQCba$SAA-NH.sub.2 264 1685.92 844.71 1686.93 843.97
562.98 256 Ac-LTF$r8EYWAQCba$SAF-NH.sub.2 265 1819.96 911.41
1820.97 910.99 607.66 257 Ac-LTF$r8EYWAQCba$SAFa-NH.sub.2 266
1890.99 947.41 1892 946.5 631.34 258 Ac-LTF$r8AYWAQCba$SAF-NH.sub.2
267 1761.95 882.73 1762.96 881.98 588.32 259
Ac-LTF34F2$r8AYWAQCba$SAF-NH.sub.2 268 1797.93 900.87 1798.94
899.97 600.32 260 Ac-LTF$r8AF4coohWAQCba$SAF-NH.sub.2 269 1789.94
896.43 1790.95 895.98 597.65 261 Ac-LTF$r8EY6clWAQCba$SAF-NH.sub.2
270 1853.92 929.27 1854.93 927.97 618.98 262
Ac-LTF$r8AYWSQCba$SAF-NH.sub.2 271 1777.94 890.87 1778.95 889.98
593.65 263 Ac-LTF$r8AYWWQCba$SAF-NH.sub.2 272 1876.99 939.91 1878
939.5 626.67 264 Ac-LTF$r8AYWAQCba$AAIa-NH.sub.2 273 1783.01 893.19
1784.02 892.51 595.34 265 Ac-LTF34F2$r8AYWAQCba$AAIa-NH.sub.2 274
1818.99 911.23 1820 910.5 607.34 266
Ac-LTF$r8AY6clWAQCba$AAIa-NH.sub.2 275 1816.97 909.84 1817.98
909.49 606.66 267 Ac-LTF$r8AF4coohWAQCba$AAIa-NH.sub.2 276 1811
906.88 1812.01 906.51 604.67 268 Ac-LTF$r8EYWAQCba$AAFa-NH.sub.2
277 1875 938.6 1876.01 938.51 626.01 269
Ac-LTF$r8EYWAQCba$AAFa-NH.sub.2 278 iso2 1875 938.6 1876.01 938.51
626.01 270 Ac-ETF$r8AYWAQCba$AWNlea-NH.sub.2 279 1914.01 958.42
1915.02 958.01 639.01 271 Ac-LTF$r8EYWAQCba$AWNlea-NH.sub.2 280
1956.06 979.42 1957.07 979.04 653.03 272
Ac-ETF$r8EYWAQCba$AWNlea-NH.sub.2 281 1972.01 987.06 1973.02 987.01
658.34 273 Ac-ETF$r8EYWAQCba$AWNlea-NH.sub.2 282 iso2 1972.01
987.06 1973.02 987.01 658.34 274 Ac-LTF$r8AYWAQCba$SAFa-NH.sub.2
283 1832.99 917.89 1834 917.5 612 275
Ac-LTF$r8AYWAQCba$SAFa-NH.sub.2 284 iso2 1832.99 918.07 1834 917.5
612 276 Ac-ETF$r8AYWAQL$AWNlea-NH.sub.2 285 1902.01 952.22 1903.02
952.01 635.01 277 Ac-LTF$r8EYWAQL$AWNlea-NH.sub.2 286 1944.06 973.5
1945.07 973.04 649.03 278 Ac-ETF$r8EYWAQL$AWNlea-NH.sub.2 287
1960.01 981.46 1961.02 981.01 654.34 279
Dmaac-LTF$r8EYWAQhL$SAA-NH.sub.2 288 1788.98 896.06 1789.99 895.5
597.33 280 Hexac-LTF$r8EYWAQhL$SAA-NH.sub.2 289 1802 902.9 1803.01
902.01 601.67 281 Napac-LTF$r8EYWAQhL$SAA-NH.sub.2 290 1871.99
937.58 1873 937 625 282 Decac-LTF$r8EYWAQhL$SAA-NH.sub.2 291
1858.06 930.55 1859.07 930.04 620.36 283
Admac-LTF$r8EYWAQhL$SAA-NH.sub.2 292 1866.03 934.07 1867.04 934.02
623.02 284 Tmac-LTF$r8EYWAQhL$SAA-NH.sub.2 293 1787.99 895.41 1789
895 597 285 Pam-LTF$r8EYWAQhL$SAA-NH.sub.2 294 1942.16 972.08
1943.17 972.09 648.39 286 Ac-LTF$r8AYWAQCba$AANleA-NH.sub.2 295
iso2 1783.01 892.64 1784.02 892.51 595.34 287
Ac-LTF34F2$r8EYWAQCba$AAIa-NH.sub.2 296 iso2 1876.99 939.62 1878
939.5 626.67 288 Ac-LTF34F2$r8EYWAQCba$SAA-NH.sub.2 297 1779.91
892.07 1780.92 890.96 594.31 289 Ac-LTF34F2$r8EYWAQCba$SAA-NH.sub.2
298 iso2 1779.91 891.61 1780.92 890.96 594.31 290
Ac-LTF$r8EF4coohWAQCba$SAA-NH.sub.2 299 1771.92 887.54 1772.93
886.97 591.65 291 Ac-LTF$r8EF4coohWAQCba$SAA-NH.sub.2 300 iso2
1771.92 887.63 1772.93 886.97 591.65 292
Ac-LTF$r8EYWSQCba$SAA-NH.sub.2 301 1759.92 881.9 1760.93 880.97
587.65 293 Ac-LTF$r8EYWSQCba$SAA-NH.sub.2 302 iso2 1759.92 881.9
1760.93 880.97 587.65 294 Ac-LTF$r8EYWAQhL$SAA-NH.sub.2 303 1745.94
875.05 1746.95 873.98 582.99 295 Ac-LTF$r8AYWAQhL$SAF-NH.sub.2 304
1763.97 884.02 1764.98 882.99 589 296 Ac-LTF$r8AYWAQhL$SAF-NH.sub.2
305 iso2 1763.97 883.56 1764.98 882.99 589 297
Ac-LTF34F2$r8AYWAQhL$SAA-NH.sub.2 306 1723.92 863.67 1724.93 862.97
575.65 298 Ac-LTF34F2$r8AYWAQhL$SAA-NH.sub.2 307 iso2 1723.92
864.04 1724.93
862.97 575.65 299 Ac-LTF$r8AF4coohWAQhL$SAA-NH.sub.2 308 1715.93
859.44 1716.94 858.97 572.98 300 Ac-LTF$r8AF4coohWAQhL$SAA-NH.sub.2
309 iso2 1715.93 859.6 1716.94 858.97 572.98 301
Ac-LTF$r8AYWSQhL$SAA-NH.sub.2 310 1703.93 853.96 1704.94 852.97
568.98 302 Ac-LTF$r8AYWSQhL$SAA-NH.sub.2 311 iso2 1703.93 853.59
1704.94 852.97 568.98 303 Ac-LTF$r8EYWAQL$AANleA-NH.sub.2 312
1829.01 915.45 1830.02 915.51 610.68 304
Ac-LTF34F2$r8AYWAQL$AANleA-NH.sub.2 313 1806.99 904.58 1808 904.5
603.34 305 Ac-LTF$r8AF4coohWAQL$AANleA-NH.sub.2 314 1799 901.6
1800.01 900.51 600.67 306 Ac-LTF$r8AYWSQL$AANleA-NH.sub.2 315 1787
894.75 1788.01 894.51 596.67 307
Ac-LTF34F2$r8AYWAQhL$AANleA-NH.sub.2 316 1821 911.79 1822.01 911.51
608.01 308 Ac-LTF34F2$r8AYWAQhL$AANleA-NH.sub.2 317 iso2 1821
912.61 1822.01 911.51 608.01 309
Ac-LTF$r8AF4coohWAQhL$AANleA-NH.sub.2 318 1813.02 907.95 1814.03
907.52 605.35 310 Ac-LTF$r8AF4coohWAQhL$AANleA-NH.sub.2 319 iso2
1813.02 908.54 1814.03 907.52 605.35 311
Ac-LTF$r8AYWSQhL$AANleA-NH.sub.2 320 1801.02 901.84 1802.03 901.52
601.35 312 Ac-LTF$r8AYWSQhL$AANleA-NH.sub.2 321 iso2 1801.02 902.62
1802.03 901.52 601.35 313 Ac-LTF$r8AYWAQhL$AAAAa-NH.sub.2 322
1814.01 908.63 1815.02 908.01 605.68 314
Ac-LTF$r8AYWAQhL$AAAAa-NH.sub.2 323 iso2 1814.01 908.34 1815.02
908.01 605.68 315 Ac-LTF$r8AYWAQL$AAAAAa-NH.sub.2 324 1871.04
936.94 1872.05 936.53 624.69 316 Ac-LTF$r8AYWAQL$AAAAAAa-NH.sub.2
325 iso2 1942.07 972.5 1943.08 972.04 648.37 317
Ac-LTF$r8AYWAQL$AAAAAAa-NH.sub.2 326 isol 1942.07 972.5 1943.08
972.04 648.37 318 Ac-LTF$r8EYWAQhL$AANleA-NH.sub.2 327 1843.03
922.54 1844.04 922.52 615.35 319 Ac-AATF$r8AYWAQL$AANleA-NH.sub.2
328 1800 901.39 1801.01 901.01 601.01 320
Ac-LTF$r8AYWAQL$AANleAA-NH.sub.2 329 1842.04 922.45 1843.05 922.03
615.02 321 Ac-ALTF$r8AYWAQL$AANleAA-NH.sub.2 330 1913.08 957.94
1914.09 957.55 638.7 322 Ac-LTF$r8AYWAQCba$AANleAA-NH.sub.2 331
1854.04 928.43 1855.05 928.03 619.02 323
Ac-LTF$r8AYWAQhL$AANleAA-NH.sub.2 332 1856.06 929.4 1857.07 929.04
619.69 324 Ac-LTF$r8EYWAQCba$SAAA-NH.sub.2 333 1814.96 909.37
1815.97 908.49 605.99 325 Ac-LTF$r8EYWAQCba$SAAA-NH.sub.2 334 iso2
1814.96 909.37 1815.97 908.49 605.99 326
Ac-LTF$r8EYWAQCba$SAAAA-NH.sub.2 335 1886 944.61 1887.01 944.01
629.67 327 Ac-LTF$r8EYWAQCba$SAAAA-NH.sub.2 336 iso2 1886 944.61
1887.01 944.01 629.67 328 Ac-ALTF$r8EYWAQCba$SAA-NH.sub.2 337
1814.96 909.09 1815.97 908.49 605.99 329
Ac-ALTF$r8EYWAQCba$SAAA-NH.sub.2 338 1886 944.61 1887.01 944.01
629.67 330 Ac-ALTF$r8EYWAQCba$SAA-NH.sub.2 339 iso2 1814.96 909.09
1815.97 908.49 605.99 331 Ac-LTF$r8EYWAQL$AAAAAa-NH.sub.2 340 iso2
1929.04 966.08 1930.05 965.53 644.02 332
Ac-LTF$r8EY6clWAQCba$SAA-NH.sub.2 341 1777.89 890.78 1778.9 889.95
593.64 333 Ac-LTF$r8EF4cooh6clWAQCba$SANleA-NH.sub.2 342 1918.96
961.27 1919.97 960.49 640.66 334
Ac-LTF$r8EF4cooh6clWAQCba$SANleA-NH.sub.2 343 iso2 1918.96 961.27
1919.97 960.49 640.66 335 Ac-LTF$r8EF4cooh6clWAQCba$AAIa-NH.sub.2
344 1902.97 953.03 1903.98 952.49 635.33 336
Ac-LTF$r8EF4cooh6clWAQCba$AAIa-NH.sub.2 345 iso2 1902.97 953.13
1903.98 952.49 635.33 337 Ac-LTF$r8AY6clWAQL$AAAAAa-NH.sub.2 346
1905 954.61 1906.01 953.51 636.01 338
Ac-LTF$r8AY6clWAQL$AAAAAa-NH.sub.2 347 iso2 1905 954.9 1906.01
953.51 636.01 339 Ac-F$r8AY6clWEAL$AAAAAAa-NH.sub.2 348 1762.89
883.01 1763.9 882.45 588.64 340 Ac-ETF$r8EYWAQL$AAAAAa-NH.sub.2 349
1945 974.31 1946.01 973.51 649.34 341
Ac-ETF$r8EYWAQL$AAAAAa-NH.sub.2 350 iso2 1945 974.49 1946.01 973.51
649.34 342 Ac-LTF$r8EYWAQL$AAAAAAa-NH.sub.2 351 2000.08 1001.6
2001.09 1001.05 667.7 343 Ac-LTF$r8EYWAQL$AAAAAAa-NH.sub.2 352 iso2
2000.08 1001.6 2001.09 1001.05 667.7 344
Ac-LTF$r8AYWAQL$AANleAAa-NH.sub.2 353 1913.08 958.58 1914.09 957.55
638.7 345 Ac-LTF$r8AYWAQL$AANleAAa-NH.sub.2 354 iso2 1913.08 958.58
1914.09 957.55 638.7 346 Ac-LTF$r8EYWAQCba$AAAAAa-NH.sub.2 355
1941.04 972.55 1942.05 971.53 648.02 347
Ac-LTF$r8EYWAQCba$AAAAAa-NH.sub.2 356 iso2 1941.04 972.55 1942.05
971.53 648.02 348 Ac-LTF$r8EF4coohWAQCba$AAAAAa-NH.sub.2 357
1969.04 986.33 1970.05 985.53 657.35 349
Ac-LTF$r8EF4coohWAQCba$AAAAAa-NH.sub.2 358 iso2 1969.04 986.06
1970.05 985.53 657.35 350 Ac-LTF$r8EYWSQCba$AAAAAa-NH.sub.2 359
1957.04 980.04 1958.05 979.53 653.35 351
Ac-LTF$r8EYWSQCba$AAAAAa-NH.sub.2 360 iso2 1957.04 980.04 1958.05
979.53 653.35 352 Ac-LTF$r8EYWAQCba$SAAa-NH.sub.2 361 1814.96 909
1815.97 908.49 605.99 353 Ac-LTF$r8EYWAQCba$SAAa-NH.sub.2 362 iso2
1814.96 909 1815.97 908.49 605.99 354
Ac-ALTF$r8EYWAQCba$SAAa-NH.sub.2 363 1886 944.52 1887.01 944.01
629.67 355 Ac-ALTF$r8EYWAQCba$SAAa-NH.sub.2 364 iso2 1886 944.98
1887.01 944.01 629.67 356 Ac-ALTF$r8EYWAQCba$SAAAa-NH.sub.2 365
1957.04 980.04 1958.05 979.53 653.35 357
Ac-ALTF$r8EYWAQCba$SAAAa-NH.sub.2 366 iso2 1957.04 980.04 1958.05
979.53 653.35 358 Ac-AALTF$r8EYWAQCba$SAAAa-NH.sub.2 367 2028.07
1016.1 2029.08 1015.04 677.03 359
Ac-AALTF$r8EYWAQCba$SAAAa-NH.sub.2 368 iso2 2028.07 1015.57 2029.08
1015.04 677.03 360 Ac-RTF$r8EYWAQCba$SAA-NH.sub.2 369 1786.94
895.03 1787.95 894.48 596.65 361 Ac-LRF$r8EYWAQCba$SAA-NH.sub.2 370
1798.98 901.51 1799.99 900.5 600.67 362
Ac-LTF$r8EYWRQCba$SAA-NH.sub.2 371 1828.99 916.4 1830 915.5 610.67
363 Ac-LTF$r8EYWARCba$SAA-NH.sub.2 372 1771.97 887.63 1772.98
886.99 591.66 364 Ac-LTF$r8EYWAQCba$RAA-NH.sub.2 373 1812.99 908.08
1814 907.5 605.34 365 Ac-LTF$r8EYWAQCba$SRA-NH.sub.2 374 1828.99
916.12 1830 915.5 610.67 366 Ac-LTF$r8EYWAQCba$SAR-NH.sub.2 375
1828.99 916.12 1830 915.5 610.67 367
5-FAM-BaLTF$r8EYWAQCba$SAA-NH.sub.2 376 2131 1067.09 2132.01
1066.51 711.34 368 5-FAM-BaLTF$r8AYWAQL$AANleA-NH.sub.2 377 2158.08
1080.6 2159.09 1080.05 720.37 369 Ac-LAF$r8EYWAQL$AANleA-NH.sub.2
378 1799 901.05 1800.01 900.51 600.67 370
Ac-ATF$r8EYWAQL$AANleA-NH.sub.2 379 1786.97 895.03 1787.98 894.49
596.66 371 Ac-AAF$r8EYWAQL$AANleA-NH.sub.2 380 1756.96 880.05
1757.97 879.49 586.66 372 Ac-AAAF$r8EYWAQL$AANleA-NH.sub.2 381
1827.99 915.57 1829 915 610.34 373
Ac-AAAAF$r8EYWAQL$AANleA-NH.sub.2 382 1899.03 951.09 1900.04 950.52
634.02 374 Ac-AATF$r8EYWAQL$AANleA-NH.sub.2 383 1858 930.92 1859.01
930.01 620.34 375 Ac-AALTF$r8EYWAQL$AANleA-NH.sub.2 384 1971.09
987.17 1972.1 986.55 658.04 376 Ac-AAALTF$r8EYWAQL$AANleA-NH.sub.2
385 2042.12 1023.15 2043.13 1022.07 681.71 377
Ac-LTF$r8EYWAQL$AANleAA-NH.sub.2 386 1900.05 952.02 1901.06 951.03
634.36 378 Ac-ALTF$r8EYWAQL$AANleAA-NH.sub.2 387 1971.09 987.63
1972.1 986.55 658.04 379 Ac-AALTF$r8EYWAQL$AANleAA-NH.sub.2 388
2042.12 1022.69 2043.13 1022.07 681.71 380
Ac-LTF$r8EYWAQCba$AANleAA-NH.sub.2 389 1912.05 958.03 1913.06
957.03 638.36 381 Ac-LTF$r8EYWAQhL$AANleAA-NH.sub.2 390 1914.07
958.68 1915.08 958.04 639.03 382 Ac-ALTF$r8EYWAQhL$AANleAA-NH.sub.2
391 1985.1 994.1 1986.11 993.56 662.71 383
Ac-LTF$r8ANmYWAQL$AANleA-NH.sub.2 392 1785.02 894.11 1786.03 893.52
596.01 384 Ac-LTF$r8ANmYWAQL$AANleA-NH.sub.2 393 iso2 1785.02
894.11 1786.03 893.52 596.01 385 Ac-LTF$r8AYNmWAQL$AANleA-NH.sub.2
394 1785.02 894.11 1786.03 893.52 596.01 386
Ac-LTF$r8AYNmWAQL$AANleA-NH.sub.2 395 iso2 1785.02 894.11 1786.03
893.52 596.01 387 Ac-LTF$r8AYAmwAQL$AANleA-NH.sub.2 396 1785.02
894.01 1786.03 893.52 596.01 388 Ac-LTF$r8AYAmwAQL$AANleA-NH.sub.2
397 iso2 1785.02 894.01 1786.03 893.52 596.01 389
Ac-LTF$r8AYWAibQL$AANleA-NH.sub.2 398 1785.02 894.01 1786.03 893.52
596.01 390 Ac-LTF$r8AYWAibQL$AANleA-NH.sub.2 399 iso2 1785.02
894.01 1786.03 893.52 596.01 391 Ac-LTF$r8AYWAQL$AAibNleA-NH.sub.2
400 1785.02 894.38 1786.03 893.52 596.01 392
Ac-LTF$r8AYWAQL$AAibNleA-NH.sub.2 401 iso2 1785.02 894.38 1786.03
893.52 596.01 393 Ac-LTF$r8AYWAQL$AaNleA-NH.sub.2 402 1771.01
887.54 1772.02 886.51 591.34 394 Ac-LTF$r8AYWAQL$AaNleA-NH.sub.2
403 iso2 1771.01 887.54 1772.02 886.51 591.34 395
Ac-LTF$r8AYWAQL$ASarNleA-NH.sub.2 404 1771.01 887.35 1772.02 886.51
591.34 396 Ac-LTF$r8AYWAQL$ASarNleA-NH.sub.2 405 iso2 1771.01
887.35 1772.02 886.51 591.34 397 Ac-LTF$r8AYWAQL$AANleAib-NH.sub.2
406 1785.02 894.75 1786.03 893.52 596.01 398
Ac-LTF$r8AYWAQL$AANleAib-NH.sub.2 407 iso2 1785.02 894.75 1786.03
893.52 596.01 399 Ac-LTF$r8AYWAQL$AANleNmA-NH.sub.2 408 1785.02
894.6 1786.03 893.52 596.01 400 Ac-LTF$r8AYWAQL$AANleNmA-NH.sub.2
409 iso2 1785.02 894.6 1786.03 893.52 596.01 401
Ac-LTF$r8AYWAQL$AANleSar-NH.sub.2 410 1771.01 886.98 1772.02 886.51
591.34 402 Ac-LTF$r8AYWAQL$AANleSar-NH.sub.2 411 iso2 1771.01
886.98 1772.02 886.51 591.34 403 Ac-LTF$r8AYWAQL$AANleAAib-NH.sub.2
412 1856.06 1857.07 929.04 619.69 404
Ac-LTF$r8AYWAQL$AANleAAib-NH.sub.2 413 iso2 1856.06 1857.07 929.04
619.69 405 Ac-LTF$r8AYWAQL$AANleANmA-NH.sub.2 414 1856.06 930.37
1857.07 929.04 619.69 406 Ac-LTF$r8AYWAQL$AANleANmA-NH.sub.2 415
iso2 1856.06 930.37 1857.07 929.04 619.69 407
Ac-LTF$r8AYWAQL$AANleAa-NH.sub.2 416 1842.04 922.69 1843.05 922.03
615.02 408 Ac-LTF$r8AYWAQL$AANleAa-NH.sub.2 417 iso2 1842.04 922.69
1843.05 922.03 615.02 409 Ac-LTF$r8AYWAQL$AANleASar-NH.sub.2 418
1842.04 922.6 1843.05 922.03 615.02 410
Ac-LTF$r8AYWAQL$AANleASar-NH.sub.2 419 iso2 1842.04 922.6 1843.05
922.03 615.02 411 Ac-LTF$/r8AYWAQL$/AANleA-NH.sub.2 420 1799.04
901.14 1800.05 900.53 600.69 412 Ac-LTFAibAYWAQLAibAANleA-NH.sub.2
421 1648.9 826.02 1649.91 825.46 550.64 413
Ac-LTF$r8Cou4YWAQL$AANleA-NH.sub.2 422 1975.05 989.11 1976.06
988.53 659.36 414 Ac-LTF$r8Cou4YWAQL$AANleA-NH.sub.2 423 iso2
1975.05 989.11 1976.06 988.53 659.36 415
Ac-LTF$r8AYWCou4QL$AANleA-NH.sub.2 424 1975.05 989.11 1976.06
988.53 659.36 416 Ac-LTF$r8AYWAQL$Cou4ANleA-NH.sub.2 425 1975.05
989.57 1976.06 988.53 659.36 417 Ac-LTF$r8AYWAQL$Cou4ANleA-NH.sub.2
426 iso2 1975.05 989.57 1976.06 988.53 659.36 418
Ac-LTF$r8AYWAQL$ACou4NleA-NH.sub.2 427 1975.05 989.57 1976.06
988.53 659.36 419 Ac-LTF$r8AYWAQL$ACou4NleA-NH.sub.2 428 iso2
1975.05 989.57 1976.06 988.53 659.36 420 Ac-LTF$r8AYWAQL$AANleA-OH
429 1771.99 887.63 1773 887 591.67 421 Ac-LTF$r8AYWAQL$AANleA-OH
430 iso2 1771.99 887.63 1773 887 591.67 422
Ac-LTF$r8AYWAQL$AANleA-NHnPr 431 1813.05 908.08 1814.06 907.53
605.36 423 Ac-LTF$r8AYWAQL$AANleA-NHnPr 432 iso2 1813.05 908.08
1814.06 907.53 605.36 424 Ac-LTF$r8AYWAQL$AANleA-NHnBu33Me 433
1855.1 929.17 1856.11 928.56 619.37 425
Ac-LTF$r8AYWAQL$AANleA-NHnBu33Me 434 iso2 1855.1 929.17 1856.11
928.56 619.37 426 Ac-LTF$r8AYWAQL$AANleA-NHHex 435 1855.1 929.17
1856.11 928.56 619.37 427 Ac-LTF$r8AYWAQL$AANleA-NHHex 436 iso2
1855.1 929.17 1856.11 928.56 619.37
428 Ac-LTA$r8AYWAQL$AANleA-NH.sub.2 437 1694.98 849.33 1695.99
848.5 566 429 Ac-LThL$r8AYWAQL$AANleA-NH.sub.2 438 1751.04 877.09
1752.05 876.53 584.69 430 Ac-LTF$r8AYAAQL$AANleA-NH.sub.2 439
1655.97 829.54 1656.98 828.99 553 431
Ac-LTF$r8AY2NalAQL$AANleA-NH.sub.2 440 1782.01 892.63 1783.02
892.01 595.01 432 Ac-LTF$r8EYWCou4QCba$SAA-NH.sub.2 441 1947.97
975.8 1948.98 974.99 650.33 433 Ac-LTF$r8EYWCou7QCba$SAA-NH.sub.2
442 16.03 974.9 17.04 9.02 6.35 434 Ac-LTF%r8EYWAQCba%SAA-NH.sub.2
443 1745.94 874.8 1746.95 873.98 582.99 435
Dmaac-LTF$r8EYWAQCba$SAA-NH.sub.2 444 1786.97 894.8 1787.98 894.49
596.66 436 Dmaac-LTF$r8AYWAQL$AAAAAa-NH.sub.2 445 1914.08 958.2
1915.09 958.05 639.03 437 Dmaac-LTF$r8AYWAQL$AAAAAa-NH.sub.2 446
iso2 1914.08 958.2 1915.09 958.05 639.03 438
Dmaac-LTF$r8EYWAQL$AAAAAa-NH.sub.2 447 1972.08 987.3 1973.09 987.05
658.37 439 Dmaac-LTF$r8EYWAQL$AAAAAa-NH.sub.2 448 iso2 1972.08
987.3 1973.09 987.05 658.37 440
Dmaac-LTF$r8EF4coohWAQCba$AAIa-NH.sub.2 449 1912.05 957.4 1913.06
957.03 638.36 441 Dmaac-LTF$r8EF4coohWAQCba$AAIa-NH.sub.2 450 iso2
1912.05 957.4 1913.06 957.03 638.36 442
Dmaac-LTF$r8AYWAQL$AANleA-NH.sub.2 451 1814.05 908.3 1815.06 908.03
605.69 443 Dmaac-LTF$r8AYWAQL$AANleA-NH.sub.2 452 iso2 1814.05
908.3 1815.06 908.03 605.69 444 Ac-LTF%r8AYWAQL%AANleA-NH.sub.2 453
1773.02 888.37 1774.03 887.52 592.01 445
Ac-LTF%r8EYWAQL%AAAAAa-NH.sub.2 454 1931.06 966.4 1932.07 966.54
644.69 446 Cou6BaLTF$r8EYWAQhL$SAA-NH.sub.2 455 2018.05 1009.9
2019.06 1010.03 673.69 447 Cou8BaLTF$r8EYWAQhL$SAA-NH.sub.2 456
1962.96 982.34 1963.97 982.49 655.32 448
Ac-LTF4I$r8EYWAQL$AAAAAa-NH.sub.2 457 2054.93 1028.68 2055.94
1028.47 685.98 449 Ac-LTF$r8EYWAQL$AAAAAa-NH.sub.2 458 1929.04
966.17 1930.05 965.53 644.02 550 Ac-LTF$r8EYWAQL$AAAAAa-OH 459
1930.02 966.54 1931.03 966.02 644.35 551 Ac-LTF$r8EYWAQL$AAAAAa-OH
460 iso2 1930.02 965.89 1931.03 966.02 644.35 552
Ac-LTF$r8EYWAEL$AAAAAa-NH.sub.2 461 1930.02 966.82 1931.03 966.02
644.35 553 Ac-LTF$r8EYWAEL$AAAAAa-NH.sub.2 462 iso2 1930.02 966.91
1931.03 966.02 644.35 554 Ac-LTF$r8EYWAEL$AAAAAa-OH 463 1931.01
967.28 1932.02 966.51 644.68 555 Ac-LTF$r8EY6clWAQL$AAAAAa-NH.sub.2
464 1963 983.28 1964.01 982.51 655.34 556
Ac-LTF$r8EF4bOH2WAQL$AAAAAa-NH.sub.2 465 1957.05 980.04 1958.06
979.53 653.36 557 Ac-AAALTF$r8EYWAQL$AAAAAa-NH.sub.2 466 2142.15
1072.83 2143.16 1072.08 715.06 558
Ac-LTF34F2$r8EYWAQL$AAAAAa-NH.sub.2 467 1965.02 984.3 1966.03
983.52 656.01 559 Ac-RTF$r8EYWAQL$AAAAAa-NH.sub.2 468 1972.06
987.81 1973.07 987.04 658.36 560 Ac-LTA$r8EYWAQL$AAAAAa-NH.sub.2
469 1853.01 928.33 1854.02 927.51 618.68 561
Ac-LTF$r8EYWAibQL$AAAAAa-NH.sub.2 470 1943.06 973.48 1944.07 972.54
648.69 562 Ac-LTF$r8EYWAQL$AAibAAAa-NH.sub.2 471 1943.06 973.11
1944.07 972.54 648.69 563 Ac-LTF$r8EYWAQL$AAAibAAa-NH.sub.2 472
1943.06 973.48 1944.07 972.54 648.69 564
Ac-LTF$r8EYWAQL$AAAAibAa-NH.sub.2 473 1943.06 973.48 1944.07 972.54
648.69 565 Ac-LTF$r8EYWAQL$AAAAAiba-NH.sub.2 474 1943.06 973.38
1944.07 972.54 648.69 566 Ac-LTF$r8EYWAQL$AAAAAiba-NH.sub.2 475
iso2 1943.06 973.38 1944.07 972.54 648.69 567
Ac-LTF$r8EYWAQL$AAAAAAib-NH.sub.2 476 1943.06 973.01 1944.07 972.54
648.69 568 Ac-LTF$r8EYWAQL$AaAAAa-NH.sub.2 477 1929.04 966.54
1930.05 965.53 644.02 569 Ac-LTF$r8EYWAQL$AAaAAa-NH.sub.2 478
1929.04 966.35 1930.05 965.53 644.02 570
Ac-LTF$r8EYWAQL$AAAaAa-NH.sub.2 479 1929.04 966.54 1930.05 965.53
644.02 571 Ac-LTF$r8EYWAQL$AAAaAa-NH.sub.2 480 iso2 1929.04 966.35
1930.05 965.53 644.02 572 Ac-LTF$r8EYWAQL$AAAAaa-NH.sub.2 481
1929.04 966.35 1930.05 965.53 644.02 573
Ac-LTF$r8EYWAQL$AAAAAA-NH.sub.2 482 1929.04 966.35 1930.05 965.53
644.02 574 Ac-LTF$r8EYWAQL$ASarAAAa-NH.sub.2 483 1929.04 966.54
1930.05 965.53 644.02 575 Ac-LTF$r8EYWAQL$AASarAAa-NH.sub.2 484
1929.04 966.35 1930.05 965.53 644.02 576
Ac-LTF$r8EYWAQL$AAASarAa-NH.sub.2 485 1929.04 966.35 1930.05 965.53
644.02 577 Ac-LTF$r8EYWAQL$AAAASara-NH.sub.2 486 1929.04 966.35
1930.05 965.53 644.02 578 Ac-LTF$r8EYWAQL$AAAAASar-NH.sub.2 487
1929.04 966.08 1930.05 965.53 644.02 579
Ac-7LTF$r8EYWAQL$AAAAAa-NH.sub.2 488 1918.07 951.99 1919.08 960.04
640.37 581 Ac-TF$r8EYWAQL$AAAAAa-NH.sub.2 489 1815.96 929.85
1816.97 908.99 606.33 582 Ac-F$r8EYWAQL$AAAAAa-NH.sub.2 490 1714.91
930.92 1715.92 858.46 572.64 583 Ac-LVF$r8EYWAQL$AAAAAa-NH.sub.2
491 1927.06 895.12 1928.07 964.54 643.36 584
Ac-AAF$r8EYWAQL$AAAAAa-NH.sub.2 492 1856.98 859.51 1857.99 929.5
620 585 Ac-LTF$r8EYWAQL$AAAAa-NH.sub.2 493 1858 824.08 1859.01
930.01 620.34 586 Ac-LTF$r8EYWAQL$AAAa-NH.sub.2 494 1786.97 788.56
1787.98 894.49 596.66 587 Ac-LTF$r8EYWAQL$AAa-NH.sub.2 495 1715.93
1138.57 1716.94 858.97 572.98 588 Ac-LTF$r8EYWAQL$Aa-NH.sub.2 496
1644.89 1144.98 1645.9 823.45 549.3 589 Ac-LTF$r8EYWAQL$a-NH.sub.2
497 1573.85 1113.71 1574.86 787.93 525.62 590
Ac-LTF$r8EYWAQL$AAA-OH 498 1716.91 859.55 1717.92 859.46 573.31 591
Ac-LTF$r8EYWAQL$A-OH 499 1574.84 975.14 1575.85 788.43 525.95 592
Ac-LTF$r8EYWAQL$AAA-NH.sub.2 500 1715.93 904.75 1716.94 858.97
572.98 593 Ac-LTF$r8EYWAQCba$SAA-OH 501 1744.91 802.49 1745.92
873.46 582.64 594 Ac-LTF$r8EYWAQCba$S-OH 502 1602.83 913.53 1603.84
802.42 535.28 595 Ac-LTF$r8EYWAQCba$S-NH.sub.2 503 1601.85 979.58
1602.86 801.93 534.96 596 4-FBzl-LTF$r8EYWAQL$AAAAAa-NH.sub.2 504
2009.05 970.52 2010.06 1005.53 670.69 597
4-FBzl-LTF$r8EYWAQCba$SAA-NH.sub.2 505 1823.93 965.8 1824.94 912.97
608.98 598 Ac-LTF$r8RYWAQL$AAAAAa-NH.sub.2 506 1956.1 988.28
1957.11 979.06 653.04 599 Ac-LTF$r8HYWAQL$AAAAAa-NH.sub.2 507
1937.06 1003.54 1938.07 969.54 646.69 600
Ac-LTF$r8QYWAQL$AAAAAa-NH.sub.2 508 1928.06 993.92 1929.07 965.04
643.69 601 Ac-LTF$r8CitYWAQL$AAAAAa-NH.sub.2 509 1957.08 987
1958.09 979.55 653.37 602 Ac-LTF$r8GlaYWAQL$AAAAAa-NH.sub.2 510
1973.03 983 1974.04 987.52 658.68 603
Ac-LTF$r8F4gYWAQL$AAAAAa-NH.sub.2 511 2004.1 937.86 2005.11 1003.06
669.04 604 Ac-LTF$r82mRYWAQL$AAAAAa-NH.sub.2 512 1984.13 958.58
1985.14 993.07 662.38 605 Ac-LTF$r8ipKYWAQL$AAAAAa-NH.sub.2 513
1970.14 944.52 1971.15 986.08 657.72 606
Ac-LTF$r8F4NH.sub.2YWAQL$AAAAAa-NH.sub.2 514 1962.08 946 1963.09
982.05 655.03 607 Ac-LTF$r8EYWAAL$AAAAAa-NH.sub.2 515 1872.02
959.32 1873.03 937.02 625.01 608 Ac-LTF$r8EYWALL$AAAAAa-NH.sub.2
516 1914.07 980.88 1915.08 958.04 639.03 609
Ac-LTF$r8EYWAAibL$AAAAAa-NH.sub.2 517 1886.03 970.61 1887.04 944.02
629.68 610 Ac-LTF$r8EYWASL$AAAAAa-NH.sub.2 518 1888.01 980.51
1889.02 945.01 630.34 611 Ac-LTF$r8EYWANL$AAAAAa-NH.sub.2 519
1915.02 1006.41 1916.03 958.52 639.35 612
Ac-LTF$r8EYWACitL$AAAAAa-NH.sub.2 520 1958.07 1959.08 980.04 653.7
613 Ac-LTF$r8EYWAHL$AAAAAa-NH.sub.2 521 1938.04 966.24 1939.05
970.03 647.02 614 Ac-LTF$r8EYWARL$AAAAAa-NH.sub.2 522 1957.08
1958.09 979.55 653.37 615 Ac-LTF$r8EpYWAQL$AAAAAa-NH.sub.2 523
2009.01 2010.02 1005.51 670.68 616 Cbm-LTF$r8EYWAQCba$SAA-NH.sub.2
524 1590.85 1591.86 796.43 531.29 617
Cbm-LTF$r8EYWAQL$AAAAAa-NH.sub.2 525 1930.04 1931.05 966.03 644.35
618 Ac-LTF$r8EYWAQL$SAAAAa-NH.sub.2 526 1945.04 1005.11 1946.05
973.53 649.35 619 Ac-LTF$r8EYWAQL$AAAASa-NH.sub.2 527 1945.04
986.52 1946.05 973.53 649.35 620 Ac-LTF$r8EYWAQL$SAAASa-NH.sub.2
528 1961.03 993.27 1962.04 981.52 654.68 621
Ac-LTF$r8EYWAQTba$AAAAAa-NH.sub.2 529 1943.06 983.1 1944.07 972.54
648.69 622 Ac-LTF$r8EYWAQAdm$AAAAAa-NH.sub.2 530 2007.09 990.31
2008.1 1004.55 670.04 623 Ac-LTF$r8EYWAQCha$AAAAAa-NH.sub.2 531
1969.07 987.17 1970.08 985.54 657.36 624
Ac-LTF$r8EYWAQhCha$AAAAAa-NH.sub.2 532 1983.09 1026.11 1984.1
992.55 662.04 625 Ac-LTF$r8EYWAQF$AAAAAa-NH.sub.2 533 1963.02
957.01 1964.03 982.52 655.35 626 Ac-LTF$r8EYWAQhF$AAAAAa-NH.sub.2
534 1977.04 1087.81 1978.05 989.53 660.02 627
Ac-LTF$r8EYWAQL$AANleAAa-NH.sub.2 535 1971.09 933.45 1972.1 986.55
658.04 628 Ac-LTF$r8EYWAQAdm$AANleAAa-NH.sub.2 536 2049.13 1017.97
2050.14 1025.57 684.05 629 4-FBz-BaLTF$r8EYWAQL$AAAAAa-NH.sub.2 537
2080.08 2081.09 1041.05 694.37 630
4-FBz-BaLTF$r8EYWAQCba$SAA-NH.sub.2 538 1894.97 1895.98 948.49
632.66 631 Ac-LTF$r5EYWAQL$s8AAAAAa-NH.sub.2 539 1929.04 1072.68
1930.05 965.53 644.02 632 Ac-LTF$r5EYWAQCba$s8SAA-NH.sub.2 540
1743.92 1107.79 1744.93 872.97 582.31 633
Ac-LTF$r8EYWAQL$AAhhLAAa-NH.sub.2 541 1999.12 2000.13 1000.57
667.38 634 Ac-LTF$r8EYWAQL$AAAAAAAa-NH.sub.2 542 2071.11 2072.12
1036.56 691.38 635 Ac-LTF$r8EYWAQL$AAAAAAAAa-NH.sub.2 543 2142.15
778.1 2143.16 1072.08 715.06 636
Ac-LTF$r8EYWAQL$AAAAAAAAAa-NH.sub.2 544 2213.19 870.53 2214.2
1107.6 738.74 637 Ac-LTA$r8EYAAQCba$SAA-NH.sub.2 545 1552.85
1553.86 777.43 518.62 638 Ac-LTA$r8EYAAQL$AAAAAa-NH.sub.2 546
1737.97 779.45 1738.98 869.99 580.33 639
Ac-LTF$r8EPmpWAQL$AAAAAa-NH.sub.2 547 2007.03 779.54 2008.04
1004.52 670.02 640 Ac-LTF$r8EPmpWAQCba$SAA-NH.sub.2 548 1821.91
838.04 1822.92 911.96 608.31 641 Ac-ATF$r8HYWAQL$S-NH.sub.2 549
1555.82 867.83 1556.83 778.92 519.61 642 Ac-LTF$r8HAWAQL$S-NH.sub.2
550 1505.84 877.91 1506.85 753.93 502.95 643
Ac-LTF$r8HYWAQA$S-NH.sub.2 551 1555.82 852.52 1556.83 778.92 519.61
644 Ac-LTF$r8EYWAQCba$SA-NH.sub.2 552 1672.89 887.18 1673.9 837.45
558.64 645 Ac-LTF$r8EYWAQL$SAA-NH.sub.2 553 1731.92 873.32 1732.93
866.97 578.31 646 Ac-LTF$r8HYWAQCba$SAA-NH.sub.2 554 1751.94 873.05
1752.95 876.98 584.99 647 Ac-LTF$r8SYWAQCba$SAA-NH.sub.2 555
1701.91 844.88 1702.92 851.96 568.31 648
Ac-LTF$r8RYWAQCba$SAA-NH.sub.2 556 1770.98 865.58 1771.99 886.5
591.33 649 Ac-LTF$r8KYWAQCba$SAA-NH.sub.2 557 1742.98 936.57
1743.99 872.5 582 650 Ac-LTF$r8QYWAQCba$SAA-NH.sub.2 558 1742.94
930.93 1743.95 872.48 581.99 651 Ac-LTF$r8EYWAACba$SAA-NH.sub.2 559
1686.9 1032.45 1687.91 844.46 563.31 652
Ac-LTF$r8EYWAQCba$AAA-NH.sub.2 560 1727.93 895.46 1728.94 864.97
576.98 653 Ac-LTF$r8EYWAQL$AAAAA-OH 561 1858.99 824.54 1860 930.5
620.67 654 Ac-LTF$r8EYWAQL$AAAA-OH 562 1787.95 894.48 1788.96
894.98 596.99 655 Ac-LTF$r8EYWAQL$AA-OH 563 1645.88 856 1646.89
823.95 549.63 656 Ac-LTF$r8AF4bOH2WAQL$AAAAAa-NH.sub.2 564 657
Ac-LTF$r8AF4bOH2WAAL$AAAAAa-NH.sub.2 565 658
Ac-LTF$r8EF4bOH2WAQCba$SAA-NH.sub.2 566 659
Ac-LTF$r8ApYWAQL$AAAAAa-NH.sub.2 567 660
Ac-LTF$r8ApYWAAL$AAAAAa-NH.sub.2 568 661
Ac-LTF$r8EpYWAQCba$SAA-NH.sub.2 569 662
Ac-LTF$rda6AYWAQL$da5AAAAAa-NH.sub.2 570 1974.06 934.44 663
Ac-LTF$rda6EYWAQCba$da5SAA-NH.sub.2 571 1846.95 870.52 869.94 664
Ac-LTF$rda6EYWAQL$da5AAAAAa-NH.sub.2 572 665
Ac-LTF$ra9EYWAQL$a6AAAAAa-NH.sub.2 573 936.57 935.51 666
Ac-LTF$ra9EYWAQL$a6AAAAAa-NH.sub.2 574 667
Ac-LTF$ra9EYWAQCba$a6SAA-NH.sub.2 575 668
Ac-LTA$ra9EYWAQCba$a6SAA-NH.sub.2 576 669
5-FAM-BaLTF$ra9EYWAQCba$a6SAA-NH.sub.2 577 670
5-FAM-BaLTF$r8EYWAQL$AAAAAa-NH.sub.2 578 2316.11 671
5-FAM-BaLTF$/r8EYWAQL$/AAAAAa-NH.sub.2 579 2344.15 672
5-FAM-BaLTA$r8EYWAQL$AAAAAa-NH.sub.2 580 2240.08 673
5-FAM-BaLTF$r8AYWAQL$AAAAAa-NH.sub.2 581 2258.11 674
5-FAM-BaATF$r8EYWAQL$AAAAAa-NH.sub.2 582 2274.07 675
5-FAM-BaLAF$r8EYWAQL$AAAAAa-NH.sub.2 583 2286.1 676
5-FAM-BaLTF$r8EAWAQL$AAAAAa-NH.sub.2 584 2224.09 677
5-FAM-BaLTF$r8EYAAQL$AAAAAa-NH.sub.2 585 2201.07
678 5-FAM-BaLTA$r8EYAAQL$AAAAAa-NH.sub.2 586 2125.04 679
5-FAM-BaLTF$r8EYWAAL$AAAAAa-NH.sub.2 587 2259.09 680
5-FAM-BaLTF$r8EYWAQA$AAAAAa-NH.sub.2 588 2274.07 681
5-FAM-BaLTF$/r8EYWAQCba$/SAA-NH.sub.2 589 2159.03 682
5-FAM-BaLTA$r8EYWAQCba$SAA-NH.sub.2 590 2054.97 683
5-FAM-BaLTF$r8EYAAQCba$SAA-NH.sub.2 591 2015.96 684
5-FAM-BaLTA$r8EYAAQCba$SAA-NH.sub.2 592 1939.92 685
5-FAM-BaQSQQTF$r8NLWRLL$QN-NH.sub.2 593 2495.23 686
5-TAMRA-BaLTF$r8EYWAQCba$SAA-NH.sub.2 594 2186.1 687
5-TAMRA-BaLTA$r8EYWAQCba$SAA-NH.sub.2 595 2110.07 688
5-TAMRA-BaLTF$r8EYAAQCba$SAA-NH.sub.2 596 2071.06 689
5-TAMRA-BaLTA$r8EYAAQCba$SAA-NH.sub.2 597 1995.03 690
5-TAMRA-BaLTF$/r8EYWAQCba$/SAA-NH.sub.2 598 2214.13 691
5-TAMRA-BaLTF$r8EYWAQL$AAAAAa-NH.sub.2 599 2371.22 692
5-TAMRA-BaLTA$r8EYWAQL$AAAAAa-NH.sub.2 600 2295.19 693
5-TAMRA-BaLTF$/r8EYWAQL$/AAAAAa-NH.sub.2 601 2399.25 694
Ac-LTF$r8EYWCou7QCba$SAA-OH 602 1947.93 695
Ac-LTF$r8EYWCou7QCba$S-OH 603 1805.86 696
Ac-LTA$r8EYWCou7QCba$SAA-NH.sub.2 604 1870.91 697
Ac-LTF$r8EYACou7QCba$SAA-NH.sub.2 605 1831.9 698
Ac-LTA$r8EYACou7QCba$SAA-NH.sub.2 606 1755.87 699
Ac-LTF$/r8EYWCou7QCba$/SAA-NH.sub.2 607 1974.98 700
Ac-LTF$r8EYWCou7QL$AAAAAa-NH.sub.2 608 2132.06 701
Ac-LTF$/r8EYWCou7QL$/AAAAAa-NH.sub.2 609 2160.09 702
Ac-LTF$r8EYWCou7QL$AAAAA-OH 610 2062.01 703
Ac-LTF$r8EYWCou7QL$AAAA-OH 611 1990.97 704
Ac-LTF$r8EYWCou7QL$AAA-OH 612 1919.94 705 Ac-LTF$r8EYWCou7QL$AA-OH
613 1848.9 706 Ac-LTF$r8EYWCou7QL$A-OH 614 1777.86 707
Ac-LTF$r8EYWAQL$AAAASa-NH.sub.2 615 iso2 974.4 973.53 708
Ac-LTF$r8AYWAAL$AAAAAa-NH.sub.2 616 iso2 1814.01 908.82 1815.02
908.01 605.68 709 Biotin-BaLTF$r8EYWAQL$AAAAAa-NH.sub.2 617 2184.14
1093.64 2185.15 1093.08 729.05 710 Ac-LTF$r8HAWAQL$S-NH.sub.2 618
iso2 1505.84 754.43 1506.85 753.93 502.95 711
Ac-LTF$r8EYWAQCba$SA-NH.sub.2 619 iso2 1672.89 838.05 1673.9 837.45
558.64 712 Ac-LTF$r8HYWAQCba$SAA-NH.sub.2 620 iso2 1751.94 877.55
1752.95 876.98 584.99 713 Ac-LTF$r8SYWAQCba$SAA-NH.sub.2 621 iso2
1701.91 852.48 1702.92 851.96 568.31 714
Ac-LTF$r8RYWAQCba$SAA-NH.sub.2 622 iso2 1770.98 887.45 1771.99
886.5 591.33 715 Ac-LTF$r8KYWAQCba$SAA-NH.sub.2 623 iso2 1742.98
872.92 1743.99 872.5 582 716 Ac-LTF$r8EYWAQCba$AAA-NH.sub.2 624
iso2 1727.93 865.71 1728.94 864.97 576.98 717
Ac-LTF$r8EYWAQL$AAAAAaBaC-NH.sub.2 625 2103.09 1053.12 2104.1
1052.55 702.04 718 Ac-LTF$r8EYWAQL$AAAAAadPeg4C-NH.sub.2 626
2279.19 1141.46 2280.2 1140.6 760.74 719
Ac-LTA$r8AYWAAL$AAAAAa-NH.sub.2 627 1737.98 870.43 1738.99 870
580.33 720 Ac-LTF$r8AYAAAL$AAAAAa-NH.sub.2 628 1698.97 851 1699.98
850.49 567.33 721 5-FAM-BaLTF$r8AYWAAL$AAAAAa-NH.sub.2 629 2201.09
1101.87 2202.1 1101.55 734.7 722 Ac-LTA$r8AYWAQL$AAAAAa-NH.sub.2
630 1795 898.92 1796.01 898.51 599.34 723
Ac-LTF$r8AYAAQL$AAAAAa-NH.sub.2 631 1755.99 879.49 1757 879 586.34
724 Ac-LTF$rda6AYWAAL$da5AAAAAa-NH.sub.2 632 1807.97 1808.98 904.99
603.66 725 FITC-BaLTF$r8EYWAQL$AAAAAa-NH.sub.2 633 2347.1 1174.49
2348.11 1174.56 783.37 726 FITC-BaLTF$r8EYWAQCba$SAA-NH.sub.2 634
2161.99 1082.35 2163 1082 721.67 733
Ac-LTF$r8EYWAQL$EAAAAa-NH.sub.2 635 1987.05 995.03 1988.06 994.53
663.36 734 Ac-LTF$r8AYWAQL$EAAAAa-NH.sub.2 636 1929.04 966.35
1930.05 965.53 644.02 735 Ac-LTF$r8EYWAQL$AAAAAaBaKbio-NH.sub.2 637
2354.25 1178.47 2355.26 1178.13 785.76 736
Ac-LTF$r8AYWAAL$AAAAAa-NH.sub.2 638 1814.01 908.45 1815.02 908.01
605.68 737 Ac-LTF$r8AYAAAL$AAAAAa-NH.sub.2 639 iso2 1698.97 850.91
1699.98 850.49 567.33 738 Ac-LTF$r8AYAAQL$AAAAAa-NH.sub.2 640 iso2
1755.99 879.4 1757 879 586.34 739 Ac-LTF$r8EYWAQL$EAAAAa-NH.sub.2
641 iso2 1987.05 995.21 1988.06 994.53 663.36 740
Ac-LTF$r8AYWAQL$EAAAAa-NH.sub.2 642 iso2 1929.04 966.08 1930.05
965.53 644.02 741 Ac-LTF$r8EYWAQCba$SAAAAa-NH.sub.2 643 1957.04
980.04 1958.05 979.53 653.35 742 Ac-LTF$r8EYWAQLStAAA$r5AA-NH.sub.2
644 2023.12 1012.83 2024.13 1012.57 675.38 743
Ac-LTF$r8EYWAQL$A$AAA$A-NH.sub.2 645 2108.17 1055.44 2109.18
1055.09 703.73 744 Ac-LTF$r8EYWAQL$AA$AAA$A-NH.sub.2 646 2179.21
1090.77 2180.22 1090.61 727.41 745
Ac-LTF$r8EYWAQL$AAA$AAA$A-NH.sub.2 647 2250.25 1126.69 2251.26
1126.13 751.09 746 Ac-AAALTF$r8EYWAQL$AAA-OH 648 1930.02 1931.03
966.02 644.35 747 Ac-AAALTF$r8EYWAQL$AAA-NH.sub.2 649 1929.04
965.85 1930.05 965.53 644.02 748 Ac-AAAALTF$r8EYWAQL$AAA-NH.sub.2
650 2000.08 1001.4 2001.09 1001.05 667.7 749
Ac-AAAAALTF$r8EYWAQL$AAA-NH.sub.2 651 2071.11 1037.13 2072.12
1036.56 691.38 750 Ac-AAAAAALTF$r8EYWAQL$AAA-NH.sub.2 652 2142.15
2143.16 1072.08 715.06 751 Ac-LTF$rda6EYWAQCba$da6SAA-NH.sub.2 653
iso2 1751.89 877.36 1752.9 876.95 584.97 752
Ac-t$r5wya$r5f4CF3ekllr-NH.sub.2 654 844.25 753
Ac-tawy$r5nf4CF3e$r5llr-NH.sub.2 655 837.03 754
Ac-tawya$r5f4CF3ek$r5lr-NH.sub.2 656 822.97 755
Ac-tawyanf4CF3e$r5llr$r5a-NH.sub.2 657 908.35 756
Ac-t$s8wyanf4CF3e$r5llr-NH.sub.2 658 858.03 757
Ac-tawy$s8nf4CF3ekll$r5a-NH.sub.2 659 879.86 758
Ac-tawya$s8f4CF3ekllr$r5a-NH.sub.2 660 936.38 759
Ac-tawy$s8naekll$r5a-NH.sub.2 661 844.25 760
5-FAM-Batawy$s8nf4CF3ekll$r5a-NH.sub.2 662 761
5-FAM-Batawy$s8naekll$r5a-NH.sub.2 663 762
Ac-tawy$s8nf4CF3eall$r5a-NH.sub.2 664 763
Ac-tawy$s8nf4CF3ekll$r5aaaaa-NH.sub.2 665 764
Ac-tawy$s8nf4CF3eall$r5aaaaa-NH.sub.2 666
[0357] Table 3a shows a selection of peptidomimetic
macrocycles.
TABLE-US-00006 TABLE 3a SEQ ID Exact Found Calc Calc Calc Sequence
NO: Iso Mass Mass (M + 1)/1 (M + 2)/2 (M + 3)/3 244
Ac-LTF$r8EF4coohWAQCba$SANleA-NH.sub.2 667 1885 943.59 1886.01
943.51 629.34 331 Ac-LTF$r8EYWAQL$AAAAAa-NH.sub.2 668 iso2 1929.04
966.08 1930.05 965.53 644.02 555 Ac-LTF$r8EY6clWAQL$AAAAAa-NH.sub.2
669 1963 983.28 1964.01 982.51 655.34 557
Ac-AAALTF$r8EYWAQL$AAAAAa-NH.sub.2 670 2142.15 1072.83 2143.16
1072.08 715.06 558 Ac-LTF34F2$r8EYWAQL$AAAAAa-NH.sub.2 671 1965.02
984.3 1966.03 983.52 656.01 562 Ac-LTF$r8EYWAQL$AAibAAAa-NH.sub.2
672 1943.06 973.11 1944.07 972.54 648.69 564
Ac-LTF$r8EYWAQL$AAAAibAa-NH.sub.2 673 1943.06 973.48 1944.07 972.54
648.69 566 Ac-LTF$r8EYWAQL$AAAAAiba-NH.sub.2 674 iso2 1943.06
973.38 1944.07 972.54 648.69 567 Ac-LTF$r8EYWAQL$AAAAAAib-NH.sub.2
675 1943.06 973.01 1944.07 972.54 648.69 572
Ac-LTF$r8EYWAQL$AAAAaa-NH.sub.2 676 1929.04 966.35 1930.05 965.53
644.02 573 Ac-LTF$r8EYWAQL$AAAAAA-NH.sub.2 677 1929.04 966.35
1930.05 965.53 644.02 578 Ac-LTF$r8EYWAQL$AAAAASar-NH.sub.2 678
1929.04 966.08 1930.05 965.53 644.02 551 Ac-LTF$r8EYWAQL$AAAAAa-OH
679 iso2 1930.02 965.89 1931.03 966.02 644.35 662
Ac-LTF$rda6AYWAQL$da5AAAAAa-NH.sub.2 680 1974.06 934.44 933.49 367
5-FAM-BaLTF$r8EYWAQCba$SAA-NH.sub.2 681 2131 1067.09 2132.01
1066.51 711.34 349 Ac-LTF$r8EF4coohWAQCba$AAAAAa-NH.sub.2 682 iso2
1969.04 986.06 1970.05 985.53 657.35 347
Ac-LTF$r8EYWAQCba$AAAAAa-NH.sub.2 683 iso2 1941.04 972.55 1942.05
971.53 648.02
[0358] Table 3b shows a further selection of peptidomimetic
macrocycles.
TABLE-US-00007 TABLE 3b SEQ ID Exact Found Calc Calc Calc SP
Sequence NO: Iso Mass Mass (M + 1)/1 (M + 2)/2 (M + 3)/3 581
Ac-TF$r8EYWAQL$AAAAAa-NH.sub.2 684 1815.96 929.85 1816.97 908.99
606.33 582 Ac-F$r8EYWAQL$AAAAAa-NH.sub.2 685 1714.91 930.92 1715.92
858.46 572.64 583 Ac-LVF$r8EYWAQL$AAAAAa-NH.sub.2 686 1927.06
895.12 1928.07 964.54 643.36 584 Ac-AAF$r8EYWAQL$AAAAAa-NH.sub.2
687 1856.98 859.51 1857.99 929.5 620 585
Ac-LTF$r8EYWAQL$AAAAa-NH.sub.2 688 1858 824.08 1859.01 930.01
620.34 586 Ac-LTF$r8EYWAQL$AAAa-NH.sub.2 689 1786.97 788.56 1787.98
894.49 596.66 587 Ac-LTF$r8EYWAQL$AAa-NH.sub.2 690 1715.93 1138.57
1716.94 858.97 572.98 588 Ac-LTF$r8EYWAQL$Aa-NH.sub.2 691 1644.89
1144.98 1645.9 823.45 549.3 589 Ac-LTF$r8EYWAQL$a-NH.sub.2 692
1573.85 1113.71 1574.86 787.93 525.62
[0359] In the sequences shown above and elsewhere, the following
abbreviations are used: "Nle" represents norleucine, "Aib"
represents 2-aminoisobutyric acid, "Ac" represents acetyl, and "Pr"
represents propionyl. Amino acids represented as "$" are alpha-Me
S5-pentenyl-alanine olefin amino acids connected by an all-carbon
crosslinker comprising one double bond. Amino acids represented as
"$r5" are alpha-Me R5-pentenyl-alanine olefin amino acids connected
by an all-carbon comprising one double bond. Amino acids
represented as "$s8" are alpha-Me S8-octenyl-alanine olefin amino
acids connected by an all-carbon crosslinker comprising one double
bond. Amino acids represented as "$r8" are alpha-Me
R8-octenyl-alanine olefin amino acids connected by an all-carbon
crosslinker comprising one double bond. "Ahx" represents an
aminocyclohexyl linker. The crosslinkers are linear all-carbon
crosslinker comprising eight or eleven carbon atoms between the
alpha carbons of each amino acid. Amino acids represented as "$/"
are alpha-Me S5-pentenyl-alanine olefin amino acids that are not
connected by any crosslinker. Amino acids represented as "$/r5" are
alpha-Me R5-pentenyl-alanine olefin amino acids that are not
connected by any crosslinker. Amino acids represented as "$/s8" are
alpha-Me S8-octenyl-alanine olefin amino acids that are not
connected by any crosslinker. Amino acids represented as "$/r8" are
alpha-Me R8-octenyl-alanine olefin amino acids that are not
connected by any crosslinker. Amino acids represented as "Amw" are
alpha-Me tryptophan amino acids. Amino acids represented as "Aml"
are alpha-Me leucine amino acids. Amino acids represented as "Amf"
are alpha-Me phenylalanine amino acids. Amino acids represented as
"2ff" are 2-fluoro-phenylalanine amino acids. Amino acids
represented as "3ff" are 3-fluoro-phenylalanine amino acids. Amino
acids represented as "St" are amino acids comprising two
pentenyl-alanine olefin side chains, each of which is crosslinked
to another amino acid as indicated. Amino acids represented as
"St//" are amino acids comprising two pentenyl-alanine olefin side
chains that are not crosslinked. Amino acids represented as "% St"
are amino acids comprising two pentenyl-alanine olefin side chains,
each of which is crosslinked to another amino acid as indicated via
fully saturated hydrocarbon crosslinks. Amino acids represented as
"Ba" are beta-alanine. The lower-case character "e" or "z" within
the designation of a crosslinked amino acid (e.g. "$er8" or "$zr8")
represents the configuration of the double bond (E or Z,
respectively). In other contexts, lower-case letters such as "a" or
"f" represent D amino acids (e.g. D-alanine, or D-phenylalanine,
respectively). Amino acids designated as "NmW" represent
N-methyltryptophan. Amino acids designated as "NmY" represent
N-methyltyrosine. Amino acids designated as "NmA" represent
N-methylalanine. "Kbio" represents a biotin group attached to the
side chain amino group of a lysine residue. Amino acids designated
as "Sar" represent sarcosine. Amino acids designated as "Cha"
represent cyclohexyl alanine. Amino acids designated as "Cpg"
represent cyclopentyl glycine. Amino acids designated as "Chg"
represent cyclohexyl glycine. Amino acids designated as "Cba"
represent cyclobutyl alanine. Amino acids designated as "F4I"
represent 4-iodo phenylalanine. "7L" represents N15 isotopic
leucine. Amino acids designated as "F3Cl" represent 3-chloro
phenylalanine. Amino acids designated as "F4cooh" represent
4-carboxy phenylalanine. Amino acids designated as "F34F2"
represent 3,4-difluoro phenylalanine. Amino acids designated as
"6clW" represent 6-chloro tryptophan. Amino acids designated as
"$rda6" represent alpha-Me R6-hexynyl-alanine alkynyl amino acids,
crosslinked via a dialkyne bond to a second alkynyl amino acid.
Amino acids designated as "$da5" represent alpha-Me
S5-pentynyl-alanine alkynyl amino acids, wherein the alkyne forms
one half of a dialkyne bond with a second alkynyl amino acid. Amino
acids designated as "$ra9" represent alpha-Me R9-nonynyl-alanine
alkynyl amino acids, crosslinked via an alkyne metathesis reaction
with a second alkynyl amino acid. Amino acids designated as "$a6"
represent alpha-Me S6-hexynyl-alanine alkynyl amino acids,
crosslinked via an alkyne metathesis reaction with a second alkynyl
amino acid. The designation "iso1" or "iso2" indicates that the
peptidomimetic macrocycle is a single isomer.
[0360] Amino acids designated as "Cit" represent citrulline. Amino
acids designated as "Cou4", "Cou6", "Cou7" and "Cou8",
respectively, represent the following structures:
##STR00044## ##STR00045##
[0361] In some embodiments, a peptidomimetic macrocycle is obtained
in more than one isomer, for example due to the configuration of a
double bond within the structure of the crosslinker (E vs Z). Such
isomers can or cannot be separable by conventional chromatographic
methods. In some embodiments, one isomer has improved biological
properties relative to the other isomer. In one embodiment, an E
crosslinker olefin isomer of a peptidomimetic macrocycle has better
solubility, better target affinity, better in vivo or in vitro
efficacy, higher helicity, or improved cell permeability relative
to its Z counterpart. In another embodiment, a Z crosslinker olefin
isomer of a peptidomimetic macrocycle has better solubility, better
target affinity, better in vivo or in vitro efficacy, higher
helicity, or improved cell permeability relative to its E
counterpart.
[0362] Table 3c shows exemplary peptidomimetic macrocycle:
TABLE-US-00008 TABLE 3c Structure SP154 (SEQ ID NO: 163)
##STR00046## Chemical Formula: C.sub.87H.sub.125N.sub.17O.sub.21
Exact Mass: 1743.92 Molecular Weight: 1745.02 SP115 (SEQ ID NO:
124) ##STR00047## Chemical Formula:
C.sub.85H.sub.125N.sub.17O.sub.19 Exact Mass: 1687.93 Molecular
Weight: 1689.00 SP114 (SEQ ID NO: 123) ##STR00048## Chemical
Formula: C.sub.85H.sub.125N.sub.17O.sub.19 Exact Mass: 1687.93
Molecular Weight: 1689.00 SP99 (SEQ ID NO: 108) ##STR00049##
Chemical Formula: C.sub.84H.sub.122ClN.sub.17O.sub.19 Exact Mass:
1707.88 Molecular Weight: 1709.42 SP388 (SEQ ID NO: 397)
##STR00050## Chemical Formula: C.sub.91H.sub.135N.sub.18O.sub.19
Exact Mass: 1785.02 Molecular Weight: 1786.16 SP331 (SEQ ID NO:
340) ##STR00051## Chemical Formula:
C.sub.95H.sub.140N.sub.20O.sub.23 Exact Mass: 1929.04 Molecular
Weight: 1930.25 SP445 (SEQ ID NO: 454) ##STR00052## Chemical
Formula: C.sub.95H.sub.142N.sub.20O.sub.23 Exact Mass: 1931.06
Molecular Weight: 1932.26 SP351 (SEQ ID NO: 360) ##STR00053##
Chemical Formula: C.sub.96H.sub.140N.sub.20O.sub.24 Exact Mass:
1957.03 Molecular Weight: 1958.26 SP71 (SEQ ID NO: 80) ##STR00054##
Chemical Formula: C.sub.90H.sub.134N.sub.18O.sub.19 Exact Mass:
1771.01 Molecular Weight: 1772.14 SP69 (SEQ ID NO: 78) ##STR00055##
Chemical Formula: C.sub.90H.sub.134N.sub.18O.sub.19 Exact Mass:
1771.01 Molecular Weight: 1772.14 SP7 (SEQ ID NO: 16) ##STR00056##
Chemical Formula: C.sub.90H.sub.127N.sub.17O.sub.19 Exact Mass:
1749.95 Molecular Weight: 1751.07 SP160 (SEQ ID NO: 169)
##STR00057## Chemical Formula:
C.sub.87H.sub.125F.sub.2N.sub.17O.sub.21 Exact Mass: 1781.92
Molecular Weight: 1783.02 SP315 (SEQ ID NO: 324) ##STR00058##
Chemical Formula: C.sub.93H.sub.138N.sub.20O.sub.21 Exact Mass:
1871.03 Molecular Weight: 1872.21 SP249 (SEQ ID NO: 258)
##STR00059## Chemical Formula: C.sub.94H.sub.136N.sub.18O.sub.22
Exact Mass: 1869.01 Molecular Weight: 1870.19 SP437 (SEQ ID NO:
446) ##STR00060## Chemical Formula:
C.sub.95H.sub.143N.sub.21O.sub.21 Exact Mass: 1914.08 Molecular
Weight: 1915.28 SP349 (SEQ ID NO: 358) ##STR00061## Chemical
Formula: C.sub.97H.sub.140N.sub.20O.sub.24 Exact Mass: 1969.03
Molecular Weight: 1970.27 SP555 (SEQ ID NO: 464) ##STR00062##
Chemical Formula: C.sub.95H.sub.139ClN.sub.20O.sub.23 Exact Mass:
1963.00 Molecular Weight: 1964.69 SP557 (SEQ ID NO: 466)
##STR00063## Chemical Formula: C.sub.104H.sub.155N.sub.23O.sub.26
Exact Mass: 2142.15 Molecular Weight: 2143.48 SP558 (SEQ ID NO:
467) ##STR00064## Chemical Formula:
C.sub.95H.sub.138F.sub.2N.sub.20O.sub.23 Exact Mass: 1965.02
Molecular Weight: 1966.23 SP367 (SEQ ID NO: 376) ##STR00065## SP562
(SEQ ID NO: 471) ##STR00066## Chemical Formula:
C.sub.96H.sub.142N.sub.20O.sub.23 Exact Mass: 1943.06 Molecular
Weight: 1944.27 SP564 (SEQ ID NO: 473) ##STR00067## Chemical
Formula: C.sub.96H.sub.142N.sub.20O.sub.23 Exact Mass: 1943.06
Molecular Weight: 1944.27 SP566 (SEQ ID NO: 475) ##STR00068## SP567
(SEQ ID NO: 476) ##STR00069## Chemical Formula:
C.sub.96H.sub.142N.sub.20O.sub.23 Exact Mass: 1943.06 Molecular
Weight: 1944.27 SP572 (SEQ ID NO: 481) ##STR00070## Chemical
Formula: C.sub.95H.sub.140N.sub.20O.sub.23 Exact Mass: 1929.04
Molecular Weight: 1930.25 SP573 (SEQ ID NO: 482) ##STR00071##
Chemical Formula: C.sub.95H.sub.140N.sub.20O.sub.23 Exact Mass:
1929.04 Molecular Weight: 1930.25 SP578 (SEQ ID NO: 487)
##STR00072## Chemical Formula: C.sub.95H.sub.140N.sub.20O.sub.23
Exact Mass: 1929.04 Molecular Weight: 1930.25 SP664 (SEQ ID NO:
572) ##STR00073## Chemical Formula:
C.sub.95H.sub.134N.sub.20O.sub.23 Exact Mass: 1922.99 Molecular
Weight: 1924.20 SP664 (SEQ ID NO: 572) ##STR00074## Chemical
Formula: C.sub.95H.sub.134N.sub.20O.sub.23 Exact Mass: 1922.99
Molecular Weight: 1924.20 (SEQ ID NO: 1500) ##STR00075## Chemical
Formula: C.sub.96H.sub.136N.sub.20O.sub.23 Exact Mass: 1937.01
Molecular Weight: 1938.23
[0363] In some embodiments, peptidomimetic macrocycles exclude
peptidomimetic macrocycles shown in Table 4a:
TABLE-US-00009 TABLE 4a Number Sequence SEQ ID NO: 765
L$r5QETFSD$s8WKLLPEN 693 766 LSQ$r5TFSDLW$s8LLPEN 694 767
LSQE$r5FSDLWK$s8LPEN 695 768 LSQET$r5SDLWKL$s8PEN 696 769
LSQETF$r5DLWKLL$s8EN 697 770 LXQETFS$r5LWKLLP$s8N 698 771
LSQETFSD$r5WKLLPE$s8 699 772 LSQQTF$r5DLWKLL$s8EN 700 773
LSQETF$r5DLWKLL$s8QN 701 774 LSQQTF$r5DLWKLL$s8QN 702 775
LSQETF$r5NLWKLL$s8QN 703 776 LSQQTF$r5NLWKLL$s8QN 704 777
LSQQTF$r5NLWRLL$s8QN 705 778 QSQQTF$r5NLWKLL$s8QN 706 779
QSQQTF$r5NLWRLL$s8QN 707 780 QSQQTA$r5NLWRLL$s8QN 708 781
L$r8QETFSD$WKLLPEN 709 782 LSQ$r8TFSDLW$LLPEN 710 783
LSQE$r8FSDLWK$LPEN 711 784 LSQET$r8SDLWKL$PEN 712 785
LSQETF$r8DLWKLL$EN 713 786 LXQETFS$r8LWKLLP$N 714 787
LSQETFSD$r8WKLLPE$ 715 788 LSQQTF$r8DLWKLL$EN 716 789
LSQETF$r8DLWKLL$QN 717 790 LSQQTF$r8DLWKLL$QN 718 791
LSQETF$r8NLWKLL$QN 719 792 LSQQTF$r8NLWKLL$QN 720 793
LSQQTF$r8NLWRLL$QN 721 794 QSQQTF$r8NLWKLL$QN 722 795
QSQQTF$r8NLWRLL$QN 723 796 QSQQTA$r8NLWRLL$QN 724 797
QSQQTF$r8NLWRKK$QN 725 798 QQTF$r8DLWRLL$EN 726 799 QQTF$r8DLWRLL$
727 800 LSQQTF$DLW$LL 728 801 QQTF$DLW$LL 729 802 QQTA$r8DLWRLL$EN
730 803 QSQQTF$r5NLWRLL$s8QN 731 (dihydroxylated olefin) 804
QSQQTA$r5NLWRLL$s8QN 732 (dihydroxylated olefin) 805
QSQQTF$r8DLWRLL$QN 733 806 QTF$r8NLWRLL$ 734 807 QSQQTF$NLW$LLPQN
735 808 QS$QTF$NLWRLLPQN 736 809 $TFS$LWKLL 737 810 ETF$DLW$LL 738
811 QTF$NLW$LL 739 812 $SQE$FSNLWKLL 740
In Table 4a, X represents S or any amino acid. Peptides shown can
comprise an N-terminal capping group such as acetyl or an
additional linker such as beta-alanine between the capping group
and the start of the peptide sequence.
[0364] In some embodiments, peptidomimetic macrocycles do not
comprise a peptidomimetic macrocycle structure as shown in Table
4a.
[0365] In other embodiments, peptidomimetic macrocycles exclude
peptidomimetic macrocycles shown in Table 4b.
TABLE-US-00010 TABLE 4b Observed SEQ Exact mass SP# Sequence ID NO:
Mass M + 2 (m/e) 813 Ac-LSQETF$r8DLWKLL$EN-NH.sub.2 741 2068.13
1035.07 1035.36 814 Ac-LSQETF$r8NLWKLL$QN-NH.sub.2 742 2066.16
1034.08 1034.31 815 Ac-LSQQTF$r8NLWRLL$QN-NH.sub.2 743 2093.18
1047.59 1047.73 816 Ac-QSQQTF$r8NLWKLL$QN-NH.sub.2 744 2080.15
1041.08 1041.31 817 Ac-QSQQTF$r8NLWRLL$QN-NH.sub.2 745 2108.15
1055.08 1055.32 818 Ac-QSQQTA$r8NLWRLL$QN-NH.sub.2 746 2032.12
1017.06 1017.24 819 Ac-QAibQQTF$r8NLWRLL$QN-NH.sub.2 747 2106.17
1054.09 1054.34 820 Ac-QSQQTFSNLWRLLPQN-NH.sub.2 748 2000.02
1001.01 1001.26 821 Ac-QSQQTF$/r8NLWRLL$/QN-NH.sub.2 749 2136.18
1069.09 1069.37 822 Ac-QSQAibTF$r8NLWRLL$QN-NH.sub.2 750 2065.15
1033.58 1033.71 823 Ac-QSQQTF$r8NLWRLL$AN-NH.sub.2 751 2051.13
1026.57 1026.70 824 Ac-ASQQTF$r8NLWRLL$QN-NH.sub.2 752 2051.13
1026.57 1026.90 825 Ac-QSQQTF$r8ALWRLL$QN-NH.sub.2 753 2065.15
1033.58 1033.41 826 Ac-QSQETF$r8NLWRLL$QN-NH.sub.2 754 2109.14
1055.57 1055.70 827 Ac-RSQQTF$r8NLWRLL$QN-NH.sub.2 755 2136.20
1069.10 1069.17 828 Ac-RSQQTF$r8NLWRLL$EN-NH.sub.2 756 2137.18
1069.59 1069.75 829 Ac-LSQETFSDLWKLLPEN-NH.sub.2 757 1959.99 981.00
981.24 830 Ac-QSQ$TFS$LWRLLPQN-NH.sub.2 758 2008.09 1005.05 1004.97
831 Ac-QSQQ$FSN$WRLLPQN-NH.sub.2 759 2036.06 1019.03 1018.86 832
Ac-QSQQT$SNL$RLLPQN-NH.sub.2 760 1917.04 959.52 959.32 833
Ac-QSQQTF$NLW$LLPQN-NH.sub.2 761 2007.06 1004.53 1004.97 834
Ac-RTQATF$r8NQWAibANle$TNAibTR-NH.sub.2 762 2310.26 1156.13 1156.52
835 Ac-QSQQTF$r8NLWRLL$RN-NH.sub.2 763 2136.20 1069.10 1068.94 836
Ac-QSQRTF$r8NLWRLL$QN-NH.sub.2 764 2136.20 1069.10 1068.94 837
Ac-QSQQTF$r8NNleWRLL$QN-NH.sub.2 765 2108.15 1055.08 1055.44 838
Ac-QSQQTF$r8NLWRNleL$QN-NH.sub.2 766 2108.15 1055.08 1055.84 839
Ac-QSQQTF$r8NLWRLNle$QN-NH.sub.2 767 2108.15 1055.08 1055.12 840
Ac-QSQQTY$r8NLWRLL$QN-NH.sub.2 768 2124.15 1063.08 1062.92 841
Ac-RAibQQTF$r8NLWRLL$QN-NH.sub.2 769 2134.22 1068.11 1068.65 842
Ac-MPRFMDYWEGLN-NH.sub.2 770 1598.70 800.35 800.45 843
Ac-RSQQRF$r8NLWRLL$QN-NH.sub.2 771 2191.25 1096.63 1096.83 844
Ac-QSQQRF$r8NLWRLL$QN-NH.sub.2 772 2163.21 1082.61 1082.87 845
Ac-RAibQQRF$r8NLWRLL$QN-NH.sub.2 773 2189.27 1095.64 1096.37 846
Ac-RSQQRF$r8NFWRLL$QN-NH.sub.2 774 2225.23 1113.62 1114.37 847
Ac-RSQQRF$r8NYWRLL$QN-NH.sub.2 775 2241.23 1121.62 1122.37 848
Ac-RSQQTF$r8NLWQLL$QN-NH.sub.2 776 2108.15 1055.08 1055.29 849
Ac-QSQQTF$r8NLWQAmlL$QN-NH.sub.2 777 2094.13 1048.07 1048.32 850
Ac-QSQQTF$r8NAmlWRLL$QN-NH.sub.2 778 2122.17 1062.09 1062.35 851
Ac-NlePRF$r8DYWEGL$QN-NH.sub.2 779 1869.98 935.99 936.20 852
Ac-NlePRF$r8NYWRLL$QN-NH.sub.2 780 1952.12 977.06 977.35 853
Ac-RF$r8NLWRLL$Q-NH.sub.2 781 1577.96 789.98 790.18 854
Ac-QSQQTF$r8N2ffWRLL$QN-NH.sub.2 782 2160.13 1081.07 1081.40 855
Ac-QSQQTF$r8N3ffWRLL$QN-NH.sub.2 783 2160.13 1081.07 1081.34 856
Ac-QSQQTF#r8NLWRLL#QN-NH.sub.2 784 2080.12 1041.06 1041.34 857
Ac-RSQQTA$r8NLWRLL$QN-NH.sub.2 785 2060.16 1031.08 1031.38 858
Ac-QSQQTF%r8NLWRLL%QN-NH.sub.2 786 2110.17 1056.09 1056.55 859
HepQSQ$TFSNLWRLLPQN-NH.sub.2 787 2051.10 1026.55 1026.82 860
HepQSQ$TF$r8NLWRLL$QN-NH.sub.2 788 2159.23 1080.62 1080.89 861
Ac-QSQQTF$r8NL6clWRLL$QN-NH.sub.2 789 2142.11 1072.06 1072.35 862
Ac-QSQQTF$r8NLMe6clwRLL$QN-NH.sub.2 790 2156.13 1079.07 1079.27 863
Ac-LTFEHYWAQLTS-NH.sub.2 791 1535.74 768.87 768.91 864
Ac-LTF$HYW$QLTS-NH.sub.2 792 1585.83 793.92 794.17 865
Ac-LTFE$YWA$LTS-NH.sub.2 793 1520.79 761.40 761.67 866
Ac-LTF$zr8HYWAQL$zS-NH.sub.2 794 1597.87 799.94 800.06 867
Ac-LTF$r8HYWRQL$S-NH.sub.2 795 1682.93 842.47 842.72 868
Ac-QS$QTFStNLWRLL$s8QN-NH.sub.2 796 2145.21 1073.61 1073.90 869
Ac-QSQQTASNLWRLLPQN-NH.sub.2 797 1923.99 963.00 963.26 870
Ac-QSQQTA$/r8NLWRLL$/QN-NH.sub.2 798 2060.15 1031.08 1031.24 871
Ac-ASQQTF$/r8NLWRLL$/QN-NH.sub.2 799 2079.16 1040.58 1040.89 872
Ac-$SQQ$FSNLWRLLAibQN-NH.sub.2 800 2009.09 1005.55 1005.86 873
Ac-QS$QTF$NLWRLLAibQN-NH.sub.2 801 2023.10 1012.55 1012.79 874
Ac-QSQQ$FSN$WRLLAibQN-NH.sub.2 802 2024.06 1013.03 1013.31 875
Ac-QSQQTF$NLW$LLAibQN-NH.sub.2 803 1995.06 998.53 998.87 876
Ac-QSQQTFS$LWR$LAibQN-NH.sub.2 804 2011.06 1006.53 1006.83 877
Ac-QSQQTFSNLW$LLA$N-NH.sub.2 805 1940.02 971.01 971.29 878
Ac-$/SQQ$/FSNLWRLLAibQN-NH.sub.2 806 2037.12 1019.56 1019.78 879
Ac-QS$/QTF$/NLWRLLAibQN-NH.sub.2 807 2051.13 1026.57 1026.90 880
Ac-QSQQ$/FSN$/WRLLAibQN-NH.sub.2 808 2052.09 1027.05 1027.36 881
Ac-QSQQTF$/NLW$/LLAibQN-NH.sub.2 809 2023.09 1012.55 1013.82 882
Ac-QSQ$TFS$LWRLLAibQN-NH.sub.2 810 1996.09 999.05 999.39 883
Ac-QSQ$/TFS$/LWRLLAibQN-NH.sub.2 811 2024.12 1013.06 1013.37 884
Ac-QS$/QTFSt//NLWRLL$/s8QN-NH.sub.2 812 2201.27 1101.64 1102.00 885
Ac-$r8SQQTFS$LWRLLAibQN-NH.sub.2 813 2038.14 1020.07 1020.23 886
Ac-QSQ$r8TFSNLW$LLAibQN-NH.sub.2 814 1996.08 999.04 999.32 887
Ac-QSQQTFS$r8LWRLLA$N-NH.sub.2 815 2024.12 1013.06 1013.37 888
Ac-QS$r5QTFStNLW$LLAibQN-NH.sub.2 816 2032.12 1017.06 1017.39 889
Ac-$/r8SQQTFS$/LWRLLAibQN-NH.sub.2 817 2066.17 1034.09 1034.80 890
Ac-QSQ$/r8TFSNLW$/LLAibQN-NH.sub.2 818 2024.11 1013.06 1014.34 891
Ac-QSQQTFS$/r8LWRLLA$/N-NH.sub.2 819 2052.15 1027.08 1027.16 892
Ac-QS$/r5QTFSt//NLW$/LLAibQN-NH.sub.2 820 2088.18 1045.09 1047.10
893 Ac-QSQQTFSNLWRLLAibQN-NH.sub.2 821 1988.02 995.01 995.31 894
Hep/QSQ$/TF$/r8NLWRLL$/QN-NH.sub.2 822 2215.29 1108.65 1108.93 895
Ac-ASQQTF$r8NLRWLL$QN-NH.sub.2 823 2051.13 1026.57 1026.90 896
Ac-QSQQTF$/r8NLWRLL$/Q-NH.sub.2 824 2022.14 1012.07 1012.66 897
Ac-QSQQTF$r8NLWRLL$Q-NH.sub.2 825 1994.11 998.06 998.42 898
Ac-AAARAA$r8AAARAA$AA-NH.sub.2 826 1515.90 758.95 759.21 899
Ac-LTFEHYWAQLTSA-NH.sub.2 827 1606.78 804.39 804.59 900
Ac-LTF$r8HYWAQL$SA-NH.sub.2 828 1668.90 835.45 835.67 901
Ac-ASQQTFSNLWRLLPQN-NH.sub.2 829 1943.00 972.50 973.27 902
Ac-QS$QTFStNLW$r5LLAibQN-NH.sub.2 830 2032.12 1017.06 1017.30 903
Ac-QSQQTFAibNLWRLLAibQN-NH.sub.2 831 1986.04 994.02 994.19 904
Ac-QSQQTFNleNLWRLLNleQN-NH.sub.2 832 2042.11 1022.06 1022.23 905
Ac-QSQQTF$/r8NLWRLLAibQN-NH.sub.2 833 2082.14 1042.07 1042.23 906
Ac-QSQQTF$/r8NLWRLLNleQN-NH.sub.2 834 2110.17 1056.09 1056.29 907
Ac-QSQQTFAibNLWRLL$/QN-NH.sub.2 835 2040.09 1021.05 1021.25 908
Ac-QSQQTFNleNLWRLL$/QN-NH.sub.2 836 2068.12 1035.06 1035.31 909
Ac-QSQQTF%r8NL6clWRNleL%QN-NH.sub.2 837 2144.13 1073.07 1073.32 910
Ac-QSQQTF%r8NLMe6clWRLL%QN-NH.sub.2 838 2158.15 1080.08 1080.31 911
Ac-FNle$YWE$L-NH.sub.2 839 1160.63 -- 1161.70 912
Ac-F$r8AYWELL$A-NH.sub.2 840 1344.75 -- 1345.90 913
Ac-F$r8AYWQLL$A-NH.sub.2 841 1343.76 -- 1344.83 914
Ac-NlePRF$r8NYWELL$QN-NH.sub.2 842 1925.06 963.53 963.69 915
Ac-NlePRF$r8DYWRLL$QN-NH.sub.2 843 1953.10 977.55 977.68 916
Ac-NlePRF$r8NYWRLL$Q-NH.sub.2 844 1838.07 920.04 920.18 917
Ac-NlePRF$r8NYWRLL$-NH.sub.2 845 1710.01 856.01 856.13 918
Ac-QSQQTF$r8DLWRLL$QN-NH.sub.2 846 2109.14 1055.57 1055.64 919
Ac-QSQQTF$r8NLWRLL$EN-NH.sub.2 847 2109.14 1055.57 1055.70 920
Ac-QSQQTF$r8NLWRLL$QD-NH.sub.2 848 2109.14 1055.57 1055.64 921
Ac-QSQQTF$r8NLWRLL$S-NH.sub.2 849 1953.08 977.54 977.60 922
Ac-ESQQTF$r8NLWRLL$QN-NH.sub.2 850 2109.14 1055.57 1055.70 923
Ac-LTF$r8NLWRNleL$Q-NH.sub.2 851 1635.99 819.00 819.10 924
Ac-LRF$r8NLWRNleL$Q-NH.sub.2 852 1691.04 846.52 846.68 925
Ac-QSQQTF$r8NWWRNleL$QN-NH.sub.2 853 2181.15 1091.58 1091.64 926
Ac-QSQQTF$r8NLWRNleL$Q-NH.sub.2 854 1994.11 998.06 998.07 927
Ac-QTF$r8NLWRNleL$QN-NH.sub.2 855 1765.00 883.50 883.59 928
Ac-NlePRF$r8NWWRLL$QN-NH.sub.2 856 1975.13 988.57 988.75 929
Ac-NlePRF$r8NWWRLL$A-NH.sub.2 857 1804.07 903.04 903.08 930
Ac-TSFAEYWNLLNH.sub.2 858 1467.70 734.85 734.90 931
Ac-QTF$r8HWWSQL$S-NH.sub.2 859 1651.85 826.93 827.12 932
Ac-FM$YWE$L-NH.sub.2 860 1178.58 -- 1179.64 933
Ac-QTFEHWWSQLLS-NH.sub.2 861 1601.76 801.88 801.94 934
Ac-QSQQTF$r8NLAmwRLNle$QN-NH.sub.2 862 2122.17 1062.09 1062.24 935
Ac-FMAibY6clWEAc3cL-NH.sub.2 863 1130.47 -- 1131.53 936
Ac-FNle$Y6clWE$L-NH.sub.2 864 1194.59 -- 1195.64 937
Ac-F$zr8AY6clWEAc3cL$z-NH.sub.2 865 1277.63 639.82 1278.71 938
Ac-F$r8AY6clWEAc3cL$A-NH.sub.2 866 1348.66 -- 1350.72 939
Ac-NlePRF$r8NY6clWRLL$QN-NH.sub.2 867 1986.08 994.04 994.64 940
Ac-AF$r8AAWALA$A-NH.sub.2 868 1223.71 -- 1224.71 941
Ac-TF$r8AAWRLA$Q-NH.sub.2 869 1395.80 698.90 399.04 942
Pr-TF$r8AAWRLA$Q-NH.sub.2 870 1409.82 705.91 706.04 943
Ac-QSQQTF%r8NLWRNleL%QN-NH.sub.2 871 2110.17 1056.09 1056.22 944
Ac-LTF%r8HYWAQL%SA-NH.sub.2 872 1670.92 836.46 836.58 945
Ac-NlePRF%r8NYWRLL%QN-NH.sub.2 873 1954.13 978.07 978.19 946
Ac-NlePRF%r8NY6clWRLL%QN-NH.sub.2 874 1988.09 995.05 995.68 947
Ac-LTF%r8HY6clWAQL%S-NH.sub.2 875 1633.84 817.92 817.93 948
Ac-QS%QTF%StNLWRLL%s8QN-NH.sub.2 876 2149.24 1075.62 1075.65 949
Ac-LTF%r8HY6clWRQL%S-NH.sub.2 877 1718.91 860.46 860.54 950
Ac-QSQQTF%r8NL6clWRLL%QN-NH.sub.2 878 2144.13 1073.07 1073.64 951
Ac-%r8SQQTFS%LWRLLAibQN-NH.sub.2 879 2040.15 1021.08 1021.13 952
Ac-LTF%r8HYWAQL%S-NH.sub.2 880 1599.88 800.94 801.09 953
Ac-TSF%r8QYWNLL%P-NH.sub.2 881 1602.88 802.44 802.58 954
Ac-LTFEHYWAQLTS-NH.sub.2 882 1535.74 768.87 769.5 955
Ac-F$er8AY6clWEAc3cL$e-NH.sub.2 883 1277.63 639.82 1278.71 956
Ac-AF$r8AAWALA$A-NH.sub.2 884 1277.63 639.82 1277.84 957
Ac-TF$r8AAWRLA$Q-NH.sub.2 885 1395.80 698.90 699.04 958
Pr-TF$r8AAWRLA$Q-NH.sub.2 886 1409.82 705.91 706.04 959
Ac-LTF$er8HYWAQL$eS-NH.sub.2 887 1597.87 799.94 800.44 960
Ac-CCPGCCBaQSQQTF$r8NLWRLL$QN-NH.sub.2 888 2745.30 1373.65 1372.99
961 Ac-CCPGCCBaQSQQTA$r8NLWRLL$QN-NH.sub.2 889 2669.27 1335.64
1336.09 962 Ac-CCPGCCBaNlePRF$r8NYWRLL$QN-NH.sub.2 890 2589.26
1295.63 1296.2 963 Ac-LTF$/r8HYWAQLS/S-NH.sub.2 891 1625.90 813.95
814.18 964 Ac-F%r8HY6clWRAc3cL%-NH.sub.2 892 1372.72 687.36 687.59
965 Ac-QTF%r8HWWSQL%S-NH.sub.2 893 1653.87 827.94 827.94 966
Ac-LTA$r8HYWRQL$S-NH.sub.2 894 1606.90 804.45 804.66 967
Ac-Q$r8QQTFSN$WRLLAibQN-NH.sub.2 895 2080.12 1041.06 1041.61 968
Ac-QSQQ$r8FSNLWR$LAibQN-NH.sub.2 896 2066.11 1034.06 1034.58 969
Ac-F$r8AYWEAc3cL$A-NH.sub.2 897 1314.70 658.35 1315.88 970
Ac-F$r8AYWEAc3cL$S-NH.sub.2 898 1330.70 666.35 1331.87 971
Ac-F$r8AYWEAc3cL$Q-NH.sub.2 899 1371.72 686.86 1372.72 972
Ac-F$r8AYWEAibL$S-NH.sub.2 900 1332.71 667.36 1334.83 973
Ac-F$r8AYWEAL$S-NH.sub.2 901 1318.70 660.35 1319.73 974
Ac-F$r8AYWEQL$S-NH.sub.2 902 1375.72 688.86 1377.53 975
Ac-F$r8HYWEQL$S-NH.sub.2 903 1441.74 721.87 1443.48 976
Ac-F$r8HYWAQL$S-NH.sub.2 904 1383.73 692.87 1385.38 977
Ac-F$r8HYWAAc3cL$S-NH.sub.2 905 1338.71 670.36 1340.82 978
Ac-F$r8HYWRAc3cL$S-NH.sub.2 906 1423.78 712.89 713.04 979
Ac-F$r8AYWEAc3cL#A-NH.sub.2 907 1300.69 651.35 1302.78 980
Ac-NlePTF%r8NYWRLL%QN-NH.sub.2 908 1899.08 950.54 950.56 981
Ac-TF$r8AAWRAL$Q-NH.sub.2 909 1395.80 698.90 699.13 982
Ac-TSF%r8HYWAQL%S-NH.sub.2 910 1573.83 787.92 787.98 983
Ac-F%r8AY6clWEAc3cL%A-NH.sub.2 911 1350.68 676.34 676.91 984
Ac-LTF$r8HYWAQI$S-NH.sub.2 912 1597.87 799.94 800.07 985
Ac-LTF$r8HYWAQNle$S-NH.sub.2 913 1597.87 799.94 800.07 986
Ac-LTF$r8HYWAQL$A-NH.sub.2 914 1581.87 791.94 792.45 987
Ac-LTF$r8HYWAQL$Abu-NH.sub.2 915 1595.89 798.95 799.03 988
Ac-LTF$r8HYWAbuQL$S-NH.sub.2 916 1611.88 806.94 807.47 989
Ac-LTF$er8AYWAQL$eS-NH.sub.2 917 1531.84 766.92 766.96 990
Ac-LAF$r8HYWAQL$S-NH.sub.2 918 1567.86 784.93 785.49 991
Ac-LAF$r8AYWAQL$S-NH.sub.2 919 1501.83 751.92 752.01 992
Ac-LTF$er8AYWAQL$eA-NH.sub.2 920 1515.85 758.93 758.97 993
Ac-LAF$r8AYWAQL$A-NH.sub.2 921 1485.84 743.92 744.05 994
Ac-LTF$r8NLWANleL$Q-NH.sub.2 922 1550.92 776.46 776.61 995
Ac-LTF$r8NLWANleL$A-NH.sub.2 923 1493.90 747.95 1495.6 996
Ac-LTF$r8ALWANleL$Q-NH.sub.2 924 1507.92 754.96 755 997
Ac-LAF$r8NLWANleL$Q-NH.sub.2 925 1520.91 761.46 761.96 998
Ac-LAF$r8ALWANleL$A-NH.sub.2 926 1420.89 711.45 1421.74 999
Ac-A$r8AYWEAc3cL$A-NH.sub.2 927 1238.67 620.34 1239.65 1000
Ac-F$r8AYWEAc3cL$AA-NH.sub.2 928 1385.74 693.87 1386.64 1001
Ac-F$r8AYWEAc3cL$Abu-NH.sub.2 929 1328.72 665.36 1330.17 1013
Ac-F$r8AYWEAc3cL$Nle-NH.sub.2 930 1356.75 679.38 1358.22 1014
Ac-F$r5AYWEAc3cL$s8A-NH.sub.2 931 1314.70 658.35 1315.51 1015
Ac-F$AYWEAc3cL$r8A-NH.sub.2 932 1314.70 658.35 1315.66 1016
Ac-F$r8AYWEAc3cl$A-NH.sub.2 933 1314.70 658.35 1316.18 1017
Ac-F$r8AYWEAc3cNle$A-NH.sub.2 934 1314.70 658.35 1315.66 1018
Ac-F$r8AYWEAmlL$A-NH.sub.2 935 1358.76 680.38 1360.21 1019
Ac-F$r8AYWENleL$A-NH.sub.2 936 1344.75 673.38 1345.71 1020
Ac-F$r8AYWQAc3cL$A-NH.sub.2 937 1313.72 657.86 1314.7 1021
Ac-F$r8AYWAAc3cL$A-NH.sub.2 938 1256.70 629.35 1257.56 1022
Ac-F$r8AYWAbuAc3cL$A-NH.sub.2 939 1270.71 636.36 1272.14 1023
Ac-F$r8AYWNleAc3cL$A-NH.sub.2 940 1298.74 650.37 1299.67 1024
Ac-F$r8AbuYWEAc3cL$A-NH.sub.2 941 1328.72 665.36 1329.65 1025
Ac-F$r8NleYWEAc3cL$A-NH.sub.2 942 1356.75 679.38 1358.66 1026
5-FAM-BaLTFEHYWAQLTS-NH.sub.2 943 1922.82 962.41 962.87 1027
5-FAM-BaLTF%r8HYWAQL%S-NH.sub.2 944 1986.96 994.48 994.97 1028
Ac-LTF$r8HYWAQhL$S-NH.sub.2 945 1611.88 806.94 807 1029
Ac-LTF$r8HYWAQTle$S-NH.sub.2 946 1597.87 799.94 799.97 1030
Ac-LTF$r8HYWAQAdm$S-NH.sub.2 947 1675.91 838.96 839.09 1031
Ac-LTF$r8HYWAQhCha$S-NH.sub.2 948 1651.91 826.96 826.98 1032
Ac-LTF$r8HYWAQCha$S-NH.sub.2 949 1637.90 819.95 820.02 1033
Ac-LTF$r8HYWAc6cQL$S-NH.sub.2 950 1651.91 826.96 826.98 1034
Ac-LTF$r8HYWAc5cQL$S-NH.sub.2 951 1637.90 819.95 820.02 1035
Ac-LThF$r8HYWAQL$S-NH.sub.2 952 1611.88 806.94 807 1036
Ac-LTIgl$r8HYWAQL$S-NH.sub.2 953 1625.90 813.95 812.99 1037
Ac-LTF$r8HYWAQChg$S-NH.sub.2 954 1623.88 812.94 812.99 1038
Ac-LTF$r8HYWAQF$S-NH.sub.2 955 1631.85 816.93 816.99 1039
Ac-LTF$r8HYWAQIgl$S-NH.sub.2 956 1659.88 830.94 829.94 1040
Ac-LTF$r8HYWAQCba$S-NH.sub.2 957 1609.87 805.94 805.96 1041
Ac-LTF$r8HYWAQCpg$S-NH.sub.2 958 1609.87 805.94 805.96 1042
Ac-LTF$r8HhYWAQL$S-NH.sub.2 959 1611.88 806.94 807 1043
Ac-F$r8AYWEAc3chL$A-NH.sub.2 960 1328.72 665.36 665.43 1044
Ac-F$r8AYWEAc3cTle$A-NH.sub.2 961 1314.70 658.35 1315.62 1045
Ac-F$r8AYWEAc3cAdm$A-NH.sub.2 962 1392.75 697.38 697.47 1046
Ac-F$r8AYWEAc3chCha$A-NH.sub.2 963 1368.75 685.38 685.34 1047
Ac-F$r8AYWEAc3cCha$A-NH.sub.2 964 1354.73 678.37 678.38 1048
Ac-F$r8AYWEAc6cL$A-NH.sub.2 965 1356.75 679.38 679.42 1049
Ac-F$r8AYWEAc5cL$A-NH.sub.2 966 1342.73 672.37 672.46 1050
Ac-hF$r8AYWEAc3cL$A-NH.sub.2 967 1328.72 665.36 665.43 1051
Ac-Igl$r8AYWEAc3cL$A-NH.sub.2 968 1342.73 672.37 671.5 1052
Ac-F$r8AYWEAc3cF$A-NH.sub.2 969 1348.69 675.35 675.35 1053
Ac-F$r8AYWEAc3cIgl$A-NH.sub.2 970 1376.72 689.36 688.37 1054
Ac-F$r8AYWEAc3cCba$A-NH.sub.2 971 1326.70 664.35 664.47 1055
Ac-F$r8AYWEAc3cCpg$A-NH.sub.2 972 1326.70 664.35 664.39 1056
Ac-F$r8AhYWEAc3cL$A-NH.sub.2 973 1328.72 665.36 665.43 1057
Ac-F$r8AYWEAc3cL$Q-NH.sub.2 974 1371.72 686.86 1372.87 1058
Ac-F$r8AYWEAibL$A-NH.sub.2 975 1316.72 659.36 1318.18 1059
Ac-F$r8AYWEAL$A-NH.sub.2 976 1302.70 652.35 1303.75 1060
Ac-LAF$r8AYWAAL$A-NH.sub.2 977 1428.82 715.41 715.49 1061
Ac-LTF$r8HYWAAc3cL$S-NH.sub.2 978 1552.84 777.42 777.5 1062
Ac-NleTF$r8HYWAQL$S-NH.sub.2 979 1597.87 799.94 800.04 1063
Ac-VTF$r8HYWAQL$S-NH.sub.2 980 1583.85 792.93 793.04 1064
Ac-FTF$r8HYWAQL$S-NH.sub.2 981 1631.85 816.93 817.02 1065
Ac-WTF$r8HYWAQL$S-NH.sub.2 982 1670.86 836.43 836.85 1066
Ac-RTF$r8HYWAQL$S-NH.sub.2 983 1640.88 821.44 821.9
1067 Ac-KTF$r8HYWAQL$S-NH.sub.2 984 1612.88 807.44 807.91 1068
Ac-LNleF$r8HYWAQL$S-NH.sub.2 985 1609.90 805.95 806.43 1069
Ac-LVF$r8HYWAQL$S-NH.sub.2 986 1595.89 798.95 798.93 1070
Ac-LFF$r8HYWAQL$S-NH.sub.2 987 1643.89 822.95 823.38 1071
Ac-LWF$r8HYWAQL$S-NH.sub.2 988 1682.90 842.45 842.55 1072
Ac-LRF$r8HYWAQL$S-NH.sub.2 989 1652.92 827.46 827.52 1073
Ac-LKF$r8HYWAQL$S-NH.sub.2 990 1624.91 813.46 813.51 1074
Ac-LTF$r8NleYWAQL$S-NH.sub.2 991 1573.89 787.95 788.05 1075
Ac-LTF$r8VYWAQL$S-NH.sub.2 992 1559.88 780.94 780.98 1076
Ac-LTF$r8FYWAQL$S-NH.sub.2 993 1607.88 804.94 805.32 1077
Ac-LTF$r8WYWAQL$S-NH.sub.2 994 1646.89 824.45 824.86 1078
Ac-LTF$r8RYWAQL$S-NH.sub.2 995 1616.91 809.46 809.51 1079
Ac-LTF$r8KYWAQL$S-NH.sub.2 996 1588.90 795.45 795.48 1080
Ac-LTF$r8HNleWAQL$S-NH.sub.2 997 1547.89 774.95 774.98 1081
Ac-LTF$r8HVWAQL$S-NH.sub.2 998 1533.87 767.94 767.95 1082
Ac-LTF$r8HFWAQL$S-NH.sub.2 999 1581.87 791.94 792.3 1083
Ac-LTF$r8HWWAQL$S-NH.sub.2 1000 1620.88 811.44 811.54 1084
Ac-LTF$r8HRWAQL$S-NH.sub.2 1001 1590.90 796.45 796.52 1085
Ac-LTF$r8HKWAQL$S-NH.sub.2 1002 1562.90 782.45 782.53 1086
Ac-LTF$r8HYWNleQL$S-NH.sub.2 1003 1639.91 820.96 820.98 1087
Ac-LTF$r8HYWVQL$S-NH.sub.2 1004 1625.90 813.92 814.03 1088
Ac-LTF$r8HYWFQL$S-NH.sub.2 1005 1673.90 837.95 838.03 1089
Ac-LTF$r8HYWWQL$S-NH.sub.2 1006 1712.91 857.46 857.5 1090
Ac-LTF$r8HYWKQL$S-NH.sub.2 1007 1654.92 828.46 828.49 1091
Ac-LTF$r8HYWANleL$S-NH.sub.2 1008 1582.89 792.45 792.52 1092
Ac-LTF$r8HYWAVL$S-NH.sub.2 1009 1568.88 785.44 785.49 1093
Ac-LTF$r8HYWAFL$S-NH.sub.2 1010 1616.88 809.44 809.47 1094
Ac-LTF$r8HYWAWL$S-NH.sub.2 1011 1655.89 828.95 829 1095
Ac-LTF$r8HYWARL$S-NH.sub.2 1012 1625.91 813.96 813.98 1096
Ac-LTF$r8HYWAQL$Nle-NH.sub.2 1013 1623.92 812.96 813.39 1097
Ac-LTF$r8HYWAQL$V-NH.sub.2 1014 1609.90 805.95 805.99 1098
Ac-LTF$r8HYWAQL$F-NH.sub.2 1015 1657.90 829.95 830.26 1099
Ac-LTF$r8HYWAQL$W-NH.sub.2 1016 1696.91 849.46 849.5 1100
Ac-LTF$r8HYWAQL$R-NH.sub.2 1017 1666.94 834.47 834.56 1101
Ac-LTF$r8HYWAQL$K-NH.sub.2 1018 1638.93 820.47 820.49 1102
Ac-Q$r8QQTFSN$WRLLAibQN-NH.sub.2 1019 2080.12 1041.06 1041.54 1103
Ac-QSQQ$r8FSNLWR$LAibQN-NH.sub.2 1020 2066.11 1034.06 1034.58 1104
Ac-LT2Pal$r8HYWAQL$S-NH.sub.2 1021 1598.86 800.43 800.49 1105
Ac-LT3Pal$r8HYWAQL$S-NH.sub.2 1022 1598.86 800.43 800.49 1106
Ac-LT4Pal$r8HYWAQL$S-NH.sub.2 1023 1598.86 800.43 800.49 1107
Ac-LTF2CF3$r8HYWAQL$S-NH.sub.2 1024 1665.85 833.93 834.01 1108
Ac-LTF2CN$r8HYWAQL$S-NH.sub.2 1025 1622.86 812.43 812.47 1109
Ac-LTF2Me$r8HYWAQL$S-NH.sub.2 1026 1611.88 806.94 807 1110
Ac-LTF3Cl$r8HYWAQL$S-NH.sub.2 1027 1631.83 816.92 816.99 1111
Ac-LTF4CF3$r8HYWAQL$S-NH.sub.2 1028 1665.85 833.93 833.94 1113
Ac-LTF4tBu$r8HYWAQL$S-NH.sub.2 1029 1653.93 827.97 828.02 1114
Ac-LTF5F$r8HYWAQL$S-NH.sub.2 1030 1687.82 844.91 844.96 1115
Ac-LTF$r8HY3BthAAQL$S-NH.sub.2 1031 1614.83 808.42 808.48 1116
Ac-LTF2Br$r8HYWAQL$S-NH.sub.2 1032 1675.78 838.89 838.97 1117
Ac-LTF4Br$r8HYWAQL$S-NH.sub.2 1033 1675.78 838.89 839.86 1118
Ac-LTF2Cl$r8HYWAQL$S-NH.sub.2 1034 1631.83 816.92 816.99 1119
Ac-LTF4Cl$r8HYWAQL$S-NH.sub.2 1035 1631.83 816.92 817.36 1120
Ac-LTF3CN$r8HYWAQL$S-NH.sub.2 1036 1622.86 812.43 812.47 1121
Ac-LTF4CN$r8HYWAQL$S-NH.sub.2 1037 1622.86 812.43 812.47 1122
Ac-LTF34Cl2$r8HYWAQL$S-NH.sub.2 1038 1665.79 833.90 833.94 1123
Ac-LTF34F2$r8HYWAQL$S-NH.sub.2 1039 1633.85 817.93 817.95 1124
Ac-LTF35F2$r8HYWAQL$S-NH.sub.2 1040 1633.85 817.93 817.95 1125
Ac-LTDip$r8HYWAQL$S-NH.sub.2 1041 1673.90 837.95 838.01 1126
Ac-LTF2F$r8HYWAQL$S-NH.sub.2 1042 1615.86 808.93 809 1127
Ac-LTF3F$r8HYWAQL$S-NH.sub.2 1043 1615.86 808.93 809 1128
Ac-LTF4F$r8HYWAQL$S-NH.sub.2 1044 1615.86 808.93 809 1129
Ac-LTF4I$r8HYWAQL$S-NH.sub.2 1045 1723.76 862.88 862.94 1130
Ac-LTF3Me$r8HYWAQL$S-NH.sub.2 1046 1611.88 806.94 807.07 1131
Ac-LTF4Me$r8HYWAQL$S-NH.sub.2 1047 1611.88 806.94 807 1132
Ac-LT1Nal$r8HYWAQL$S-NH.sub.2 1048 1647.88 824.94 824.98 1133
Ac-LT2Nal$r8HYWAQL$S-NH.sub.2 1049 1647.88 824.94 825.06 1134
Ac-LTF3CF3$r8HYWAQL$S-NH.sub.2 1050 1665.85 833.93 834.01 1135
Ac-LTF4NO2$r8HYWAQL$S-NH.sub.2 1051 1642.85 822.43 822.46 1136
Ac-LTF3NO2$r8HYWAQL$S-NH.sub.2 1052 1642.85 822.43 822.46 1137
Ac-LTF$r82ThiYWAQL$S-NH.sub.2 1053 1613.83 807.92 807.96 1138
Ac-LTF$r8HBipWAQL$S-NH.sub.2 1054 1657.90 829.95 830.01 1139
Ac-LTF$r8HF4tBuWAQL$S-NH.sub.2 1055 1637.93 819.97 820.02 1140
Ac-LTF$r8HF4CF3WAQL$S-NH.sub.2 1056 1649.86 825.93 826.02 1141
Ac-LTF$r8HF4ClWAQL$S-NH.sub.2 1057 1615.83 808.92 809.37 1142
Ac-LTF$r8HF4MeWAQL$S-NH.sub.2 1058 1595.89 798.95 799.01 1143
Ac-LTF$r8HF4BrWAQL$S-NH.sub.2 1059 1659.78 830.89 830.98 1144
Ac-LTF$r8HF4CNWAQL$S-NH.sub.2 1060 1606.87 804.44 804.56 1145
Ac-LTF$r8HF4NO2WAQL$S-NH.sub.2 1061 1626.86 814.43 814.55 1146
Ac-LTF$r8H1NalWAQL$S-NH.sub.2 1062 1631.89 816.95 817.06 1147
Ac-LTF$r8H2NalWAQL$S-NH.sub.2 1063 1631.89 816.95 816.99 1148
Ac-LTF$r8HWAQL$S-NH.sub.2 1064 1434.80 718.40 718.49 1149
Ac-LTF$r8HY1NalAQL$S-NH.sub.2 1065 1608.87 805.44 805.52 1150
Ac-LTF$r8HY2NalAQL$S-NH.sub.2 1066 1608.87 805.44 805.52 1151
Ac-LTF$r8HYWAQI$S-NH.sub.2 1067 1597.87 799.94 800.07 1152
Ac-LTF$r8HYWAQNle$S-NH.sub.2 1068 1597.87 799.94 800.44 1153
Ac-LTF$er8HYWAQL$eA-NH.sub.2 1069 1581.87 791.94 791.98 1154
Ac-LTF$r8HYWAQL$Abu-NH.sub.2 1070 1595.89 798.95 799.03 1155
Ac-LTF$r8HYWAbuQL$S-NH.sub.2 1071 1611.88 806.94 804.47 1156
Ac-LAF$r8HYWAQL$S-NH.sub.2 1072 1567.86 784.93 785.49 1157
Ac-LTF$r8NLWANleL$Q-NH.sub.2 1073 1550.92 776.46 777.5 1158
Ac-LTF$r8ALWANleL$Q-NH.sub.2 1074 1507.92 754.96 755.52 1159
Ac-LAF$r8NLWANleL$Q-NH.sub.2 1075 1520.91 761.46 762.48 1160
Ac-F$r8AYWAAc3cL$A-NH.sub.2 1076 1256.70 629.35 1257.56 1161
Ac-LTF$r8AYWAAL$S-NH.sub.2 1077 1474.82 738.41 738.55 1162
Ac-LVF$r8AYWAQL$S-NH.sub.2 1078 1529.87 765.94 766 1163
Ac-LTF$r8AYWAbuQL$S-NH.sub.2 1079 1545.86 773.93 773.92 1164
Ac-LTF$r8AYWNleQL$S-NH.sub.2 1080 1573.89 787.95 788.17 1165
Ac-LTF$r8AbuYWAQL$S-NH.sub.2 1081 1545.86 773.93 773.99 1166
Ac-LTF$r8AYWHQL$S-NH.sub.2 1082 1597.87 799.94 799.97 1167
Ac-LTF$r8AYWKQL$S-NH.sub.2 1083 1588.90 795.45 795.53 1168
Ac-LTF$r8AYWOQL$S-NH.sub.2 1084 1574.89 788.45 788.5 1169
Ac-LTF$r8AYWRQL$S-NH.sub.2 1085 1616.91 809.46 809.51 1170
Ac-LTF$r8AYWSQL$S-NH.sub.2 1086 1547.84 774.92 774.96 1171
Ac-LTF$r8AYWRAL$S-NH.sub.2 1087 1559.89 780.95 780.95 1172
Ac-LTF$r8AYWRQL$A-NH.sub.2 1088 1600.91 801.46 801.52 1173
Ac-LTF$r8AYWRAL$A-NH.sub.2 1089 1543.89 772.95 773.03 1174
Ac-LTF$r5HYWAQL$s8S-NH.sub.2 1090 1597.87 799.94 799.97 1175
Ac-LTF$HYWAQL$r8S-NH.sub.2 1091 1597.87 799.94 799.97 1176
Ac-LTF$r8HYWAAL$S-NH.sub.2 1092 1540.84 771.42 771.48 1177
Ac-LTF$r8HYWAAbuL$S-NH.sub.2 1093 1554.86 778.43 778.51 1178
Ac-LTF$r8HYWALL$S-NH.sub.2 1094 1582.89 792.45 792.49 1179
Ac-F$r8AYWHAL$A-NH.sub.2 1095 1310.72 656.36 656.4 1180
Ac-F$r8AYWAAL$A-NH.sub.2 1096 1244.70 623.35 1245.61 1181
Ac-F$r8AYWSAL$A-NH.sub.2 1097 1260.69 631.35 1261.6 1182
Ac-F$r8AYWRAL$A-NH.sub.2 1098 1329.76 665.88 1330.72 1183
Ac-F$r8AYWKAL$A-NH.sub.2 1099 1301.75 651.88 1302.67 1184
Ac-F$r8AYWOAL$A-NH.sub.2 1100 1287.74 644.87 1289.13 1185
Ac-F$r8VYWEAc3cL$A-NH.sub.2 1101 1342.73 672.37 1343.67 1186
Ac-F$r8FYWEAc3cL$A-NH.sub.2 1102 1390.73 696.37 1392.14 1187
Ac-F$r8WYWEAc3cL$A-NH.sub.2 1103 1429.74 715.87 1431.44 1188
Ac-F$r8RYWEAc3cL$A-NH.sub.2 1104 1399.77 700.89 700.95 1189
Ac-F$r8KYWEAc3cL$A-NH.sub.2 1105 1371.76 686.88 686.97 1190
Ac-F$r8ANleWEAc3cL$A-NH.sub.2 1106 1264.72 633.36 1265.59 1191
Ac-F$r8AVWEAc3cL$A-NH.sub.2 1107 1250.71 626.36 1252.2 1192
Ac-F$r8AFWEAc3cL$A-NH.sub.2 1108 1298.71 650.36 1299.64 1193
Ac-F$r8AWWEAc3cL$A-NH.sub.2 1109 1337.72 669.86 1338.64 1194
Ac-F$r8ARWEAc3cL$A-NH.sub.2 1110 1307.74 654.87 655 1195
Ac-F$r8AKWEAc3cL$A-NH.sub.2 1111 1279.73 640.87 641.01 1196
Ac-F$r8AYWVAc3cL$A-NH.sub.2 1112 1284.73 643.37 643.38 1197
Ac-F$r8AYWFAc3cL$A-NH.sub.2 1113 1332.73 667.37 667.43 1198
Ac-F$r8AYWWAc3cL$A-NH.sub.2 1114 1371.74 686.87 686.97 1199
Ac-F$r8AYWRAc3cL$A-NH.sub.2 1115 1341.76 671.88 671.94 1200
Ac-F$r8AYWKAc3cL$A-NH.sub.2 1116 1313.75 657.88 657.88 1201
Ac-F$r8AYWEVL$A-NH.sub.2 1117 1330.73 666.37 666.47 1202
Ac-F$r8AYWEFL$A-NH.sub.2 1118 1378.73 690.37 690.44 1203
Ac-F$r8AYWEWL$A-NH.sub.2 1119 1417.74 709.87 709.91 1204
Ac-F$r8AYWERL$A-NH.sub.2 1120 1387.77 694.89 1388.66 1205
Ac-F$r8AYWEKL$A-NH.sub.2 1121 1359.76 680.88 1361.21 1206
Ac-F$r8AYWEAc3cL$V-NH.sub.2 1122 1342.73 672.37 1343.59 1207
Ac-F$r8AYWEAc3cL$F-NH.sub.2 1123 1390.73 696.37 1392.58 1208
Ac-F$r8AYWEAc3cL$W-NH.sub.2 1124 1429.74 715.87 1431.29 1209
Ac-F$r8AYWEAc3cL$R-NH.sub.2 1125 1399.77 700.89 700.95 1210
Ac-F$r8AYWEAc3cL$K-NH.sub.2 1126 1371.76 686.88 686.97 1211
Ac-F$r8AYWEAc3cL$AV-NH.sub.2 1127 1413.77 707.89 707.91 1212
Ac-F$r8AYWEAc3cL$AF-NH.sub.2 1128 1461.77 731.89 731.96 1213
Ac-F$r8AYWEAc3cL$AW-NH.sub.2 1129 1500.78 751.39 751.5 1214
Ac-F$r8AYWEAc3cL$AR-NH.sub.2 1130 1470.80 736.40 736.47 1215
Ac-F$r8AYWEAc3cL$AK-NH.sub.2 1131 1442.80 722.40 722.41 1216
Ac-F$r8AYWEAc3cL$AH-NH.sub.2 1132 1451.76 726.88 726.93 1217
Ac-LTF2NO2$r8HYWAQL$S-NH.sub.2 1133 1642.85 822.43 822.54 1218
Ac-LTA$r8HYAAQL$S-NH.sub.2 1134 1406.79 704.40 704.5 1219
Ac-LTF$r8HYAAQL$S-NH.sub.2 1135 1482.82 742.41 742.47 1220
Ac-QSQQTF$r8NLWALL$AN-NH.sub.2 1136 1966.07 984.04 984.38 1221
Ac-QAibQQTF$r8NLWALL$AN-NH.sub.2 1137 1964.09 983.05 983.42 1222
Ac-QAibQQTF$r8ALWALL$AN-NH.sub.2 1138 1921.08 961.54 961.59 1223
Ac-AAAATF$r8AAWAAL$AA-NH.sub.2 1139 1608.90 805.45 805.52 1224
Ac-F$r8AAWRAL$Q-NH.sub.2 1140 1294.76 648.38 648.48 1225
Ac-TF$r8AAWAAL$Q-NH.sub.2 1141 1310.74 656.37 1311.62 1226
Ac-TF$r8AAWRAL$A-NH.sub.2 1142 1338.78 670.39 670.46 1227
Ac-VF$r8AAWRAL$Q-NH.sub.2 1143 1393.82 697.91 697.99 1228
Ac-AF$r8AAWAAL$A-NH.sub.2 1144 1223.71 612.86 1224.67 1229
Ac-TF$r8AAWKAL$Q-NH.sub.2 1145 1367.80 684.90 684.97 1230
Ac-TF$r8AAWOAL$Q-NH.sub.2 1146 1353.78 677.89 678.01 1231
Ac-TF$r8AAWSAL$Q-NH.sub.2 1147 1326.73 664.37 664.47 1232
Ac-LTF$r8AAWRAL$Q-NH.sub.2 1148 1508.89 755.45 755.49 1233
Ac-F$r8AYWAQL$A-NH.sub.2 1149 1301.72 651.86 651.96 1234
Ac-F$r8AWWAAL$A-NH.sub.2 1150 1267.71 634.86 634.87 1235
Ac-F$r8AWWAQL$A-NH.sub.2 1151 1324.73 663.37 663.43 1236
Ac-F$r8AYWEAL$-NH.sub.2 1152 1231.66 616.83 1232.93 1237
Ac-F$r8AYWAAL$-NH.sub.2 1153 1173.66 587.83 1175.09 1238
Ac-F$r8AYWKAL$-NH.sub.2 1154 1230.72 616.36 616.44 1239
Ac-F$r8AYWOAL$-NH.sub.2 1155 1216.70 609.35 609.48 1240
Ac-F$r8AYWQAL$-NH.sub.2 1156 1230.68 616.34 616.44 1241
Ac-F$r8AYWAQL$-NH.sub.2 1157 1230.68 616.34 616.37 1242
Ac-F$r8HYWDQL$S-NH.sub.2 1158 1427.72 714.86 714.86 1243
Ac-F$r8HFWEQL$S-NH.sub.2 1159 1425.74 713.87 713.98 1244
Ac-F$r8AYWHQL$S-NH.sub.2 1160 1383.73 692.87 692.96 1245
Ac-F$r8AYWKQL$S-NH.sub.2 1161 1374.77 688.39 688.45 1246
Ac-F$r8AYWOQL$S-NH.sub.2 1162 1360.75 681.38 681.49 1247
Ac-F$r8HYWSQL$S-NH.sub.2 1163 1399.73 700.87 700.95 1248
Ac-F$r8HWWEQL$S-NH.sub.2 1164 1464.76 733.38 733.44 1249
Ac-F$r8HWWAQL$S-NH.sub.2 1165 1406.75 704.38 704.43 1250
Ac-F$r8AWWHQL$S-NH.sub.2 1166 1406.75 704.38 704.43 1251
Ac-F$r8AWWKQL$S-NH.sub.2 1167 1397.79 699.90 699.92 1252
Ac-F$r8AWWOQL$S-NH.sub.2 1168 1383.77 692.89 692.96 1253
Ac-F$r8HWWSQL$S-NH.sub.2 1169 1422.75 712.38 712.42 1254
Ac-LTF$r8NYWANleL$Q-NH.sub.2 1170 1600.90 801.45 801.52 1255
Ac-LTF$r8NLWAQL$Q-NH.sub.2 1171 1565.90 783.95 784.06 1256
Ac-LTF$r8NYWANleL$A-NH.sub.2 1172 1543.88 772.94 773.03 1257
Ac-LTF$r8NLWAQL$A-NH.sub.2 1173 1508.88 755.44 755.49 1258
Ac-LTF$r8AYWANleL$Q-NH.sub.2 1174 1557.90 779.95 780.06 1259
Ac-LTF$r8ALWAQL$Q-NH.sub.2 1175 1522.89 762.45 762.45 1260
Ac-LAF$r8NYWANleL$Q-NH.sub.2 1176 1570.89 786.45 786.5 1261
Ac-LAF$r8NLWAQL$Q-NH.sub.2 1177 1535.89 768.95 769.03 1262
Ac-LAF$r8AYWANleL$A-NH.sub.2 1178 1470.86 736.43 736.47 1263
Ac-LAF$r8ALWAQL$A-NH.sub.2 1179 1435.86 718.93 719.01 1264
Ac-LAF$r8AYWAAL$A-NH.sub.2 1180 1428.82 715.41 715.41 1265
Ac-F$r8AYWEAc3cL$AAib-NH.sub.2 1181 1399.75 700.88 700.95 1266
Ac-F$r8AYWAQL$AA-NH.sub.2 1182 1372.75 687.38 687.78 1267
Ac-F$r8AYWAAc3cL$AA-NH.sub.2 1183 1327.73 664.87 664.84 1268
Ac-F$r8AYWSAc3cL$AA-NH.sub.2 1184 1343.73 672.87 672.9 1269
Ac-F$r8AYWEAc3cL$AS-NH.sub.2 1185 1401.73 701.87 701.84 1270
Ac-F$r8AYWEAc3cL$AT-NH.sub.2 1186 1415.75 708.88 708.87 1271
Ac-F$r8AYWEAc3cL$AL-NH.sub.2 1187 1427.79 714.90 714.94 1272
Ac-F$r8AYWEAc3cL$AQ-NH.sub.2 1188 1442.76 722.38 722.41 1273
Ac-F$r8AFWEAc3cL$AA-NH.sub.2 1189 1369.74 685.87 685.93 1274
Ac-F$r8AWWEAc3cL$AA-NH.sub.2 1190 1408.75 705.38 705.39 1275
Ac-F$r8AYWEAc3cL$SA-NH.sub.2 1191 1401.73 701.87 701.99 1276
Ac-F$r8AYWEAL$AA-NH.sub.2 1192 1373.74 687.87 687.93 1277
Ac-F$r8AYWENleL$AA-NH.sub.2 1193 1415.79 708.90 708.94 1278
Ac-F$r8AYWEAc3cL$AbuA-NH.sub.2 1194 1399.75 700.88 700.95 1279
Ac-F$r8AYWEAc3cL$NleA-NH.sub.2 1195 1427.79 714.90 714.86 1280
Ac-F$r8AYWEAibL$NleA-NH.sub.2 1196 1429.80 715.90 715.97 1281
Ac-F$r8AYWEAL$NleA-NH.sub.2 1197 1415.79 708.90 708.94 1282
Ac-F$r8AYWENleL$NleA-NH.sub.2 1198 1457.83 729.92 729.96 1283
Ac-F$r8AYWEAibL$Abu-NH.sub.2 1199 1330.73 666.37 666.39 1284
Ac-F$r8AYWENleL$Abu-NH.sub.2 1200 1358.76 680.38 680.39 1285
Ac-F$r8AYWEAL$Abu-NH.sub.2 1201 1316.72 659.36 659.36 1286
Ac-LTF$r8AFWAQL$S-NH.sub.2 1202 1515.85 758.93 759.12 1287
Ac-LTF$r8AWWAQL$S-NH.sub.2 1203 1554.86 778.43 778.51 1288
Ac-LTF$r8AYWAQI$S-NH.sub.2 1204 1531.84 766.92 766.96 1289
Ac-LTF$r8AYWAQNle$S-NH.sub.2 1205 1531.84 766.92 766.96 1290
Ac-LTF$r8AYWAQL$SA-NH.sub.2 1206 1602.88 802.44 802.48 1291
Ac-LTF$r8AWWAQL$A-NH.sub.2 1207 1538.87 770.44 770.89 1292
Ac-LTF$r8AYWAQI$A-NH.sub.2 1208 1515.85 758.93 759.42 1293
Ac-LTF$r8AYWAQNle$A-NH.sub.2 1209 1515.85 758.93 759.42 1294
Ac-LTF$r8AYWAQL$AA-NH.sub.2 1210 1586.89 794.45 794.94 1295
Ac-LTF$r8HWWAQL$S-NH.sub.2 1211 1620.88 811.44 811.47 1296
Ac-LTF$r8HRWAQL$S-NH.sub.2 1212 1590.90 796.45 796.52 1297
Ac-LTF$r8HKWAQL$S-NH.sub.2 1213 1562.90 782.45 782.53 1298
Ac-LTF$r8HYWAQL$W-NH.sub.2 1214 1696.91 849.46 849.5 1299
Ac-F$r8AYWAbuAL$A-NH.sub.2 1215 1258.71 630.36 630.5 1300
Ac-F$r8AbuYWEAL$A-NH.sub.2 1216 1316.72 659.36 659.51 1301
Ac-NlePRF%r8NYWRLL%QN-NH.sub.2 1217 1954.13 978.07 978.54 1302
Ac-TSF%r8HYWAQL%S-NH.sub.2 1218 1573.83 787.92 787.98 1303
Ac-LTF%r8AYWAQL%S-NH.sub.2 1219 1533.86 767.93 768 1304
Ac-HTF$r8HYWAQL$S-NH.sub.2 1220 1621.84 811.92 811.96 1305
Ac-LHF$r8HYWAQL$S-NH.sub.2 1221 1633.88 817.94 818.02 1306
Ac-LTF$r8HHWAQL$S-NH.sub.2 1222 1571.86 786.93 786.94 1307
Ac-LTF$r8HYWHQL$S-NH.sub.2 1223 1663.89 832.95 832.38 1308
Ac-LTF$r8HYWAHL$S-NH.sub.2 1224 1606.87 804.44 804.48 1309
Ac-LTF$r8HYWAQL$H-NH.sub.2 1225 1647.89 824.95 824.98 1310
Ac-LTF$r8HYWAQL$S-NHPr 1226 1639.91 820.96 820.98 1311
Ac-LTF$r8HYWAQL$S-NHsBu 1227 1653.93 827.97 828.02 1312
Ac-LTF$r8HYWAQL$S-NHiBu 1228 1653.93 827.97 828.02 1313
Ac-LTF$r8HYWAQL$S-NHBn 1229 1687.91 844.96 844.44 1314
Ac-LTF$r8HYWAQL$S-NHPe 1230 1700.92 851.46 851.99 1315
Ac-LTF$r8HYWAQL$S-NHChx 1231 1679.94 840.97 841.04 1316
Ac-ETF$r8AYWAQL$S-NH.sub.2 1232 1547.80 774.90 774.96 1317
Ac-STF$r8AYWAQL$S-NH.sub.2 1233 1505.79 753.90 753.94 1318
Ac-LEF$r8AYWAQL$S-NH.sub.2 1234 1559.84 780.92 781.25
1319 Ac-LSF$r8AYWAQL$S-NH.sub.2 1235 1517.83 759.92 759.93 1320
Ac-LTF$r8EYWAQL$S-NH.sub.2 1236 1589.85 795.93 795.97 1321
Ac-LTF$r8SYWAQL$S-NH.sub.2 1237 1547.84 774.92 774.96 1322
Ac-LTF$r8AYWEQL$S-NH.sub.2 1238 1589.85 795.93 795.9 1323
Ac-LTF$r8AYWAEL$S-NH.sub.2 1239 1532.83 767.42 766.96 1324
Ac-LTF$r8AYWASL$S-NH.sub.2 1240 1490.82 746.41 746.46 1325
Ac-LTF$r8AYWAQL$E-NH.sub.2 1241 1573.85 787.93 787.98 1326
Ac-LTF2CN$r8HYWAQL$S-NH.sub.2 1242 1622.86 812.43 812.47 1327
Ac-LTF3Cl$r8HYWAQL$S-NH.sub.2 1243 1631.83 816.92 816.99 1328
Ac-LTDip$r8HYWAQL$S-NH.sub.2 1244 1673.90 837.95 838.01 1329
Ac-LTF$r8HYWAQTle$S-NH.sub.2 1245 1597.87 799.94 800.04 1330
Ac-F$r8AY6clWEAL$A-NH.sub.2 1246 1336.66 669.33 1338.56 1331
Ac-F$r8AYdl6brWEAL$A-NH.sub.2 1247 1380.61 691.31 692.2 1332
Ac-F$r8AYdl6fWEAL$A-NH.sub.2 1248 1320.69 661.35 1321.61 1333
Ac-F$r8AYdl4mWEAL$A-NH.sub.2 1249 1316.72 659.36 659.36 1334
Ac-F$r8AYdl5clWEAL$A-NH.sub.2 1250 1336.66 669.33 669.35 1335
Ac-F$r8AYdl7mWEAL$A-NH.sub.2 1251 1316.72 659.36 659.36 1336
Ac-LTF%r8HYWAQL%A-NH.sub.2 1252 1583.89 792.95 793.01 1337
Ac-LTF$r8HCouWAQL$S-NH.sub.2 1253 1679.87 840.94 841.38 1338
Ac-LTFEHCouWAQLTS-NH.sub.2 1254 1617.75 809.88 809.96 1339
Ac-LTA$r8HCouWAQL$S-NH.sub.2 1255 1603.84 802.92 803.36 1340
Ac-F$r8AYWEAL$AbuA-NH.sub.2 1256 1387.75 694.88 694.88 1341
Ac-F$r8AYWEAI$AA-NH.sub.2 1257 1373.74 687.87 687.93 1342
Ac-F$r8AYWEANle$AA-NH.sub.2 1258 1373.74 687.87 687.93 1343
Ac-F$r8AYWEAmlL$AA-NH.sub.2 1259 1429.80 715.90 715.97 1344
Ac-F$r8AYWQAL$AA-NH.sub.2 1260 1372.75 687.38 687.48 1345
Ac-F$r8AYWAAL$AA-NH.sub.2 1261 1315.73 658.87 658.92 1346
Ac-F$r8AYWAbuAL$AA-NH.sub.2 1262 1329.75 665.88 665.95 1347
Ac-F$r8AYWNleAL$AA-NH.sub.2 1263 1357.78 679.89 679.94 1348
Ac-F$r8AbuYWEAL$AA-NH.sub.2 1264 1387.75 694.88 694.96 1349
Ac-F$r8NleYWEAL$AA-NH.sub.2 1265 1415.79 708.90 708.94 1350
Ac-F$r8FYWEAL$AA-NH.sub.2 1266 1449.77 725.89 725.97 1351
Ac-LTF$r8HYWAQhL$S-NH.sub.2 1267 1611.88 806.94 807 1352
Ac-LTF$r8HYWAQAdm$S-NH.sub.2 1268 1675.91 838.96 839.04 1353
Ac-LTF$r8HYWAQIgl$S-NH.sub.2 1269 1659.88 830.94 829.94 1354
Ac-F$r8AYWAQL$AA-NH.sub.2 1270 1372.75 687.38 687.48 1355
Ac-LTF$r8ALWAQL$Q-NH.sub.2 1271 1522.89 762.45 762.52 1356
Ac-F$r8AYWEAL$AA-NH.sub.2 1272 1373.74 687.87 687.93 1357
Ac-F$r8AYWENleL$AA-NH.sub.2 1273 1415.79 708.90 708.94 1358
Ac-F$r8AYWEAibL$Abu-NH.sub.2 1274 1330.73 666.37 666.39 1359
Ac-F$r8AYWENleL$Abu-NH.sub.2 1275 1358.76 680.38 680.38 1360
Ac-F$r8AYWEAL$Abu-NH.sub.2 1276 1316.72 659.36 659.36 1361
Ac-F$r8AYWEAc3cL$AbuA-NH.sub.2 1277 1399.75 700.88 700.95 1362
Ac-F$r8AYWEAc3cL$NleA-NH.sub.2 1278 1427.79 714.90 715.01 1363
H-LTF$r8AYWAQL$S-NH.sub.2 1279 1489.83 745.92 745.95 1364
mdPEG3-LTF$r8AYWAQL$S-NH.sub.2 1280 1679.92 840.96 840.97 1365
mdPEG7-LTF$r8AYWAQL$S-NH.sub.2 1281 1856.02 929.01 929.03 1366
Ac-F$r8ApmpEt6clWEAL$A-NH.sub.2 1282 1470.71 736.36 788.17 1367
Ac-LTF3Cl$r8AYWAQL$S-NH.sub.2 1283 1565.81 783.91 809.18 1368
Ac-LTF3Cl$r8HYWAQL$A-NH.sub.2 1284 1615.83 808.92 875.24 1369
Ac-LTF3Cl$r8HYWWQL$S-NH.sub.2 1285 1746.87 874.44 841.65 1370
Ac-LTF3Cl$r8AYWWQL$S-NH.sub.2 1286 1680.85 841.43 824.63 1371
Ac-LTF$r8AYWWQL$S-NH.sub.2 1287 1646.89 824.45 849.98 1372
Ac-LTF$r8HYWWQL$A-NH.sub.2 1288 1696.91 849.46 816.67 1373
Ac-LTF$r8AYWWQL$A-NH.sub.2 1289 1630.89 816.45 776.15 1374
Ac-LTF4F$r8AYWAQL$S-NH.sub.2 1290 1549.83 775.92 776.15 1375
Ac-LTF2F$r8AYWAQL$S-NH.sub.2 1291 1549.83 775.92 776.15 1376
Ac-LTF3F$r8AYWAQL$S-NH.sub.2 1292 1549.83 775.92 785.12 1377
Ac-LTF34F2$r8AYWAQL$S-NH.sub.2 1293 1567.83 784.92 785.12 1378
Ac-LTF35F2$r8AYWAQL$S-NH.sub.2 1294 1567.83 784.92 1338.74 1379
Ac-F3Cl$r8AYWEAL$A-NH.sub.2 1295 1336.66 669.33 705.28 1380
Ac-F3Cl$r8AYWEAL$AA-NH.sub.2 1296 1407.70 704.85 680.11 1381
Ac-F$r8AY6clWEAL$AA-NH.sub.2 1297 1407.70 704.85 736.83 1382
Ac-F$r8AY6clWEAL$-NH.sub.2 1298 1265.63 633.82 784.1 1383
Ac-LTF$r8HYWAQLSt/S-NH.sub.2 1299 16.03 9.02 826.98 1384
Ac-LTF$r8HYWAQL$S-NHsBu 1300 1653.93 827.97 828.02 1385
Ac-STF$r8AYWAQL$S-NH.sub.2 1301 1505.79 753.90 753.94 1386
Ac-LTF$r8AYWAEL$S-NH.sub.2 1302 1532.83 767.42 767.41 1387
Ac-LTF$r8AYWAQL$E-NH.sub.2 1303 1573.85 787.93 787.98 1388
mdPEG3-LTF$r8AYWAQL$S-NH.sub.2 1304 1679.92 840.96 840.97 1389
Ac-LTF$r8AYWAQhL$S-NH.sub.2 1305 1545.86 773.93 774.31 1390
Ac-LTF$r8AYWAQCha$S-NH.sub.2 1306 1571.88 786.94 787.3 1391
Ac-LTF$r8AYWAQChg$S-NH.sub.2 1307 1557.86 779.93 780.4 1392
Ac-LTF$r8AYWAQCba$S-NH.sub.2 1308 1543.84 772.92 780.13 1393
Ac-LTF$r8AYWAQF$S-NH.sub.2 1309 1565.83 783.92 784.2 1394
Ac-LTF4F$r8HYWAQhL$S-NH.sub.2 1310 1629.87 815.94 815.36 1395
Ac-LTF4F$r8HYWAQCha$S-NH.sub.2 1311 1655.89 828.95 828.39 1396
Ac-LTF4F$r8HYWAQChg$S-NH.sub.2 1312 1641.87 821.94 821.35 1397
Ac-LTF4F$r8HYWAQCba$S-NH.sub.2 1313 1627.86 814.93 814.32 1398
Ac-LTF4F$r8AYWAQhL$S-NH.sub.2 1314 1563.85 782.93 782.36 1399
Ac-LTF4F$r8AYWAQCha$S-NH.sub.2 1315 1589.87 795.94 795.38 1400
Ac-LTF4F$r8AYWAQChg$S-NH.sub.2 1316 1575.85 788.93 788.35 1401
Ac-LTF4F$r8AYWAQCba$S-NH.sub.2 1317 1561.83 781.92 781.39 1402
Ac-LTF3Cl$r8AYWAQhL$S-NH.sub.2 1318 1579.82 790.91 790.35 1403
Ac-LTF3Cl$r8AYWAQCha$S-NH.sub.2 1319 1605.84 803.92 803.67 1404
Ac-LTF3Cl$r8AYWAQChg$S-NH.sub.2 1320 1591.82 796.91 796.34 1405
Ac-LTF3Cl$r8AYWAQCba$S-NH.sub.2 1321 1577.81 789.91 789.39 1406
Ac-LTF$r8AYWAQhF$S-NH.sub.2 1322 1579.84 790.92 791.14 1407
Ac-LTF$r8AYWAQF3CF3$S-NH.sub.2 1323 1633.82 817.91 818.15 1408
Ac-LTF$r8AYWAQF3Me$S-NH.sub.2 1324 1581.86 791.93 791.32 1409
Ac-LTF$r8AYWAQ1Nal$S-NH.sub.2 1325 1615.84 808.92 809.18 1413
Ac-LTF$r8AYWAQBip$S-NH.sub.2 1326 1641.86 821.93 822.13 1414
Ac-LTF$r8FYWAQL$A-NH.sub.2 1327 1591.88 796.94 797.33 1415
Ac-LTF$r8HYWAQL$S-NHAm 1328 1667.94 834.97 835.92 1416
Ac-LTF$r8HYWAQL$S-NHiAm 1329 1667.94 834.97 835.55 1417
Ac-LTF$r8HYWAQL$S-NHnPr3Ph 1330 1715.94 858.97 859.79 1418
Ac-LTF$r8HYWAQL$S-NHnBu3,3Me 1331 1681.96 841.98 842.49 1419
Ac-LTF$r8HYWAQL$S-NHnPr 1332 1639.91 820.96 821.58 1420
Ac-LTF$r8HYWAQL$S-NHnEt2Ch 1333 1707.98 854.99 855.35 1421
Ac-LTF$r8HYWAQL$S-NHHex 1334 1681.96 841.98 842.4 1422
Ac-LTF$r8AYWAQL$S-NHmdPeg2 1335 1633.91 817.96 818.35 1423
Ac-LTF$r8AYWAQL$A-NHmdPeg2 1336 1617.92 809.96 810.3 1424
Ac-LTF$r8AYWAQL$A-NHmdPeg4 1337 1705.97 853.99 854.33 1425
Ac-F$r8AYdl4mWEAL$A-NH.sub.2 1338 1316.72 659.36 659.44 1426
Ac-F$r8AYdl5clWEAL$A-NH.sub.2 1339 1336.66 669.33 669.43 1427
Ac-LThF$r8AYWAQL$S-NH.sub.2 1340 1545.86 773.93 774.11 1428
Ac-LT2Nal$r8AYWAQL$S-NH.sub.2 1341 1581.86 791.93 792.43 1429
Ac-LTA$r8AYWAQL$S-NH.sub.2 1342 1455.81 728.91 729.15 1430
Ac-LTF$r8AYWVQL$S-NH.sub.2 1343 1559.88 780.94 781.24 1431
Ac-LTF$r8HYWAAL$A-NH.sub.2 1344 1524.85 763.43 763.86 1432
Ac-LTF$r8VYWAQL$A-NH.sub.2 1345 1543.88 772.94 773.37 1433
Ac-LTF$r8IYWAQL$S-NH.sub.2 1346 1573.89 787.95 788.17 1434
Ac-FTF$r8VYWSQL$S-NH.sub.2 1347 1609.85 805.93 806.22 1435
Ac-ITF$r8FYWAQL$S-NH.sub.2 1348 1607.88 804.94 805.2 1436
Ac-2NalTF$r8VYWSQL$S-NH.sub.2 1349 1659.87 830.94 831.2 1437
Ac-ITF$r8LYWSQL$S-NH.sub.2 1350 1589.89 795.95 796.13 1438
Ac-FTF$r8FYWAQL$S-NH.sub.2 1351 1641.86 821.93 822.13 1439
Ac-WTF$r8VYWAQL$S-NH.sub.2 1352 1632.87 817.44 817.69 1440
Ac-WTF$r8WYWAQL$S-NH.sub.2 1353 1719.88 860.94 861.36 1441
Ac-VTF$r8AYWSQL$S-NH.sub.2 1354 1533.82 767.91 768.19 1442
Ac-WTF$r8FYWSQL$S-NH.sub.2 1355 1696.87 849.44 849.7 1443
Ac-FTF$r8IYWAQL$S-NH.sub.2 1356 1607.88 804.94 805.2 1444
Ac-WTF$r8VYWSQL$S-NH.sub.2 1357 1648.87 825.44 824.8 1445
Ac-FTF$r8LYWSQL$S-NH.sub.2 1358 1623.87 812.94 812.8 1446
Ac-YTF$r8FYWSQL$S-NH.sub.2 1359 1673.85 837.93 837.8 1447
Ac-LTF$r8AY6clWEAL$A-NH.sub.2 1360 1550.79 776.40 776.14 1448
Ac-LTF$r8AY6clWSQL$S-NH.sub.2 1361 1581.80 791.90 791.68 1449
Ac-F$r8AY6clWSAL$A-NH.sub.2 1362 1294.65 648.33 647.67 1450
Ac-F$r8AY6clWQAL$AA-NH.sub.2 1363 1406.72 704.36 703.84 1451
Ac-LHF$r8AYWAQL$S-NH.sub.2 1364 1567.86 784.93 785.21 1452
Ac-LTF$r8AYWAQL$S-NH.sub.2 1365 1531.84 766.92 767.17 1453
Ac-LTF$r8AHWAQL$S-NH.sub.2 1366 1505.84 753.92 754.13 1454
Ac-LTF$r8AYWAHL$S-NH.sub.2 1367 1540.84 771.42 771.61 1455
Ac-LTF$r8AYWAQL$H-NH.sub.2 1368 1581.87 791.94 792.15 1456
H-LTF$r8AYWAQL$A-NH.sub.2 1369 1473.84 737.92 737.29 1457
Ac-HHF$r8AYWAQL$S-NH.sub.2 1370 1591.83 796.92 797.35 1458
Ac-aAibWTF$r8VYWSQL$S-NH.sub.2 1371 1804.96 903.48 903.64 1459
Ac-AibWTF$r8HYWAQL$S-NH.sub.2 1372 1755.91 878.96 879.4 1460
Ac-AibAWTF$r8HYWAQL$S-NH.sub.2 1373 1826.95 914.48 914.7 1461
Ac-fWTF$r8HYWAQL$S-NH.sub.2 1374 1817.93 909.97 910.1 1462
Ac-AibWWTF$r8HYWAQL$S-NH.sub.2 1375 1941.99 972.00 972.2 1463
Ac-WTF$r8LYWSQL$S-NH.sub.2 1376 1662.88 832.44 832.8 1464
Ac-WTF$r8NleYWSQL$S-NH.sub.2 1377 1662.88 832.44 832.6 1465
Ac-LTF$r8AYWSQL$a-NH.sub.2 1378 1531.84 766.92 767.2 1466
Ac-LTF$r8EYWARL$A-NH.sub.2 1379 1601.90 801.95 802.1 1467
Ac-LTF$r8EYWAHL$A-NH.sub.2 1380 1582.86 792.43 792.6 1468
Ac-aTF$r8AYWAQL$S-NH.sub.2 1381 1489.80 745.90 746.08 1469
Ac-AibTF$r8AYWAQL$S-NH.sub.2 1382 1503.81 752.91 753.11 1470
Ac-AmfTF$r8AYWAQL$S-NH.sub.2 1383 1579.84 790.92 791.14 1471
Ac-AmwTF$r8AYWAQL$S-NH.sub.2 1384 1618.86 810.43 810.66 1472
Ac-NmLTF$r8AYWAQL$S-NH.sub.2 1385 1545.86 773.93 774.11 1473
Ac-LNmTF$r8AYWAQL$S-NH.sub.2 1386 1545.86 773.93 774.11 1474
Ac-LSarF$r8AYWAQL$S-NH.sub.2 1387 1501.83 751.92 752.18 1475
Ac-LGF$r8AYWAQL$S-NH.sub.2 1388 1487.82 744.91 745.15 1476
Ac-LTNmF$r8AYWAQL$S-NH.sub.2 1389 1545.86 773.93 774.2 1477
Ac-TF$r8AYWAQL$S-NH.sub.2 1390 1418.76 710.38 710.64 1478
Ac-ETF$r8AYWAQL$A-NH.sub.2 1391 1531.81 766.91 767.2 1479
Ac-LTF$r8EYWAQL$A-NH.sub.2 1392 1573.85 787.93 788.1 1480
Ac-LT2Nal$r8AYWSQL$S-NH.sub.2 1393 1597.85 799.93 800.4 1481
Ac-LTF$r8AYWAAL$S-NH.sub.2 1394 1474.82 738.41 738.68 1482
Ac-LTF$r8AYWAQhCha$S-NH.sub.2 1395 1585.89 793.95 794.19 1483
Ac-LTF$r8AYWAQChg$S-NH.sub.2 1396 1557.86 779.93 780.97 1484
Ac-LTF$r8AYWAQCba$S-NH.sub.2 1397 1543.84 772.92 773.19 1485
Ac-LTF$r8AYWAQF3CF3$S-NH.sub.2 1398 1633.82 817.91 818.15 1486
Ac-LTF$r8AYWAQ1Nal$S-NH.sub.2 1399 1615.84 808.92 809.18 1487
Ac-LTF$r8AYWAQBip$S-NH.sub.2 1400 1641.86 821.93 822.32 1488
Ac-LT2Nal$r8AYWAQL$S-NH.sub.2 1401 1581.86 791.93 792.15 1489
Ac-LTF$r8AYWVQL$S-NH.sub.2 1402 1559.88 780.94 781.62 1490
Ac-LTF$r8AWWAQL$S-NH.sub.2 1403 1554.86 778.43 778.65 1491
Ac-FTF$r8VYWSQL$S-NH.sub.2 1404 1609.85 805.93 806.12 1492
Ac-ITF$r8FYWAQL$S-NH.sub.2 1405 1607.88 804.94 805.2 1493
Ac-ITF$r8LYWSQL$S-NH.sub.2 1406 1589.89 795.95 796.22 1494
Ac-FTF$r8FYWAQL$S-NH.sub.2 1407 1641.86 821.93 822.41 1495
Ac-VTF$r8AYWSQL$S-NH.sub.2 1408 1533.82 767.91 768.19 1496
Ac-LTF$r8AHWAQL$S-NH.sub.2 1409 1505.84 753.92 754.31 1497
Ac-LTF$r8AYWAQL$H-NH.sub.2 1410 1581.87 791.94 791.94 1498
Ac-LTF$r8AYWAHL$S-NH.sub.2 1411 1540.84 771.42 771.61 1499
Ac-aAibWTF$r8VYWSQL$S-NH.sub.2 1412 1804.96 903.48 903.9 1500
Ac-AibWTF$r8HYWAQL$S-NH.sub.2 1413 1755.91 878.96 879.5 1501
Ac-AibAWTF$r8HYWAQL$S-NH.sub.2 1414 1826.95 914.48 914.7 1502
Ac-fWTF$r8HYWAQL$S-NH.sub.2 1415 1817.93 909.97 910.2 1503
Ac-AibWWTF$r8HYWAQL$S-NH.sub.2 1416 1941.99 972.00 972.7 1504
Ac-WTF$r8LYWSQL$S-NH.sub.2 1417 1662.88 832.44 832.7 1505
Ac-WTF$r8NleYWSQL$S-NH.sub.2 1418 1662.88 832.44 832.7 1506
Ac-LTF$r8AYWSQL$a-NH.sub.2 1419 1531.84 766.92 767.2 1507
Ac-LTF$r8EYWARL$A-NH.sub.2 1420 1601.90 801.95 802.2 1508
Ac-LTF$r8EYWAHL$A-NH.sub.2 1421 1582.86 792.43 792.6 1509
Ac-aTF$r8AYWAQL$S-NH.sub.2 1422 1489.80 745.90 746.1 1510
Ac-AibTF$r8AYWAQL$S-NH.sub.2 1423 1503.81 752.91 753.2 1513
Ac-AmfTF$r8AYWAQL$S-NH.sub.2 1424 1579.84 790.92 791.2 1514
Ac-AmwTF$r8AYWAQL$S-NH.sub.2 1425 1618.86 810.43 810.7 1515
Ac-NmLTF$r8AYWAQL$S-NH.sub.2 1426 1545.86 773.93 774.1 1516
Ac-LNmTF$r8AYWAQL$S-NH.sub.2 1427 1545.86 773.93 774.4 1517
Ac-LSarF$r8AYWAQL$S-NH.sub.2 1428 1501.83 751.92 752.1 1518
Ac-TF$r8AYWAQL$S-NH.sub.2 1429 1418.76 710.38 710.8 1519
Ac-ETF$r8AYWAQL$A-NH.sub.2 1430 1531.81 766.91 767.4 1520
Ac-LTF$r8EYWAQL$A-NH.sub.2 1431 1573.85 787.93 788.2 1521
Ac-WTF$r8VYWSQL$S-NH.sub.2 1432 1648.87 825.44 825.2 1522
Ac-YTF$r8FYWSQL$S-NH.sub.2 1433 1673.85 837.93 837.3 1523
Ac-F$r8AY6clWSAL$A-NH.sub.2 1434 1294.65 648.33 647.74 1524
Ac-ETF$r8EYWVQL$S-NH.sub.2 1435 1633.84 817.92 817.36 1525
Ac-ETF$r8EHWAQL$A-NH.sub.2 1436 1563.81 782.91 782.36 1526
Ac-ITF$r8EYWAQL$S-NH.sub.2 1437 1589.85 795.93 795.38 1527
Ac-ITF$r8EHWVQL$A-NH.sub.2 1438 1575.88 788.94 788.42 1528
Ac-ITF$r8EHWAQL$S-NH.sub.2 1439 1563.85 782.93 782.43 1529
Ac-LTF4F$r8AYWAQCba$S-NH.sub.2 1440 1561.83 781.92 781.32 1530
Ac-LTF3Cl$r8AYWAQhL$S-NH.sub.2 1441 1579.82 790.91 790.64 1531
Ac-LTF3Cl$r8AYWAQCha$S-NH.sub.2 1442 1605.84 803.92 803.37 1532
Ac-LTF3Cl$r8AYWAQChg$S-NH.sub.2 1443 1591.82 796.91 796.27 1533
Ac-LTF3Cl$r8AYWAQCba$S-NH.sub.2 1444 1577.81 789.91 789.83 1534
Ac-LTF$r8AY6clWSQL$S-NH.sub.2 1445 1581.80 791.90 791.75 1535
Ac-LTF4F$r8HYWAQhL$S-NH.sub.2 1446 1629.87 815.94 815.36 1536
Ac-LTF4F$r8HYWAQCba$S-NH.sub.2 1447 1627.86 814.93 814.32 1537
Ac-LTF4F$r8AYWAQhL$S-NH.sub.2 1448 1563.85 782.93 782.36 1538
Ac-LTF4F$r8AYWAQChg$S-NH.sub.2 1449 1575.85 788.93 788.35 1539
Ac-ETF$r8EYWVAL$S-NH.sub.2 1450 1576.82 789.41 788.79 1540
Ac-ETF$r8EHWAAL$A-NH.sub.2 1451 1506.79 754.40 754.8 1541
Ac-ITF$r8EYWAAL$S-NH.sub.2 1452 1532.83 767.42 767.75 1542
Ac-ITF$r8EHWVAL$A-NH.sub.2 1453 1518.86 760.43 760.81 1543
Ac-ITF$r8EHWAAL$S-NH.sub.2 1454 1506.82 754.41 754.8 1544
Pam-LTF$r8EYWAQL$S-NH.sub.2 1455 1786.07 894.04 894.48 1545
Pam-ETF$r8EYWAQL$S-NH.sub.2 1456 1802.03 902.02 902.34 1546
Ac-LTF$r8AYWLQL$S-NH.sub.2 1457 1573.89 787.95 787.39 1547
Ac-LTF$r8EYWLQL$S-NH.sub.2 1458 1631.90 816.95 817.33 1548
Ac-LTF$r8EHWLQL$S-NH.sub.2 1459 1605.89 803.95 804.29 1549
Ac-LTF$r8VYWAQL$S-NH.sub.2 1460 1559.88 780.94 781.34 1550
Ac-LTF$r8AYWSQL$S-NH.sub.2 1461 1547.84 774.92 775.33 1551
Ac-ETF$r8AYWAQL$S-NH.sub.2 1462 1547.80 774.90 775.7 1552
Ac-LTF$r8EYWAQL$S-NH.sub.2 1463 1589.85 795.93 796.33 1553
Ac-LTF$r8HYWAQL$S-NHAm 1464 1667.94 834.97 835.37 1554
Ac-LTF$r8HYWAQL$S-NHiAm 1465 1667.94 834.97 835.27 1555
Ac-LTF$r8HYWAQL$S-NHnPr3Ph 1466 1715.94 858.97 859.42 1556
Ac-LTF$r8HYWAQL$S-NHnBu3,3Me 1467 1681.96 841.98 842.67 1557
Ac-LTF$r8HYWAQL$S-NHnBu 1468 1653.93 827.97 828.24 1558
Ac-LTF$r8HYWAQL$S-NHnPr 1469 1639.91 820.96 821.31 1559
Ac-LTF$r8HYWAQL$S-NHnEt2Ch 1470 1707.98 854.99 855.35 1560
Ac-LTF$r8HYWAQL$S-NHHex 1471 1681.96 841.98 842.4 1561
Ac-LTF$r8AYWAQL$S-NHmdPeg2 1472 1633.91 817.96 855.35 1562
Ac-LTF$r8AYWAQL$A-NHmdPeg2 1473 1617.92 809.96 810.58 1563
Ac-LTF$r5AYWAAL$s8S-NH.sub.2 1474 1474.82 738.41 738.79 1564
Ac-LTF$r8AYWCouQL$S-NH.sub.2 1475 1705.88 853.94 854.61 1565
Ac-LTF$r8CouYWAQL$S-NH.sub.2 1476 1705.88 853.94 854.7 1566
Ac-CouTF$r8AYWAQL$S-NH.sub.2 1477 1663.83 832.92 833.33 1567
H-LTF$r8AYWAQL$A-NH.sub.2 1478 1473.84 737.92 737.29 1568
Ac-HHF$r8AYWAQL$S-NH.sub.2 1479 1591.83 796.92 797.72 1569
Ac-LT2Nal$r8AYWSQL$S-NH.sub.2 1480 1597.85 799.93 800.68 1570
Ac-LTF$r8HCouWAQL$S-NH.sub.2 1481 1679.87 840.94 841.38 1571
Ac-LTF$r8AYWCou2QL$S-NH.sub.2 1482 1789.94 895.97 896.51 1572
Ac-LTF$r8Cou2YWAQL$S-NH.sub.2 1483 1789.94 895.97 896.5 1573
Ac-Cou2TF$r8AYWAQL$S-NH.sub.2 1484 1747.90 874.95 875.42 1574
Ac-LTF$r8ACou2WAQL$S-NH.sub.2 1485 1697.92 849.96 850.82
1575 Dmaac-LTF$r8AYWAQL$S-NH.sub.2 1486 1574.89 788.45 788.82 1576
Hexac-LTF$r8AYWAQL$S-NH.sub.2 1487 1587.91 794.96 795.11 1577
Napac-LTF$r8AYWAQL$S-NH.sub.2 1488 1657.89 829.95 830.36 1578
Pam-LTF$r8AYWAQL$S-NH.sub.2 1489 1728.06 865.03 865.45 1579
Ac-LT2Nal$r8HYAAQL$S-NH.sub.2 1490 1532.84 767.42 767.61 1580
Ac-LT2Nal$/r8HYWAQL$/S-NH.sub.2 1491 1675.91 838.96 839.1 1581
Ac-LT2Nal$r8HYFAQL$S-NH.sub.2 1492 1608.87 805.44 805.9 1582
Ac-LT2Nal$r8HWAAQL$S-NH.sub.2 1493 1555.86 778.93 779.08 1583
Ac-LT2Nal$r8HYAWQL$S-NH.sub.2 1494 1647.88 824.94 825.04 1584
Ac-LT2Nal$r8HYAAQW$S-NH.sub.2 1495 1605.83 803.92 804.05 1585
Ac-LTW$r8HYWAQL$S-NH.sub.2 1496 1636.88 819.44 819.95 1586
Ac-LT1Nal$r8HYWAQL$S-NH.sub.2 1497 1647.88 824.94 825.41
[0366] Observed mass was measured by electrospray ionization-mass
spectrometry.
[0367] In some embodiments, a peptidomimetic macrocycles disclosed
herein do not comprise a peptidomimetic macrocycle structure as
shown in Table 4b.
[0368] Table 4c shows examples of non-crosslinked polypeptides
comprising D-amino acids.
TABLE-US-00011 TABLE 4c SEQ ID Exact Found Calc Calc Calc SP
Sequence NO: Isomer Mass Mass (M + 1)/1 (M + 2)/2 (M + 3)/3 1587
Ac-tawyanfekllr-NH.sub.2 1498 777.46 1588
Ac-tawyanf4CF3ekllr-NH.sub.2 1499 811.41
Example 2: Cell Viability Assay
[0369] Cells were thawed from a liquid nitrogen preserved state.
Once cells were expanded and divide at their expected doubling
times, screening began. Cells were seeded in growth media in black
384-well tissue culture treated plates at 500 cells/well. Cells
were equilibrated in assay plates via centrifugation and placed in
incubators attached to the Dosing Modules at 37.degree. C. for 24
hr before treatment, resulting in cell densities of approximately
500 cells/plate. At the time of treatment, a set of assay plates
(which did not receive treatment) were collected and ATP levels
were measured by adding ATPLite (Perkin Elmer). These T-zero (To)
plates were read using ultra-sensitive luminescence on Envision
Plate Readers. Using an automated acoustic dispensing system, assay
plates were treated with compound or peptide from a 1000.times.DMSO
stock, achieving a 1:1000 working dilution. Final treatment
concentrations in the plates were 0 (vehicle), 0.001, 0.003, 0.01,
0.03, 0.1, 0.3, 1, 3, 10 and 30 M. The assay plates (4 replicates
per treatment) were incubated with compound or peptide for 72 hr.
After 72 hr, plates were developed for endpoint analysis using
ATPLite. All data points were collected via automated processes;
quality controlled; and analyzed using Zalicus proprietary
software. Assay plates were accepted if they passed the following
quality control standards: relative luciferase values were
consistent throughout the entire experiment, Z-factor scores were
greater than 0.6, and untreated/vehicle controls behaved
consistently on the plate.
[0370] Growth Inhibition (GI), used as a measure of cell viability,
was measured at the time of dosing (To) and after 72 hr (T.sub.72).
A GI reading of 0% represents no growth inhibition; a GI 100%
represents complete growth inhibition, a cytostatic effect. A GI
200% represents complete death of all cells in the culture well.
Compounds reaching an activity plateau of GI 200% were considered
cytotoxic. GI was calculated by the following test and
equation:
If T < V 0 : 100 * ( 1 - T - V 0 V 0 ) ##EQU00001## If T
.gtoreq. V 0 : 100 * ( 1 - T - V 0 V - V 0 ) ##EQU00001.2##
[0371] Where T is the signal measure for a test article, V is the
vehicle-treated control measure, and Vo is the vehicle control
measure at time zero. This formula was derived from the Growth
Inhibition calculation used in the National Cancer Institute's
NCI-60 high throughput screen
[0372] Cell lines were assigned as p53 wild-type, mutant, or null
from information available from the Cancer Cell Line Encyclopedia.
Results for exemplary p53 peptidomimetic macrocycles are shown in
Table 5 below
TABLE-US-00012 TABLE 5 Cell Viability in p53.sup.WT/p53.sup.MUT
cancer cell lines SP-154 SP-763 EC.sub.50 EC.sub.50 Cell lines
Cancer Type p53.sup.WT/p53.sup.MUT (.mu.M) (.mu.M) A2058 skin
p53.sup.MUT 18.6 30 AsPC-1 Pancreas p53.sup.MUT 30 30 Caov-3 ovary
p53.sup.MUT 12.9 30 Caov-4 ovary p53.sup.MUT 30 30 COLO-679 skin
p53.sup.MUT 13.5 30 COLO-684 endometrium p53.sup.MUT 10.1 30
COLO-741 skin p53.sup.MUT 18.5 30 EBC-1 Lung-NSCLC p53.sup.MUT 30
30 ECC10 gastric p53.sup.MUT 15.2 30 KM12 colorectal p53.sup.MUT 30
30 LS-123 colorectal p53.sup.MUT 30 30 MIA PaCa-2 pancreas
p53.sup.MUT 30 30 NCI-H508 colorectal p53.sup.MUT 30 30 OVCAR-3
ovary p53.sup.MUT 30 30 RPMI-7951 skin p53.sup.MUT 10.8 30 SF126
brain p53.sup.MUT 11 30 SK-OV-3 ovary p53.sup.MUT 14.3 30 SW480
colorectal p53.sup.MUT 30 30 HCT-116 colorectal p53.sup.WT 0.464
1.02 HEC-151 endometrium p53.sup.WT 1.19 30 HEC-265 endometrium
p53.sup.WT 1.78 30 HUH-6-clone5 liver p53.sup.WT 0.865 0.791
IST-MES1 lung p53.sup.WT 1.43 30 KP-N-RT-BM-1 nerve p53.sup.WT
0.285 0.255 KP-N-S19s nerve p53.sup.WT 0.176 0.0168 LoVo colorectal
p53.sup.WT 0.431 0.134 LS-174T colorectal p53.sup.WT 0.402 0.205
MSTO-211H lung p53.sup.WT 0.288 0.209 MV-4-11 AML p53.sup.WT 0.159
0.307 NCI-H929 multiple myeloma p53.sup.WT 0.858 0.24 PA-1 ovary
p53.sup.WT 0.339 0.592 WM-115 skin p53.sup.WT 0.425 0.429 COLO-205
colorectal p53.sup.WT 0.646 0.429 COLO-849 skin p53.sup.WT 1.66
0.501 NCI-H28 lung p53.sup.WT 3.42 30
Example 3: Safety and/or Tolerability Study--I
Study Objectives
[0373] This study was designed to (i) evaluate the safety and/or
tolerability of Aileron peptide 1, and to (ii) determine the DLTs
and the MTD of the Aileron peptide 1 in patients with advanced
solid tumors, including patients with tumors expressing WT p53
protein. Aileron peptide 1 is an alpha helical hydrocarbon
cross-linked polypeptide macrocycle, with an amino acid sequence
less than 20 amino acids long that is derived from the
transactivation domain of wild type human P53 protein and that
contains a phenylalanine, a tryptophan and a leucine amino acid in
the same positions relative to each other as in the transactivation
domain of wild type human P53 protein. Aileron peptide 1 has a
single cross link spanning amino acids in the i to the i+7 position
of the amino acid sequence and has more than three amino acids
between the i+7 position and the carboxyl terminus. Aileron peptide
1 binds to human MDM2 and MDM4 and has an observed mass of 950-975
m/e as measured by electrospray ionization-mass spectrometry.
Investigational Plan
Study Design
[0374] The study consisted of a Dose Escalation Phase (DEP) and an
Expansion Phase (EXP). The DEP was a "3+3" dose escalation design
to establish the MTD of Aileron peptide-1. The EXP enrolled
patients with specific solid tumors at the MTD to further
investigate the clinical safety profile and potential efficacy of
the dose level. The selection of patients for the EXP is finalized
based on results of the DEP, as well as data from additional
nonclinical pharmacology studies. The later includes the
investigation of multiple solid cancer cell lines (e.g., breast,
bladder, head/neck, gastrointestinal, liver, lung, pancreas,
prostate, sarcoma) that facilitates the comparison of cell line
sensitivity to Aileron peptide-1 across and within tumor types.
[0375] After completing the screening, eligible patients received a
single IV dose of Aileron peptide-1 on Days 1, 8, and 15, and
remained in the clinic for approximately 8 hours after dosing
completion for clinical evaluation, laboratory testing and
pharmacokinetic assessments. In addition, a tumor biopsy was
performed within 48 hours of Dose 3 (last dose) in Cycle 1 or 2 for
pharmacodynamic assessment, unless the biopsy posed a significant
risk to the patient. The selection of Cycle 1 or 2 was made at the
discretion of the Investigator. Patients returned to the clinic for
additional observation and laboratory assessments on Day 22 and for
end-of-cycle assessments on Day 29.
[0376] Treatment of patients in the dose escalation and the dose
expansion phases of the study continued until documentation of
disease progression, unacceptable toxicity, or patient or physician
decision to discontinue therapy.
p53 Status Determination and Tumor Sampling Requirement Prior to
Enrollment:
[0377] A central laboratory tested both archived tissue samples or
fresh biopsy samples from all patients enrolled in the study for
p53 status using Next-Generation Sequencing (NGS).
[0378] For the First 3 Dose Levels of Stage 1:
[0379] Patients were enrolled irrespective of p53 status.
Nevertheless, patients were still tested for p53 status at the
central laboratory. To this end, archived tissue was used (sample
were not older than 3 years), or alternatively, a fresh biopsy was
considered, unless the biopsy posed a significant risk to the
patient.
[0380] Starting at Dose Level 4 of Stage 1 (and for patients
enrolled in Stage 2 of the DEP):
[0381] Only patients with tumors expressing WT p53 protein were
enrolled. This key inclusion criterion was based on the proposed
mechanism of action of Aileron peptide-1, which requires WT p53
protein to be pharmacologically active. The inclusion criterion is
also supported by results of in vitro tumor growth assays, in which
Aileron peptide-1 demonstrated activity in tumor cells expressing
WT p53 protein, but not in cells with null-mutations of p53.
Patients met the p53 requirement through one of the following
scenarios: [0382] Patients were eligible based on a previous p53
gene test result done at a local lab. These patients were still
tested for p53 status using NGS at the central laboratory. To this
end, archived tissue was used (sample was not be older than 3
years), or alternatively, a fresh biopsy was considered, unless the
biopsy posed a significant risk to the patient. Patients who did
not have archived tissue and for whom a biopsy posed a significant
risk, were not enrolled. [0383] Patients were eligible based on
archived tissue tested for p53 (sample was not be older than 3
years) at the central lab, or alternatively, a fresh biopsy was
considered, unless the biopsy posed a significant risk to the
patient. Patients who didn't have archived tissue and for whom a
biopsy posed a significant risk, were enrolled.
[0384] For patients enrolling into the EXP: [0385] Only patients
with tumors expressing p53 WT were enrolled, and all patients were
tested for p53 status using NGS at the central laboratory PRIOR to
enrollment. Archived tissue were used (if sample were not older
than 1 year), or alternatively, a fresh biopsy was considered,
unless the biopsy posed a significant risk to the patient. Patients
who didn't have archived tissue and for whom a biopsy posed a
significant risk, were not enrolled.
[0386] Only patients with tumors expressing WT p53 protein were
enrolled. The determination of p53 status was performed on tumor
samples obtained during the screening period. The assay was
performed by study sites with required capabilities; otherwise it
was performed at a central laboratory. Results from archival tissue
samples, if available, could also be used to determine patient
eligibility in the DEP. The total number of patients enrolled in
the study depended on the number of dose levels and the number of
patients in each cohort before MTD is established. Approximately 27
adult patients, exclusive of replacements for patients who
discontinue for non-safety reasons, were enrolled in the DEP, and
approximately 30 additional patients were enrolled in the EXP.
Enrollment of a total of up to 60 patients was planned for the
study. Up to 6 clinical sites in the US were planned. The expected
accrual phase is approximately 24 months. The expected follow-up
phase is approximately 9 months after the last patient is enrolled,
for a total study duration of approximately 33 months.
[0387] Patients who satisfy all inclusion and exclusion criteria,
including documentation of WT p53 status, were enrolled in cohorts
of 3 to 6 patients to receive Aileron peptide 1. Aileron peptide 1
was administered by IV infusion over 1 hour (.+-.15 min) on Days 1,
8 and 15 of each 28-day cycle. Treatment continued until disease
progression, unacceptable toxicity or patient or physician
withdrawal of consent. After the MTD was established, approximately
30 additional patients were enrolled in an expansion cohort to gain
further experience at this dose level and in particular patient or
tumor types.
[0388] Safety was evaluated based on the incidence, severity,
duration, causality, seriousness, and type of AE, and changes in
the patient's physical examination, vital signs and clinical
laboratory results. Investigators used the NCI CTCAE version 4.0 to
assess the severity of AEs.
[0389] Because the primary objectives of this study were based on
safety and pharmacokinetic, statistical analyses were descriptive
in nature and accounted for all doses studied and all observed
responses, including patients who achieve a complete response (CR)
or partial response (PR) or who maintain stable disease (SD) based
on RECIST 1.1. Patients who received at least one dose of Aileron
peptide 1 constituted the safety population and were be included in
all safety analyses. Patients who completed at least one cycle of
Aileron peptide 1 and underwent a post-treatment objective disease
assessment constituted the efficacy-evaluable patient
population.
Patient Population
Inclusion Criterias
[0390] All patients were required to meet the following inclusion
criterias: (i) Male or female patients age 18 years and older,
inclusive, at the time of informed consent (ii) Histologically- or
cytologically-confirmed malignancy that is metastatic or
unresectable and for which standard curative measures do not exist
or are no longer effective; (iii) WT p53 status for the relapsing
or treatment-refractory solid neoplasm is mandatory for patients
enrolling at dose level 4 and higher in Stage 1 of the DEP, as well
as for all patients enrolled in Stage 2 of the DEP or in the EXP;
(iv) at least one target lesion that is measurable by RECIST 1.1;
(v) ECOG performance status 0-1; (vi) predicted life expectancy of
.gtoreq.3 months; (vii) adequate hematologic function, measured
within 7 days prior to the first dose of Aileron peptide 1 (defined
as: ANC .gtoreq.1.5.times.10.sup.9/L, Hemoglobin .gtoreq.9.0 g/d,
and Platelets .gtoreq.100.times.10.sup.9/L); (viii) adequate
hepatic function, measured within 7 days prior to the first dose of
Aileron peptide 1 (defined as: in the absence of disease
involvement in the liver:bilirubin .ltoreq.1.5 times institutional
ULN:AST and ALT .ltoreq.2.5 times ULN; in the presence of disease
involvement in the liver:bilirubin .ltoreq.2 times ULN:AST and ALT
.ltoreq.5 times ULN, (ix) adequate renal function, measured within
7 days prior to the first dose of Aileron peptide 1, (defined as:
urinalysis with no evidence of +2 or higher proteinuria, serum
creatinine .ltoreq.1.5 times institutional ULN or calculated
creatinine clearance .gtoreq.50 mL/min (Cockcroft-Gault formula));
(x) acceptable coagulation profile, measured within 7 days prior to
the first dose of Aileron peptide 1 (defined as: PT or INR
.ltoreq.1.5 times ULN; aPTT .ltoreq.1.5 times ULN); (Xi) at least 4
weeks since prior chemotherapy or biologic therapy, radiotherapy or
surgery (intra-thoracic, intra-abdominal or intra-pelvic) with
recovery to Grade 1 or baseline of significant toxicities,
excluding alopecia, from previous therapies. Palliative
radiotherapy for bone lesions .ltoreq.2 weeks prior to the first
dose of Aileron peptide 1 is acceptable if acute toxicity has
resolved; (xii) negative serum pregnancy test within 14 days prior
to the first dose of Aileron peptide 1 for women of child-bearing
potential, defined as a sexually mature woman who has not undergone
a hysterectomy or who has not been naturally postmenopausal for
.gtoreq.24 consecutive months (i.e., who has had menses any time in
the preceding 24 consecutive months); (xiii) all patients (males
and females) of child-bearing potential agree to use an effective
method of birth control (i.e., latex condom, diaphragm, cervical
cap, IUD, birth control pill, etc.) beginning two weeks before the
first dose of Aileron peptide 1 and for 30 days after the last dose
of Aileron peptide 1; (xiv) ability to understand and willingness
to sign a written informed consent document; and patients with
prostate cancer must continue androgen deprivation therapy, unless
such therapy was discontinued 6 months prior to first dose of
Aileron peptide 1.
Exclusion Criterias
[0391] Patients who meet any of the following criteria at screening
or Day -1 were excluded: (i) previous treatment with
investigational agents that affect MDM2 or MDMX activity; known
hypersensitivity to any study drug component; (iii) known and
untreated brain metastases. Patients with brain metastases that
have been treated and demonstrated to be clinically stable for
.gtoreq.30 days can be enrolled onto the dose escalation portion of
the study; (iv) history of coagulopathy, platelet disorder or
history of non-drug induced thrombocytopenia; (v) history of
pulmonary embolism within 6 months prior to the first dose of
Aileron peptide 1 or untreated DVT; (vi) required concurrent use of
anti-coagulants or anti-platelet medication, with the exception of
aspirin doses .ltoreq.81 mg/day, low-dose SC heparin or SC
low-molecular-weight heparin for DVT prophylaxis, or heparin
flushes to maintain IV catheter patency; (vii) patients with
pre-existing history of or known cardiovascular risk (for example:
history of acute coronary syndromes including myocardial
infarction, unstable angina, coronary artery bypass graft,
angioplasty, or stenting within 6 months prior to the first dose of
Aileron peptide 1; uncontrolled hypertension defined as a systolic
BP .gtoreq.160 mmHg and/or diastolic BP .gtoreq.100 mmHg;
pre-existing cardiac failure (New York Heart Association class
III-IV); atrial fibrillation on anti-coagulants; clinically
significant uncontrolled arrhythmias or arrhythmia requiring
treatment, with the exceptions of atrial fibrillation and
paroxysmal supraventricular tachycardia; severe valvulopathy;
corrected QTc interval on screening ECG .gtoreq.450 msec for males
and .gtoreq.470 msec for females); (viii) clinically significant
gastrointestinal bleeding within 6 months prior to the first dose
of Aileron peptide 1; (ix) clinically significant third-space fluid
accumulation (e.g., ascites requiring tapping despite the use of
diuretics, or pleural effusion that requires tapping or is
associated with shortness of breath); (x) pregnant or lactating
females; (xi) evidence of serious and/or unstable pre-existing
medical, psychiatric or other condition (including laboratory
abnormalities) that could interfere with patient safety or
provision of informed consent to participate in this study; (xii)
active uncontrolled infection, a history of HIV/AIDS, or a history
of hepatitis B or C in the absence of hepatocellular carcinoma.
Patients with primary liver cancer that have positive hepatitis
serology but are not demonstrating active viral hepatitis can be
considered for enrollment if they meet all other inclusion and no
other exclusion criteria; (xiii) starting at dose level 4 and
higher in Stage 1 of the DEP (as well as for all patients enrolling
in Stage 2 of the DEP or in the EXP): Cancers with known Human
Papilloma Virus (HPV)-association such as HPV-positive cervical
cancers, HPV-positive oropharyngeal cancers or HPV-positive anal
cancers; (xiv)known history of another primary malignancy that has
not been in remission for .gtoreq.2 years. Non-melanoma skin cancer
and cervical carcinoma in situ or squamous intraepithelial lesions
(e.g., CIN or PIN) are allowed; (xv) any psychological,
sociological, or geographical condition that could potentially
interfere with compliance with the study protocol and follow-up
schedule; (xvi) the required use of any concomitant medications
that are predominantly cleared by hepatobiliary transporters (e.g.,
OATP members OATP1B1 and OATP1B3) within 24 hours of Aileron
peptide 1 infusion; (xvii) the use of any investigational agents
within 4 weeks or 5 circulating half-lives prior to the first dose
of Aileron peptide 1.
Patient Removal/Replacement from Study Therapy
[0392] A patient was removed from the study therapy for a variety
of reasons, including: (i) disease progression; (ii) unacceptable
adverse event(s); (iii) intercurrent illness that prevents further
participation; (iv) clinically significant toxicity despite a
2-week dosing delay or after two dose reductions; (v) patient
refusal to continue treatment through the study and/or consent
withdrawal for study participation; (vi) patient unable or
unwilling to comply with study requirements; (vii) pregnancy or
failure to use adequate birth control; (viii) general or specific
changes in the patient's condition that render the patient
unacceptable for further treatment in this study in the judgment of
the investigator
[0393] Any patient who completed enrollment and did not receive a
dose of Aileron peptide 1 was replaced. A patient in the dose
escalation portion of the study who discontinued the study prior to
completion of the first cycle for reasons other than safety was
replaced. A patient in the dose expansion portion of the study who
discontinued the study participation prior to the completion of the
first cycle of treatment for any reason was replaced.
Treatment Plan
Drug Administration Study--1
[0394] The study drug was the investigational agent Aileron peptide
1. This investigational agent was be distributed to clinical sites.
Patients began treatment with Aileron peptide 1 within 21 days
following the start of screening. Aileron peptide 1 drug was be a
frozen liquid product supplied in single-use glass vials. The
peptidomimetic macrocycle for injection was stored frozen at
.ltoreq.-15.degree. C. Aileron peptide 1 was introduced into an IV
infusion bag containing D5W; known as Aileron peptide 1 dosing
solution, and was provided by the site pharmacy for administration
to the patient. Aileron peptide 1 dosing solution was labeled with
a patient identification number. An investigative staff confirmed
this information and its relevancy to the intended patient.
[0395] Aileron peptide 1 was administered by IV infusion in D5W
over 1 hour (.+-.15 min) on Days 1, 8 and 15 of each 28-day
treatment cycle. The pre-defined dose were calculated for each
patient based on body weight at the start of each cycle. Aileron
peptide 1 was not administered outside of the planned schedule
(i.e., infusions on Days 1, 8 and 15 of a 28-day cycle). Deviations
if any were noted on the eCRF. Treatment of patients in the dose
escalation and the dose expansion phases of the study continued
until documentation of disease progression, unacceptable toxicity,
or patient or physician decision to discontinue therapy.
[0396] In case of infusion-related reactions, Aileron peptide 1
infusion were temporarily discontinued. Pharmacologic agents and
other therapeutic interventions were administered per institutional
guidelines. The decision to re-start Aileron peptide 1 infusion was
made after a careful assessment of the patient.
Starting Dose, Dose Escalation and Dose Reduction
Dose Levels for the Dose Escalation Portion of Study
[0397] In the Dose Escalation portion of the study, increasing dose
levels of Aileron peptide 1 was evaluated in cohorts of 3-6
patients. Aileron peptide 1 was administered by IV infusion over 1
hour (.+-.15 min) on Days 1, 8 and 15 of each 28-day cycle.
Patients enrolled in Cohort 1 received Aileron peptide 1 at Dose
Level 1 (0.16 mg/kg). Based on allometric scaling, the projected
AUC in humans at 0.16 mg/kg (50 .mu.ghr/mL) is approximately 9% of
the rat AUC at STD.sub.10 and approximately 6% of the AUC at the
monkey HNSTD.
[0398] In the absence of DLT, subsequent cohorts of 3 to 6 patients
received escalated doses until the MTD was established.
[0399] A 2-stage dose escalation design was employed. During the
initial Stage 1 Escalation Phase (Table 6), 100% dose increments
was utilized until .gtoreq.1 of 3 patients in a cohort experiences
any Grade .gtoreq.2 AE that is at least possibly related to study
drug. Subsequent dose escalation continued using 3-patient cohorts
and the modified Fibonacci sequence (i.e., Stage 2 Escalation
Phase; Table 7), until the MTD was established.
TABLE-US-00013 TABLE 6 Stage 1 Dose escalation schedule Dose Level
Aileron peptide 1 Dose (mg/kg) Level -2 0.04 Level -1 0.08 Level 1
0.16 Level 2 0.32 Level 3 0.64 Level 4 1.25 Level 5 2.5 Level 6 5
Continue 100% dose increase in subsequent cohorts until the MTD is
established or Stage 2 escalation schedule is initiated
TABLE-US-00014 TABLE 7 Stage 2 Dose escalation Schedule Dose Level
Aileron peptide 1 Dose (mg/kg) Level 1 Last Stage 1 Dose x 1.67
Level 2 Previous Dose x 1.5 Level 3 Previous Dose x 1.4 Level 4
Previous Dose x 1.33 In the absence of DLT, continue dose
escalation at 33% increments after Stage 2 Level 4 until the MTD is
established
[0400] The escalation scheme were switched to the Stage 2
Escalation Schedule at any point that the Investigators, Sponsor's
Medical Monitor and Safety Physician representative agreed on a
more conservative progression.
[0401] The observation of DLT(s) was used to make individual
patient determinations regarding dose reductions, interruptions or
discontinuation throughout the course of the trial, but DLTs
occurring during Cycle 1 were used to inform safety and
tolerability assessments for dose escalation decisions.
[0402] If DLTs was observed in the first cohort, the dose was
de-escalated to Dose Level -1. If DLTs were observed at Dose Level
-1, the dose was de-escalated to Dose Level -2. If DLTs was
observed at Dose Level -2, other dose levels were considered and
implemented after discussions among the Investigators, Sponsor's
Medical Monitor and Safety Physician representative.
[0403] At least three patients were treated at each dose level. If
no patients experience a DLT, then the subsequent 3 patients were
treated at the next planned dose level.
[0404] If DLT was observed in .gtoreq.2 of 3 patients in a cohort,
then no further dose escalation occurred, and the current dose was
defined as the MAD.
[0405] If DLT was observed in 1 of 3 patients, then up to 3
additional patients were enrolled at that same dose level. If DLT
was observed in .gtoreq.2 patients in the expanded cohort, then no
further dose escalation will occur, and the current dose was
defined as the MAD.
[0406] After the MAD was defined, either the previously
administered lower dose was expanded to a total of 6 patients, or
an intermediate (between the MAD and the next lower dose level) was
investigated in up to six patients. The highest dose tolerated
without DLT in at least 5 of 6 patients in a cohort was defined as
the MTD.
Dose Level for the Expansion Portion of Study
[0407] After the MTD was defined, approximately 30 additional
patients were enrolled in an expansion study to gain further
experience at this dose level and investigate the effect of Aileron
peptide 1 in specific patient or tumor types. Two disease types
were selected for evaluation, and 15 patients of each disease type
were enrolled in each of two cohorts in the expansion study. The
dose of Aileron peptide 1 administered to patients in the expansion
cohort was derived from evaluation of available safety and other
information from patients in the dose escalation portion of the
study.
Intra-Patient Dose Escalation
[0408] Intra-patient dose escalation was not permitted.
Dose and Schedule Adjustments for Toxicity
[0409] Toxicity that occurred during a cycle was required to
recover as outlined below for treatment to continue.
[0410] Hemoglobin .gtoreq.8.5 g/dL; ANC .gtoreq.1.0 10.sup.9/L;
platelet count .gtoreq.75.times.10.sup.9/L; liver function tests
back to grade prior to previous cycle (includes PT/INR); other
toxicities must return to Grade .ltoreq.1 or to baseline level if
Grade >1 was acceptable for inclusion in the trial.
[0411] If a clinically significant AE was observed in a patient
during a treatment cycle, further dosing was be delayed until the
toxicity has resolved to an acceptable level. Treatment can be
delayed by up to 2 weeks to allow for the resolution of AEs, and a
dose reduction to the preceding level can be made at the discretion
of the Investigator in consultation with Sponsor's Medical Monitor
and Safety Physician representative. If a patient experiences
multiple AEs, decisions on dosing delay or dose reduction was based
on the most severe AE. Any patient who experienced recurrent,
clinically significant AE after one dose reduction underwent one
additional dose reduction. Patients who continued to experience
clinically significant toxicity after a 2-week delay or two dose
reductions were discontinued from the study.
[0412] Adverse events considered for dose reduction did not include
the events assessed by the investigator as exclusively related to
underlying disease or other medical condition or concomitant
treatment. A patient who experienced an AE considered related to
Aileron peptide 1 continued on study if the patient was receiving
clinical benefit and/or the Investigator felt continued
participation was in the best interest of the patient. In such
cases, at the Investigator's discretion and in agreement with
Sponsor's Medical Monitor and Safety Physician representative, the
dose for a patient was reduced to the preceding lower level.
[0413] Up to two dose reductions for a patient were permitted,
after which the patient were discontinued from the study.
[0414] A patient who experienced a DLT continued treatment at the
preceding lower level at the discretion of the Investigator and in
agreement with Sponsor's Medical Monitor and Safety Physician
representative until disease progression or unacceptable toxicity.
Once the dose had been reduced for a patient, it was not
re-escalated.
[0415] Toxicity grading was based on NCI CTCAE v4.0.
Statistical Methods
[0416] Statistical analyses of safety and efficacy for DEP and EXP
were primarily descriptive in nature because the objectives of the
study were to determine the DLTs and MTD. These objectives were
achieved by the results of a deterministic algorithm; thus,
statistical hypothesis testing was neither intended nor appropriate
within this context. Continuous variables were summarized using
descriptive statistics [n, mean, standard deviation, median,
minimum, and maximum]. Categorical variables were summarized
showing the number and percentage (n, %) of patients within each
classification.
Study Procedures
Schedule of Study Events
[0417] The schedule of study activities, including assessments,
tests, exams, disease assessments, submission of tissue specimens,
and study drug administration) that will be conducted, beginning
with screening and continuing through Cycle 1 [day 1, day 8, and
day 15 of a 28 day cycle] are outlined in Table 8. Study that will
be conducted beginning with Cycle 2 [day 29 of cycle 1=day 1 of
cycle 2] are listed on Table 9.
TABLE-US-00015 TABLE 8 Schedule of study activities through Cycle 1
Day 29/ Day 1, Cycle Day 8 .+-. Day 15 .+-. 2 .+-. Clinical Day 1 1
d 1 d 3 d Molecular Screen -21 pre- post- Day 2 .+-. Day 3 .+-.
pre- post- pre- post- Day 16 .+-. Day 22 .+-. Refer to Screen days
Day -1 dose dose 4 h 4 h dose dose dose dose 2 d 1 d Table 9
Written informed X X consent Medical history X Demographics X Tumor
biopsy or X X archive tissue sample for p53 WT confirmation and
biomarker assessment Confirm X X eligibility Blood test for X HIV,
hepatitis B and C Serum or urine X pregnancy Vital signs: X X X X X
X X X X X X X Blood pressure, pulse, respiration rate, body
temperature Physical exam X X X X 12-lead ECG X X X Laboratory X X
X X X X X X assessments: Clinical chemistry (glucose, calcium,
albumin, total protein, sodium, potassium, CO.sub.2, chloride, BUN
[blood urea nitrogen], serum creatinine, ALP, ALT, AST, total and
direct bilirubin), hematology (complete blood count, platelets and
differential), urinalysis (dipstick measurement [pH, specific
gravity, protein, glucose, ketones, nitrite, leukocyte esterase]
with microscopic analysis, if results of the dipstick indicate
additional testing required), coagulation (PT, INR, aPTT).
Collection of X blood for immunogenicity Collection of X X X X X X
X X X blood for biomarker assessments Collection of X X X X X X X X
X blood for pharmacokinetic assessments ECOG X X X X Performance
Status Needle biopsy X X for biomarker assessments Tumor X
Assessment Aileron peptide 1 X X X dosing Concomitant X X X X X X X
X X X X X medications AE assessment X X X X X X X X X
TABLE-US-00016 TABLE 9 Schedule of study activities through Cycle 2
Day 29 of prior cycle/ Day 1 of next cycle for End-of-Study
patients continuing Day 8 .+-. Day 15 .+-. 30 .+-. 3 d treatment
.+-. 3 d 1 d 1 d After even after last Pre- post- pre- Post- pre-
post- Day 16 .+-. numbered dose or study dose dose dose dose dose
dose 2 d cycles withdrawal Serum pregnancy X Vital signs: X X X X X
X X X Blood pressure, pulse, respiration rate, body temperature.
Physical exam X X X X 12-lead ECG X X At X pre-dose pre-dose and
and EOI (+10 EOI (+10 min) min) Laboratory X X X X X assessments:
(Hematology Clinical chemistry, only) hematology, urinalysis,
coagulation (PT, INR, aPTT). Collection of blood X X X for
immunogenicity Collection of blood X X X X X for biomarker
assessments (each cycle) Collection of blood X X X X X X for
pharmacokinetic assessments (Cycle 2 and End-of-Study only) ECOG
Performance X X X X status Needle Biopsy for X biomarker
assessments Tumor assessment X At X end of even- numbered cycles.
Prior to start of the next treatment cycle Aileron peptide 1 X X X
dosing Concomitant X X X X X X X X medications AE assessment X X X
X X X X X (begins at the point of the first Aileron peptide 1
infusion and continues until 30 days after last infusion)
Pharmacokinetic Analysis
[0418] Levels of Aileron peptide 1 and its metabolites will be
measured in blood samples collected at specific time points
described below. Pharmacokinetic data will be tabulated and
summarized by individual patient and collectively by dose level.
Graphical displays will be provided where useful in the
interpretation of results.
[0419] Blood samples for pharmacokinetic assessment will be
collected at the following time points:
TABLE-US-00017 TABLE 10 Time points for collection of blood samples
for pharmacokinetic assessment Cycle 1 Day 1 within one hour before
SOI EOI (+5 min) 30 min after EOI (.+-.5 min) 1 hr after EOI (.+-.5
min) 2 hr after EOI (.+-.10 min) 4 hr after EOI (.+-.10 min) 8 hr
after EOI (.+-.10 min) Day 2 24 hours (.+-.4 hr) after the start of
Day 1 infusion Day 3 48 hours (.+-.4 hr) after the start of Day 1
infusion Day 8 within one hour before SOI EOI (+5 min) 30 min after
EOI (.+-.5 min) 1 hr after EOI (.+-.5 min) 2 hr after EOI (.+-.10
min) 4 hr after EOI (.+-.10 min) Day 15 within one hour before SOI
EOI (+5 min) 30 min after EOI (.+-.5 min) 1 hr after EOI (.+-.5
min) 2 hr after EOI (.+-.10 min) 4 hr after EOI (.+-.10 min) 8 hr
after EOI (.+-.10 min) Day 16 24 hours (.+-.4 hrs) after the start
of Day 15 infusion Cycle 2 Cycle 1 Day 29/ within one hour before
SOI Cycle 2 Day 1 EOI (+5 min) 30 min after EOI (.+-.5 min) 1 hr
after EOI (.+-.5 min) 2 hr after EOI (.+-.10 min) 4 hr after EOI
(.+-.10 min) Day 15 within one hour SOI EOI (.+-.5 min) 30 min
after EOI (.+-.5 min) Day 15 (cont.) 1 hr after EOI (.+-.5 min) 2
hr after EOI (.+-.10 min) 4 hr after EOI (.+-.10 min) Day 16 24
hours (.+-.4 hrs) after the start of Day 15 infusion End of study
visit During end of study visit
[0420] SOI stands for start of infusion of the Aileron peptide 1;
EOI stands for the end of infusion of the Aileron peptide 1.
Pharmacodynamic Analysis
[0421] Levels of p53, MDM2, MDMX, p21 and caspase will be measured
in tumor specimens collected before beginning treatment and at the
end of Cycle 1 or Cycle 2. MIC-1 will be measured in blood samples.
The specific time points for blood and tissue collection for
pharmacodynamic assessments are described below. Pharmacodynamic
data will be tabulated and summarized by individual patient and
collectively by dose level. Graphical displays will be provided
where useful in the interpretation of results.
[0422] Results available from previous genetic and biomarker tests,
and additional tests of the blood and tumor samples for biomarkers
relevant to the safety and efficacy of Aileron peptide-1 can be
investigated for possible correlation with patient outcome.
[0423] Blood samples for pharmacodynamic assessments will be
collected at the following time points:
TABLE-US-00018 TABLE 11 Time points for collection of blood samples
for pharmacodynamic assessments Cycle 1 Day 1 within 1 hour before
the start of infusion (SOI) Day 2 24 hours (.+-.4 hr) after the
start of Day 1 infusion Day 3 48 hours (.+-.4 hr) after the start
of Day 1 infusion Day 8 within 1 hour before SOI within 1 hour
after the end of infusion (EOI) Day 15 within 1 hour before SOI
within 1 hour after EOI Day 16 24 hours (.+-.4 hrs) after the start
of Day 15 infusion Day 22 During Day 22 visit Each Subsequent Cycle
Starting in Cycle 2 Cycle 1 Day 29/ within 1 hour before SOI Cycle
2 Day 1 within 1 hour after EOI Day 15 within 1 hour before SOI Day
16 24 hours (.+-.4 hrs) after the start of Day 15 infusion End of
study visit During end of study visit
Assessment of Clinical Activity of the Peptidomimetic
Macrocycle
[0424] To evaluate clinical activity, response rates and duration
of response based on RECIST 1.1 or other appropriate criteria will
be provided with a case-by-case description of all patients who
exhibit CR, PR or SD. A descriptive analysis of other evidence of
anti-tumor activity or other clinical benefit will be provided
based on clinical, radiographic or other appropriate assessment of
efficacy or clinical anti-tumor activity. Analysis of clinical
activity will be conducted on two patient populations: (1) the
subset of patients who receive at least one cycle of therapy and
have at least one post-baseline disease assessment (the
efficacy-evaluable population) and (2) a larger group of patients
that includes the efficacy-evaluable population as well as patients
who exhibit objective disease progression or experience a DLT
and/or unacceptable toxicity prior to the end of Cycle 1.
[0425] Imaging scans, physical examination, and/or laboratory-based
assays (e.g., prostate specific antigen) for patients with relevant
disease indications will be obtained at baseline (within 21 days of
Cycle 1 Day 1) and for objective tumor assessment after the second
cycle of treatment and after every second treatment cycle
thereafter (Cycle 4, Cycle 6, etc.). The same type of imaging,
physical examination, or laboratory-based assay procedure will be
used for each assessment for a patient. RECIST 1.1 will be used to
assess tumor response and duration of response. Scheduled scans
(and/or other laboratory-based assay) will be interpreted prior to
the start of the next treatment cycle. If the criteria for a CR or
PR are met, then the scan will be repeated no earlier than within 4
weeks to confirm the response. A responding patient (CR, PR or SD)
will continue on study, with disease assessment after every second
cycle, until disease progression, withdrawal of informed consent,
or unacceptable toxicity.
[0426] Films or other records from imaging procedures, including
those procedures performed at a regional or other facility outside
of the primary institutions, will be read and reviewed by the
radiology staff at the corresponding primary study institution for
the patient.
Drug Administration Study--II
Study Objectives
[0427] This Phase I open-label, multi-center, dose-escalation,
2-arm study was designed to evaluate the safety, tolerability,
pharmacokinetic, pharmacodynamic, and anti-tumor effects of Aileron
peptide-1 administered by IV infusion using 2 different dosing
regimens of a 28- or 21-day cycle, in patients with advanced solid
tumors or lymphomas expressing WT p53 protein (see p53 Status
Determination below). Patients received Aileron peptide-1 either
once weekly for three consecutive weeks for a 28-day cycle or twice
weekly for two consecutive weeks for a 21-day cycle. Many patients
with a solid tumor or lymphoma present circulating tumor cells
(CTC) in peripheral blood, which can be detected and analyzed using
flow cytometry. This enabled the detection of study drug-specific
target engagement in these cells.
[0428] This study consisted of a DEP and a EXP. The DEP was a "3+3"
dose escalation designed to establish the MTD or the OBD of Aileron
peptide-1. The EXP enrolled up to 2 distinct groups of patients
with specific solid tumors to further investigate the clinical
safety profile and potential efficacy of Aileron peptide-1 at the
MTD or OBD.
Starting Dose, Dose Escalation, and Dose Reduction
[0429] All subjects were dosed at a pre-defined level based on body
weight. Starting at Dose Level (DL) 3, patients was sequentially
assigned to one of two treatment arms: Dose Regimen (DR) A testing
administration of Aileron peptide-1 once per week, or Dose Regimen
(DR) B testing administration of Aileron peptide-1 twice per week.
For Dose Level 3, DR-A were enrolled first, DR-B were enrolled
second. The starting dose (DL-1) in DEP, based on results from
nonclinical toxicology assessments, was 0.16 mg/kg.
[0430] During the first 2 dose levels, patients received Aileron
peptide-1 on Days 1, 8, and 15 of a 28-day cycle. Starting with DL
3, patients in DR-A continued being treated once a week on Days 1,
8, and 15 of a 28-day cycle, whereas patients in DR-B were treated
twice a week, on Days 1 and 4, 8 and 11 of a 21-day cycle. This
dosing schedule is summarized in FIG. 2.
[0431] Doses were doubled in subsequent dose levels until .gtoreq.1
of 3 patients in a cohort experienced any drug-related Grade
.gtoreq.2 adverse event (AE). A drug-related AE is an event that is
possibly, probably or definitely attributed to Aileron peptide-1.
Grading of AEs was defined by the NCI Common Terminology Criteria
for Adverse Events (CTCAE) version 4.03. Subsequent dose escalation
continued using modified Fibonacci sequence (i.e., 67%, 50%, 40%,
and 33%; FIGS. 3 and 4).
[0432] Escalation to the next dose level within each DR proceeded
in the absence of DLT at the completion of Cycle 1 (treatment
cycle=28 days for DR-A and 21 days for DR-B). Escalation to the
next dose level within each DR was decided by a Safety Review
Committee (SRC), consisting of the Principal Investigators,
Sponsor's Medical Monitor, and Safety Physician representative,
which reviewed all available safety information from all
patients.
[0433] Within each Dose Regimen cohort, if no DLT was observed in a
cohort, the subsequent patient group was enrolled at the next
planned dose level of that dose regimen. If a DLT was observed in
.gtoreq.2 of 3 patients at any dose level no further dose
escalation occurred in that DR, and the current dose was defined as
the maximum administered dose (MAD). If a DLT was observed in 1 of
3 patients in a cohort at any dose level, then up to 3 additional
patients were enrolled in the same DR at that dose level. If a DLT
was observed in 2 or more patients in the expanded cohort, then no
further dose escalation occurred, and the current dose was defined
as the MAD. After the MAD was defined, either the previously
administered lower dose was expanded to a total of 6 patients, or
an intermediate dose (between the MAD and the previous dose level)
was investigated in a total of 6 patients. The highest dose
tolerated in at least 5 of 6 patients was defined as the MTD or
OBD.
[0434] The selection of dose regimen and dose level for up to 2 EXP
cohorts was based on the MTD determination in Cycle 1, as well as
the cumulative safety, efficacy and pharmacokinetic/pharmacodynamic
profile of Aileron peptide-1 in subsequent treatment cycles in
DEP.
[0435] Dose levels were not increased between cycles within each
cohort, and patients were assigned only one dose level (i.e., no
intra-patient dose escalation).
Statistical Methods
[0436] Results from DR-A and DR-B will be compared for all dose
levels and patient groups.
Screening Assessments and Other Requirements Prior to Day 1 of
Cycle 1
[0437] Molecular Screening Prior to Day 1 of Cycle 1: Molecular
screening encompassed the following prior to the first
administration of Aileron peptide-1 (Day 1 of Cycle 1): (i)
collection of signed informed consent for molecular screening; (ii)
collection of an archived tumor sample or a fresh tumor biopsy
(unless a biopsy poses significant clinical risk) for p53 testing;
(i) if confirmed to be p53 WT, the remainder of the tissue sample
from enrolled patients was used to test for pharmacodynamic
biomarkers. Confirmation of p53 WT status before administration of
the first dose of Aileron peptide-1 was mandatory for enrollment in
stage 1 of DEP for patients starting at Dose Level 4 and higher and
stage 2 (if necessary) of DEP and EXP for all patients.
[0438] Molecular Screening Prior At Dose Level 4 and higher in
Stage 1 of the DEP (as well as for all patients enrolled in Stage 2
of the DEP), molecular screening in patients with unknown p53
status was done prior to initiating the clinical screening. If the
p53 status was known to be WT, these patients proceeded to clinical
screening and were enrolled and receive Aileron peptide-1 before
confirmation of p53 WT by the central laboratory.
[0439] In the EXP, patients had completed molecular screening at
the central laboratory prior to proceeding to enrolment. These
patients were only enrolled and received Aileron peptide-1 after
confirmation of p53 WT by the central laboratory.
Clinical Screening within 21 Calendar Days Prior to Day 1 of Cycle
1 for DR-A and DR-B, all Dose Levels
[0440] The screening assessments and procedures performed within 21
calendar days (or as noted) prior to the first administration of
Aileron peptide-1 (Day 1 of Cycle 1) included collection of signed
informed consent, medical history (evaluation of baseline signs and
symptoms), demographics, eligibility assessment, blood test for
HIV, hepatitis B and C, vital signs (includes blood pressure,
pulse, respiration rate, body temperature), physical examination,
ECG, laboratory assessments including clinical chemistry (glucose,
calcium, albumin, total protein, sodium, potassium, CO.sub.2,
chloride, phosphate, BUN [blood urea nitrogen], serum creatinine,
uric acid, ALP, ALT, AST, total and direct bilirubin), hematology
(complete blood count, platelets and differential), urinalysis
(dipstick measurement [pH, specific gravity, protein, glucose,
ketones, nitrite, leukocyte esterase] with microscopic analysis, if
results of the dipstick indicate additional testing required),
coagulation (PT, INR, aPTT), ECOG performance status, RECIST--(for
solid tumor patients) or IWG--(for lymphoma patients) compliant
imaging for disease assessment and tumor measurements as well as
laboratory-based assays (e.g., prostate specific antigen) for
patients with relevant disease indications, including a baseline
PET-FDG and possibly FLT-PET scan(s), concomitant medications
(current medications and those taken within 28 days of Cycle 1, Day
1).
Within 7 Calendar Days Prior to Day 1 of Cycle 1 for DR-A and DR-B,
all Dose Levels
[0441] The screening assessments that was completed within 7
calendar days prior to the first administration of Aileron
peptide-1 (Day 1 of Cycle 1) included serum or urine pregnancy test
(.beta.-hCG) for women of child-bearing potential: performed within
2 days prior to first dose of Aileron peptide-1, confirm
eligibility, vital signs, laboratory assessments--can be omitted if
screening tests were performed within 7 days prior, ECOG
performance status, and concomitant medications.
Requirements During Cycle 1
Day 1 of Cycle 1, for DR-A and DR-B, all Dose Levels
[0442] The study procedures that were performed prior to
administration of Aileron peptide-1 included vital signs: within 30
minutes prior to SOI, physical exam, ECG: within 30 minutes prior
to SOI (performed in triplicate (5-10 min between readings)),
collection of blood for immunogenicity within 1 hr prior to SOI,
collection of blood for all biomarker assessments within 1 hr prior
to SOI, collection of blood for pharmacokinetic assessments: within
1 hr prior to SOI, and concomitant medications.
[0443] The study procedures that were performed after
administration of Aileron peptide-1 included vital signs: (During
infusion) 30 min (.+-.3 min); (Post-infusion) At EOI (.+-.5 min),
and 1 (.+-.5 min) and 2 hrs (.+-.10 min) following EOI; ECG: At EOI
(.+-.5 min) and 1 hr (.+-.5 min) and 2 hrs (.+-.10 min) following
EOI. Perform in triplicate (5-10 min between readings) only if
patient has a QTc that is a) >500 msec; b) increased by 60 msec
over pre-dose; or c) decreased by 50 msec below pre-dose recording;
collection of blood for pharmacokinetic assessments: At EOI (.+-.5
min), 30 min (.+-.5 min) and 1 hr (.+-.5 min), 2 (.+-.10 min), 4
(.+-.10 min) and 8 hrs (.+-.10 min) following EOI; collection of
blood for all biomarker assessments, EOI (.+-.5 min) and 1 hr
(.+-.5 min) and 2, 4, and 8 hrs (.+-.10 min) following EOI;
concomitant medications; and adverse event (AE) assessment
Day 2 of Cycle 1, for DR-A and DR-B, all Dose Levels
[0444] The study procedures that were performed included vital
signs, laboratory assessments, collection of blood for all
biomarker assessments at 24 hr (.+-.4 hr) after Day 1 SOI,
collection of blood for pharmacokinetic assessments at 24 hr (.+-.4
hr) after Day 1 SOI, concomitant medications, AE assessment, and
TLS monitoring (via routine laboratory assessment sample)
Day 3 of Cycle 1, for DR-A and DR-B, all Dose Levels
[0445] The study procedures performed included vital signs,
laboratory assessments (collection of blood for all biomarker
assessments at 48 hr (.+-.4 hr) after Day 1 SOI), collection of
blood for pharmacokinetic assessments at 48 hr (.+-.4 hr) after Day
1 SOI, concomitant medications and AE assessment.
Day 4 of Cycle 1, for DR-B ONLY, all Dose Levels
[0446] The study procedures performed prior to administration of
Aileron peptide-1 included vital signs: within 30 minutes prior to
SOI, physical exam, ECG: within 30 minutes prior to SOI. Perform in
triplicate (5-10 min between readings), laboratory assessments,
collection of blood for immunogenicity within 1 hr prior to SOI,
collection of blood for all biomarker assessments within 1 hr prior
to SOI, collection of blood for pharmacokinetic assessments: within
1 hr prior to SOI, concomitant medications, and dverse Event (AE)
Assessments
[0447] The study procedures performed after administration of
Aileron peptide-1 included vital signs: (During infusion) 30 min
(.+-.3 min); (Post-infusion) At EOI (.+-.5 min), and 1 and 2 hrs
(.+-.10 min) following EOI; ECG: At EOI (.+-.5 min) and 1 hr (.+-.5
min) and 2 hr (.+-.10 min) following EOI. Perform in triplicate
(5-10 min between readings) only if patient has a QTc that is a)
>500 msec; b) increased by 60 msec over pre-dose; or c)
decreased by 50 msec below pre-dose recording; laboratory
assessments; collection of blood for all biomarker assessments
within 1 hr after EOI; ollection of blood for pharmacokinetic
assessments at EOI (.+-.5 min), 30 min (.+-.5 min) and 1 hr (.+-.5
min), 2 (.+-.10 min), 4 (.+-.10 min) after EOI; concomitant
medications and adverse event (AE) assessment
Day 8 of Cycle 1 for DR-A and DR-B, all Dose Levels
[0448] The study procedures performed prior to administration of
Aileron peptide-1 included vital signs: within 30 minutes prior to
SOI; physical exam; laboratory assessments, collection of blood for
all biomarker assessments within 1 hr prior to SOI, collection of
blood for pharmacokinetic assessments within 1 hr prior to SOI,
ECOG performance status, concomitant medications and AE
assessment.
[0449] The study procedures performed after administration of
Aileron peptide-1 include vital signs: (During infusion) 30 min
(.+-.3 min); (Post-infusion) At EOI (.+-.5 min), and 1 hr (.+-.5
min) and 2 hrs (.+-.10 min) following EOI; collection of blood for
all biomarker assessments within 1 hr after EOI; collection of
blood for pharmacokinetic assessments at EOI (.+-.5 min) and 30 min
(.+-.5 min), 1 hr (.+-.5 min), 2 and 4 hrs (.+-.10 min) after EOI;
concomitant medications and AE assessment.
Day 15 for DR-A and Day 11 for DR-B of Cycle 1
[0450] The study procedures performed prior to administration of
Aileron peptide-1 included vital signs: within 30 minutes prior to
SOI; physical exam; laboratory assessments; collection of blood for
all biomarker assessments within 1 hr prior to SOI; collection of
blood for pharmacokinetic assessments within 1 hr prior to SOI;
ECOG performance status; concomitant medication and AE
assessment.
[0451] The study procedures performed after administration of
Aileron peptide-1 included vital signs: (During infusion) 30 min
(.+-.3 min); (Post-infusion) At EOI (.+-.5 min), and 1 hr (.+-.5
min) and 2 hrs (.+-.10 min) following EOI; collection of blood for
pharmacokinetic assessments At EOI (.+-.5 min) and 30 min (.+-.5
min), 1 hr (.+-.5 min); 2, 4, and 8 hrs (.+-.10 min) after EOI;
collection of blood for all biomarker assessments within 1 hr at
EOI (.+-.5 min); EOI plus 1 hr (.+-.5 min); 4, and 8 hr (.+-.10
min) after EOI; concomitant medications; and AE assessment.
Day 16 DR-A and Day 12 DR-B for Cycle 1
[0452] The study procedures performed included vital signs;
laboratory assessments; collection of blood for biomarker
assessments: at 24 hr (.+-.4 hr) after SOI on the day prior; only
for patients with a successful study biopsy performed prior to
start of study medication: Needle biopsy for biomarker assessments-
to be performed within 48 hours of Cycle 1, Day 15 (DR-A) or Day 11
(DR-B) infusion OR Cycle 2, Day 15 (DR-A) or Day 11 (DR-B)
infusion, at the discretion of the Investigator (unless the biopsy
poses significant risk to the patient); collection of blood for
pharmacokinetic assessments at 24 hr (.+-.4 hr) after Day 15 (DR-A)
or Day 11 (DR-B) SOI; collection of blood for all pharmacodynamic
assessments at 24 hr (.+-.4 hr) after SOI on the day prior;
concomitant medications; cAE assessment; and FLT-PET for patients
who received FLT-PET at screen and have SUV .gtoreq.5.
Day 22 for DR-A and Day 18 for DR-B or of Cycle 1
[0453] The study procedures performed included vital signs;
laboratory assessments--hematology only; collection of blood for
all biomarker assessments; concomitant medications; and AE
assessment.
Day 29 for DR-A and Day 22 for DR-B of Cycle 1 (-1 Day Up to +3
Days)/Cycle 2 Day 1
[0454] Procedures listed below under Requirements During Subsequent
Cycles Beginning with Cycle 2 were performed. Note: "Day 22 or
29"=Day 1 of next cycle for patients continuing treatment. Cycle 1
Day 22 or 29/Cycle 2 Day 1 pre-dose evaluations were to be done
within 3 days prior to next cycle drug administration.
[0455] If the patient was not continuing treatment beyond Cycle 1
the procedures listed below under End-of-Study Visit section were
performed.
Requirements During Subsequent Cycles Beginning with Cycle 2
Day 29 for DR-A and Day 22 for DR-B of Prior Cycle/Day 1 of Cycle 2
and Subsequent Cycles
[0456] Note: "Day 22 or 29"=Day 1 of next cycle for patients
continuing treatment. Day 22 or 29 of prior cycle/Day 1 of current
cycle pre-dose evaluations were done within 3 days prior to drug
administration.
[0457] Note: Blood samples to evaluate CTC were not collected in
Cycle 2 or subsequent cycles.
[0458] The study procedures performed prior to administration of
Aileron peptide-1 included vital signs: within 30 minutes prior to
SOI; physical exam; ECG: within 30 minutes prior to SOI. Perform in
triplicate (5-10 min between readings); laboratory assessments;
collection of blood for immunogenicity: within 1 hr prior to SOI;
collection of blood for biomarker assessments (MIC-1 only): within
1 hr prior to SOI; collection of blood for pharmacokinetic
assessments (Cycle 2 Only): within 1 hr prior to SOI; ECOG
performance status; concomitant medications; and AE assessment.
[0459] The study procedures performed after administration of
Aileron peptide-1 included vital signs: (During infusion) 30 min
(.+-.3 min); (Post-infusion) at EOI (.+-.5 min) and as clinically
indicated following EOI; ECG: at EOI (.+-.5 min). Perform in
triplicate (5-10 min between readings) only if patient has a QTc
that is a) >500 msec; b) increased by 60 msec over pre-dose; or
c) decreased by 50 msec below pre-dose recording; collection of
blood for biomarker assessments (MIC-1 only): within 1 hr after
EOI; collection of blood for pharmacokinetic assessments (Cycle 2
Only): At EOI (.+-.5 min) and 30 min ((.+-.5 min), 1 hr (.+-.5
min), 2 and 4 hrs (.+-.10 min) after EOI; concomitant medications;
and AE assessment.
Day 8 of DR-A and Days 4 and 8 of DR-B of Cycle 2 and Beyond
[0460] The study procedures performed prior to administration of
Aileron peptide-1 included vital signs: within 30 minutes prior to
SOI; physical exam; laboratory assessments--hematology only; ECOG
performance status; concomitant medications; and AE assessment.
[0461] The study procedures performed after administration of
Aileron peptide-1 included vital signs: (During infusion) 30 min
(.+-.3 min); (Post-infusion) at EOI (.+-.5 min) and as clinically
indicated following EOI; concomitant medications; and AE
assessment.
Day 15 of DR-A and Day 11 of DR-B of Cycle 2 and Beyond
[0462] The study procedures performed prior to administration of
Aileron peptide-1 included vital signs: within 30 min prior to SOI;
physical exam; laboratory assessments; collection of blood for
biomarker assessments (MIC-1 only): within 1 hr prior to SOI;
collection of blood for pharmacokinetic assessments (Cycle 2 Only):
within 1 hr prior to SOI; ECOG performance status; concomitant
medications; and AE assessment.
[0463] The study procedures performed after administration of
Aileron peptide-1 included: vital signs: (During infusion) 30 min
(.+-.3 min); (Post-infusion) at EOI (.+-.5 min) and as clinically
indicated following EOI; collection of blood for biomarker
assessments (MIC-1 only): within 1 hr after EOI; collection of
blood for pharmacokinetic assessments (Cycle 2 Only) at EOI (.+-.5
min) and 30 min (5 min), 1 hr (.+-.5 min), 2 and 4 hrs (.+-.10 min)
after EOI; concomitant medications and AE assessment.
Day 16 DR-A and Day 12 of DR-B of Cycle 2 and Beyond
[0464] The study procedures performed included vital signs,
laboratory assessments, collection of blood for biomarker
assessments: at 24 hr (.+-.4 hr) after Day 15 or 11 SOI, collection
of blood for pharmacokinetic assessments (Cycle 2 Only): at 24 hr
(.+-.4 hr) after Day 15 or 11 SOI, concomitant medications, and AE
assessment.
After Even Numbered Cycles
[0465] Blood was collected for immunogenicity. Tumor assessment was
performed following same procedure used for baseline measurement,
for example imaging, physical exam as well as laboratory-based
assays (e.g., prostate specific antigen) for patients with relevant
disease indications.
[0466] For patients who achieved "Stable Disease" as defined by
RECIST or IWG criteria, a FDG-PET scan was indicated, provided that
an evaluable FDG-PET-scan was performed prior to starting treatment
with study drug.
CT Imaging
[0467] All patients receive a CT image prior to the first dose.
After dosing commences in Dosing Regimen--A (DR-A), CT images will
be obtained at the end of Cycle 2 and every other cycle thereafter
in DR-A, e.g., Cycles 4, 6, and 8. In Dosing Regimen-B (DR-B) CT
images will be obtained after the last infusion in Cycle 3 and
every third cycle thereafter in DR-B, e.g., Cycles 6, 9, and 12.
Images will be obtained after the last dose is administered in
those cycles but prior to the Day 18 visit.
End-of-Study Visit
[0468] The end-of-study visit was to be conducted 30 (.+-.2)
calendar days after the last administration of Aileron peptide-1 or
withdrawal from the study. The study procedures performed included
serum or urine pregnancy, vital signs, physical exam, ECG,
laboratory assessments, collection of blood for immunogenicity,
collection of blood for biomarker assessment, collection of blood
for pharmacokinetic assessment, ECOG performance status, tumor
assessment following same procedure used for baseline measurement,
for example imaging, physical exam as well as laboratory-based
assays (e.g., prostate specific antigen) for patients with relevant
disease indications, concomitant medication and AE assessment.
Pharmacodynamic Assessment
[0469] Blood samples for pharmacodynamic assessments were collected
at the following timepoints:
TABLE-US-00019 TABLE 12 Cycle 1 and Cycle 2 Dose Regimen
Pharmacodynamic Assessments Dose Regimens Assessment Blood Sample
Collection Schedule Cycle 1 DR-A, DR-B, or Both: Day 1- Both (pre)
MIC-1 and within 1 hour before the start of CTC infusion (SOI) Day
1- Both (post) Samples EOI (+5 min) & EOI + 1 hr (.+-.5 min),
2, 4, and 8 hr (.+-.10 min) Day 2- Both 24 hours (.+-.4 hr) after
SOI on Day 1 Day 3- Both 48 hours (.+-.4 hr) after SOI on Day 1 Day
8 DR-A within 1 hour before SOI and Day 4 & 8 DR-B within 1
hour after the end of infusion (EOI) Day 15 DR-A within 1 hour
before SOI and Day 11 DR-B within 1 hour after EOI Day 15 DR-A
within 1 hour before SOI and Day 11 DR-B EOI (+5 min) & EOI + 1
hr (.+-.5 min), 2, 4, and 8 hr (.+-.10 min) Day 16 DR-A 24 hours
(.+-.4 hrs) after SOI day Day 12 DR-B prior Day 22 DR-A During Day
visit Day 18 DR-B Each Subsequent Cycle Starting in Cycle (Cy) 2 Cy
1 Day 29 DR-A MIC-1 Only within 1 hour before SOI and Cy 1 Day 23
DR-B = within 1 hour after EOI Cycle 2 Day 1 Day 15 DR-A within 1
hour before SOI and Day 11 DR-B within 1 hour after EOI Day 16 DR-A
24 hours (.+-.4 hrs) after SOI day Day 12 DR-B prior End of study
visit During end of study visit NOTE: no pharmacodynamic
assessments for solid tumors or lymphoma on Day 8 DR-A or Days 4
and 8 DR-B
Pharmacokinetic (PK) Assessments
[0470] Blood samples for pharmacokinetic assessment were collected
at the following timepoints:
TABLE-US-00020 TABLE 13 Cycle 1 and Cycle 2 Dose Regimen
Pharmacokinetic Assessments Cycle 1 Day 1 within one hour before
SOI DR-A and DR-B EOI (+5 min) 30 min after EOI (.+-.5 min) 1 hr
after EOI (.+-.5 min) 2 hr after EOI (.+-.10 min) 4 hr after EOI
(.+-.10 min) 8 hr after EOI (.+-.10 min) Day 2 24 hours (.+-.4 hr)
after SOI day DR-A and DR-B prior Day 3 48 hours (.+-.4 hr) after
SOI Day 1 DR-A and DR-B Day 8, DR-A within one hour before SOI Days
4 & 8, DR-B EOI (+5 min) 30 min after EOI (.+-.5 min) 1 hr
after EOI (.+-.5 min) 2 hr after EOI (.+-.10 min) 4 hr after EOI
(.+-.10 min) Day 15, DR-A within one hour before SOI Day 11, DR-B
EOI (+5 min) 30 min after EOI (.+-.5 min) 1 hr after EOI (.+-.5
min) 2 hr after EOI (.+-.10 min) 4 hr after EOI (.+-.10 min) 8 hr
after EOI (.+-.10 min) Day 16, DR-A 24 hours (.+-.4 hrs) after SOI
day Day 12, DR-B prior Cycle 2 Cycle 1 Day 29/ within one hour
before SOI Cycle 2 Day 1, DR-A EOI (+5 min) Cycle 1 Day 23/ 30 min
after EOI (.+-.5 min) Cycle 2 Day 1, DR-B 1 hr after EOI (.+-.5
min) 2 hr after EOI (.+-.10 min) 4 hr after EOI (.+-.10 min) Cycle
2 Day 15, DR-A within one hour SOI Day 11, DR-B EOI (+5 min) 30 min
after EOI (.+-.5 min) 1 hr after EOI (.+-.5 min) 2 hr after EOI
(.+-.10 min) 4 hr after EOI (.+-.10 min) Day 16, DR-A {close
oversize brace} 24 hours (.+-.4 hrs) after SOI day Cycle 2 Only
prior Day 12, DR-B NOTE: no PK assessments on Day 8 in DR-A or Days
4 and 8 in DR-B End of study visit During end of study visit
Example 4: Further Studies
[0471] Aileron peptide-1 was evaluated for safety, tolerability,
pharmacokinetics and pharmacodynamics in adult patients with
advanced solid tumors or lymphomas expressing WT p53 that are
refractory to or intolerant of standard therapy, or for which no
standard therapy exists. FIG. 6 shows one way Aileron peptide-1 was
designed to inhibit MDMX and/or MDM2, which leads to reactivation
of WT p53.
[0472] Aileron peptide-1 was able to penetrate the cell membrane
and localize within the nucleus. Further Aileron peptide-1 can
disrupt protein-protein interactions within the cell such as the
interactions between p53 and MDM2 and MDMX.
[0473] Several in-vivo and in-vitro studies of Aileron peptide-1
were conducted. In these studies, Aileron peptide-1 binded to both
MDM2 and MDMX with nanomolar affinities and demonstrated evidence
of specific on-target mechanism in vitro by gene expression
profiling. In addition, Aileron peptide-1 demonstrated tumor growth
suppression, p53-dependent cell cycle arrest, apoptosis and
anti-tumor activity in an MDM2/MDMX-overexpressing xenograft cancer
model with clear correlation to on-target pharmacokinetic and
pharmacodynamic, or pharmacokinetic/pharmacodynamic, activity.
[0474] Dose escalation phase were designed to evaluate Aileron
peptide-1 in patients with solid tumors or lymphoma. The dose
escalation phase was not limited by type of tumor or lymphoma.
Aileron peptide-1 was administered to patients with sarcoma,
gastric cancer, non-small cell lung cancer, ovarian cancer and
thymoma. In some cases Aileron peptide-1 was used to treat tumors
and lymphomas in which WT p53 is prevalent in greater than 50% of
patients. p53 wild-type status is prevalent in greater than 50% of
patients suffering from at least 19 different tumor types. Thus,
indication potential can vary from orphan indications or large
market opportunities. See e.g., FIG. 7.
[0475] p53 signal activation study was conducted to determine if
Aileron peptide-1 had a differential effect on cancer cell lines
with mutant p53 compared to WT p53. In the study, we measured the
effect of Aileron peptide-1 in 312 cell lines across a variety of
different cancers to compare the effect of Aileron peptide-1 in
cell lines with mutant p53 and cell lines with WT p53. See FIG. 8.
In the 207 mutant p53 cell lines, Aileron peptide-1 had no
discernable effect, but in the 105 WT p53 cell lines, nearly all
showed tumor cell death. See FIG. 8. The WT p53 cell lines that did
not show tumor cell death included WT p53 cell lines associated
with Human Papilloma Virus, or HPV, related cancers, such as
cervical and head and neck. By concentrating on WT p53 and
responsive tumors, we are able to predict patient populations that
can have a better chance of response from our product
candidate.
[0476] In another study, the binding affinity of Aileron peptide-1
for MDM2 or MDMX relative to the binding affinity for MDM2 and MDMX
of WT p53 and of an MDM2 small molecule inhibitor was measured. The
affinity of a drug to a receptor is the measure of how effectively
that drug binds to its target and can provide insight on the
potential for on-target effect and off-target toxicity. Aileron
peptide-1 was designed to bind to MDM2 and/or MDMX with a higher
affinity than WT p53 so that Aileron peptide-1 disrupts the binding
of MDM2 and/or MDMX to WT p53 by binding to MDM2 and/or MDMX in
lieu of p53. Such binding can enable p53 to be released and
activated. In this study, we also measured a small molecule MDM2
inhibitor's binding affinity to MDMX, which showed no binding to
this target. Table 14 below shows Aileron peptide-1's ability to
bind to MDM2 and MDMX relative to WT p53 and the small molecule
MDM2 inhibitor.
TABLE-US-00021 TABLE 14 Aileron peptide-l's ability to bind to MDM2
and MDMX relative to WT p53 and the small molecule MDM2 inhibitor.
Kd, nM WT p53 Aileron peptide-l MDM2 Inhibitor MDM2 770 13.7 9.8
MDMX 480 8.9 >3000
In Vivo
[0477] We studied the effects of Aileron peptide-1 in both solid
tumors. In the study depicted FIGS. 9a and 9b, we evaluated the
effect of Aileron peptide-1 administered by an intravenous, or IV,
injection in an MDMX-driven MCF-7 breast cancer xenograft model. In
this study, we evaluated different doses, schedules and durations
of treatment with Aileron peptide-1 and vehicle to determine effect
on tumor volume growth. Aileron peptide-1 showed statistically
significant tumor growth inhibition at doses ranging from 2.5 mg/kg
to 5 mg/kg to 10 mg/kg and 20 mg/kg when these doses were
administered twice a week for a 28 day period. See FIGS. 11a and
11b.
Toxicology and Nonclinical Safety Experiments
[0478] The pivotal 4-week multiple-dose GLP studies in rats and
monkeys utilized twice-weekly IV dosing rather than the once-weekly
IV dosing planned as the initial clinical regimen. The studies
provided dose- and exposure-related assessments during both dosing
and recovery periods, and results were utilized to define the
maximum tolerated doses (MTD) and estimate the severely toxic dose
for 10% (STD.sub.10) of rats and the highest non-severely toxic
dose (HNSTD) in monkeys. All gross and microscopic signs of
intolerance (e.g., reduced organ weights, sporadic findings of
multi-tissue hemorrhage and hepatic necrosis) and changes in serum
chemistry parameters were considered as secondary to red blood cell
(RBC), platelet and/or white blood cell (WBC) depletions or
anorexia and dehydration in both species. Recovery assessments
revealed regenerative and compensatory changes consistent with
marrow cell survival and reversibility of all related hematologic
and secondary toxicities.
[0479] The DLT in both animal species appears to be related to the
suppression of hematopoietic cells in the bone marrow, in
particular cells of the megakaryocyte lineage, resulting in
significant decreases in peripheral blood platelets that
demonstrated recovery upon the cessation of dosing. See FIG. 7.
[0480] The STD.sub.10 in rats was defined at 10 mg/kg based on the
mortality of one animal in a satellite group for hematology
sampling during recovery. The HNSTD in monkeys was defined at 5
mg/kg, based on a complete lack of significant thrombocytopenia at
this lowest dose level. However, almost all of the monkeys at the
mid- and high-dose levels tolerated Aileron peptide-1
administration well; only one animal at each of these dose levels
developed significant thrombocytopenia
(<100,000.times.10.sup.6/ml).
[0481] Rats are more sensitive to the bone marrow and hematologic
effects of Aileron peptide-1 than monkeys on the basis of exposures
at maximally tolerated doses. Exposure at rat STD.sub.10
(AUC.sub.0-.infin.=562 .mu.ghr/mL at 10 mg/kg) was below that of
HNSTD in monkeys (AUC.sub.0-.infin.=813 .mu.ghr/mL at 5 mg/kg).
These in vivo results correlate with those obtained from in vitro
hemotoxicity assays via luminescence output (HALO). In these
investigations, Aileron peptide-1 in general inhibited the induced
proliferation of bone marrow precursor cells from rats to a greater
extent than those from monkeys or humans. IC.sub.50 values were
.about.2- to 8-fold higher for rat cells than for monkey or human
cells, with the largest difference noted for megakaryocyte colony
forming cells, the platelet precursors. These results correlate
with in vivo findings indicating that rats are more sensitive to
the bone marrow and hematologic effects of Aileron peptide-1 than
monkeys on the basis of dose and exposures at maximally tolerated
doses. These results also suggest that, in terms of projecting
potential bone marrow and hematological toxicity levels in humans,
the monkey pharmacokinetic--pharmacodynamic data can be more
clinically relevant than the rat data.
[0482] Aileron peptide-1 was negative in genetic toxicology
studies, including bacterial mutagenicity (Ames), chromosomal
aberrations (human peripheral blood lymphocyte) and in vivo
micronucleus (rat bone marrow) assays. Safety pharmacology studies
were performed to assess the effects of Aileron peptide-1 on hERG
potassium channels in vitro and on cardiac function in cynomolgus
monkeys. There were no significant adverse findings in these
studies.
[0483] Compared to the twice-weekly IV dosing schedule utilized in
the 4-week GLP toxicity studies, the first-in-human clinical trial
of Aileron peptide-1 will initially assess once-weekly IV dosing
for three weeks. In addition, the demonstrated reversibility of
Aileron test peptide-1-induced hematologic effects, the ability to
detect such findings with routine laboratory measurements, and the
availability of effective supportive therapies, all provide
additional safety margin in the clinic.
Pharmacokinetics and Absorption, Distribution, Metabolism and
Excretion
[0484] In rats, Aileron peptide-1 generally showed linear,
dose-proportional increases in C.sub.max and AUC. In the 4-week rat
GLP toxicity study, C.sub.max of Aileron peptide-1 ranged from 49.9
to 186 .mu.g/mL, AUC.sub.0-.infin. ranged from 90.5 to 562
.mu.ghr/mL, and clearance ranged from 19.2 to 28.3 mL/hr/kg.
Half-life (t.sub.1/2) values could not be calculated due to
variable coefficients of determination (r.sup.2<0.9).
[0485] In non-human primates, Aileron peptide-1 generally showed
exposures that increased proportionally with dose, although an
apparent plateau in exposure was observed at the high-dose group
(20 mg/kg) in the 4-week monkey GLP toxicity study. In the study,
C.sub.max of Aileron peptide-1 ranged from 133 to 562 .mu.g/mL,
t.sub.1/2 ranged from 3.7 to 6.0 hrs, AUC.sub.0-.infin. ranged from
813 to 1,600 .mu.ghr/mL, and clearance ranged from 6.5 to 13.8
mL/hr/kg.
[0486] No significant sex-based differences in pharmacokinetic
parameters were observed in either rats or monkeys, and no
accumulation was observed following repeated doses on a
twice-weekly schedule in the GLP toxicity studies.
[0487] Proteolysis is the expected major biotransformation pathway
of Aileron peptide-1. The predominant metabolite, Aileron peptide
metabolite-1, is a 3-amino acid truncation with the cyclic peptide
portion intact, and the same metabolite profile was noted in in
vitro stability studies with monkey, rat, mouse and human
cryopreserved hepatocytes. In a single-dose rat study,
hepatobiliary metabolism and elimination represented the
predominant clearance pathway for Aileron peptide-1, with Aileron
peptide metabolite-1 as the major excretion product observed in the
bile.
[0488] In vitro studies revealed that Aileron peptide-1 is not an
inhibitor of any cytochrome P450 (CYP) isoforms tested. In vitro
assays for CYP induction also did not indicate any significant
treatment-related effects with Aileron peptide-1. Based on these
findings, the potential of clinically relevant drug-drug
interactions for concomitant medications that are cleared through
CYP-mediated mechanisms is deemed to be low.
[0489] Aileron peptide-1 was tested in vitro against common
transporters, and >90% inhibition of organic anion transporter
polypeptide (OATP) members OATP1B1 and OATP1B3 and bile salt export
pump (BSEP) at concentrations that can be clinically relevant
(e.g., at C.sub.max of high-dose levels) was observed. Based on
these findings, the potential of clinically relevant drug-drug
interactions by Aileron peptide-1 with medications (e.g.,
methotrexate, statins) that are significantly cleared by
hepatobiliary transporters should be considered.
In Vivo
[0490] An open-label, multi-center, dose-escalation, two-arm study
was used to design to evaluate the safety, tolerability,
pharmacokinetic, pharmacodynamic and anti-tumor effects of Aileron
peptide-1 administered by intravenous (IV) infusion in patients
with advanced solid tumors or lymphomas expressing WT p53 that are
refractory to or intolerant of standard therapy, or for which no
standard therapy exists. The study included a dose escalation phase
to establish the maximum tolerated dose, or MTD, or the optimum
biologic dose, or OBD, of Aileron peptide-1, and a dose expansion
phase to investigate the clinical safety profile and potential
efficacy of Aileron peptide-1 at the MTD or OBD. In the expansion
phase of the study, Aileron peptide-1 was studied in distinct
groups of patients with specific solid tumors or lymphomas. The
selection of the solid tumors or lymphomas was finalized based on
results of the dose escalation phase, as well as data from
additional nonclinical pharmacology studies. The latter included
the investigation of multiple solid cancer cell lines such as
breast, bladder, head/neck, gastrointestinal, or GI, liver, lung,
pancreas, prostate and sarcoma to facilitate the comparison of cell
line sensitivity to Aileron peptide-1 across and within tumor
types. Treatment of patients in the dose escalation and the dose
expansion phases of the trial continued until documentation of
disease progression, unacceptable toxicity, or patient or physician
decision to discontinue therapy.
[0491] The dose escalation phase is based on a "3+3" dose
escalation design. In the dose escalation phase, patients in the
first two dose levels received Aileron peptide-1 once a week for
three weeks every 28 days. Patients in the higher dose levels
received Aileron peptide-1 either once weekly for three consecutive
weeks for a 28-day cycle or twice weekly for two consecutive weeks
for a 21-day cycle. See FIG. 10.
[0492] Patients with solid tumors or lymphoma were enrolled in the
dose escalation phase who had exhausted standard therapies or for
whom standard therapies are not available, completed enrollment up
to dose group 4b and are enrolled patients in dose group 5a.
Patients suffering from cancers with known HPV-association were
excluded from enrollment, because HPV is known to deactivate WT
p53. The tumor types included as of such date are non-small cell
lung cancer, various types of sarcoma, cholangiocarcinoma, cystic
adenoid carcinoma, follicular non-hodgkin lymphoma, thymoma,
prostate cancer, endometrial cancer, and ovarian cancer. Since our
trial was primarily safety and tolerability focused, we started
dosing at relatively low dose levels and the protocol did not
require patients in the first three dose levels to be p53-wildtype
or HPV-negative.
[0493] To identify specific p53 patients for our trial, we employed
a central laboratory to test both archived tumor tissue samples and
fresh biopsy samples from patients enrolled in the trial for p53
status using Next-Generation Sequencing. 12 of 13 patients enrolled
in those dose levels were confirmed to have WT status. Starting in
dose level 4, WT p53 status was a mandatory eligibility
criterion.
[0494] In this trial, clinical activity or response to Aileron
peptide-1 was assessed through the use of both pharmacodynamic
biomarkers and imaging assessment. Pharmacodynamic biomarkers
provided us with information as to on-target activity, specific
patient type response and early insight as to effect on tumor. As
part of the trial, we were also assessing the effect of Aileron
peptide-1 on potential Pharmacodynamic biomarkers in various
different sources of biological samples such as tumor biopsies,
circulating tumor cells where detectable, mononuclear blood cells
and blood samples. Dependent on the sample type, those
pharmacodynamic biomarkers include measurements of MDMX, MDM2, p21,
p53, apoptosis and macrophage inhibitory cytokine-1, or MIC-1. In
addition, we received standard imaging assessments, such as
computed tomography, or CT, magnetic resonance imaging, bone scans
and PET scans from patients, depending on number cycles
administered. CT-imaging was performed at the end of cycle 2, and
every two cycles thereafter in the 28-day cycle group and at the
end of cycle 3 and every three cycles thereafter in the 21-day
cycle group. We were measuring anti-tumor activity using RECIST for
patients with solid tumors and 2014 International Working Group, or
IWG, criteria for patients with lymphomas, enabling us to
objectively evaluate whether a tumor has progressed, stabilized or
shrunk. In addition, anti-tumor effects can be determined by
physical examination or clinically validated serum tumor
markers.
Pharmacokinetic Profile
[0495] Aileron peptide-1 was delivered systemically in an IV
administration given potential advantages of avoiding metabolic
impact from hepatic and gastrointestinal enzymes as well as ability
for reproducible systemic bioavailability with dose escalation. As
shown in the FIG. 11a, drug concentration was measured in dose
levels for Cohorts 1 (0.16 mg/kg), 2 (0.32 mg/kg), 3a (0.64 mg/kg),
3b (0.32 mg/kg), 4a (1.25 mg/kg). In patients, Aileron peptide-1
has consistently produced a dose dependent increase in maximum drug
serum concentration observed, or Cmax, as well as longer
corresponding half-life of between eight and 10 hours. This
half-life is adequate to re-activate WT p53 and begin the process
of initiating the regulation of the transcription of genes.
[0496] Aileron peptide-1 shows reproducible profiles from patient
to patient and dose-to dose enabling exposure projections for
higher dose levels to predict efficacy and safety. FIG. 11b shows
the measured drug concentration in dose levels 1 (0.16 mg/kg), 2
(0.32 mg/kg) and 3 (0.64 mg/kg); and projected for dose levels 4
(1.25 mg/kg), 5 (2.5 mg/kg), and 6 (5.0 mg/kg).
[0497] FIG. 12 shows the pharmacokinetic model of Aileron
peptide-1. The peptide shows nonlinear Michaelis-Menten clearance
and linear elimination.
Safety Results
[0498] Aileron peptide-1 was considered to be well tolerated at all
dose levels by the investigators. There were no reported
dose-limiting toxicities nor study-related serious adverse events.
Looking at non-hematologic safety, the most common related adverse
events are nausea and fatigue. Looking at hematologic safety, the
first two dose levels 1 and 2 showed no cytopenias during cycles 1
and 2, whereas at dose levels 3A, 3B and 4A, patients showed
drug-related events of mild to moderate anemia, mild
thrombocytopenia and mild neutropenia. One patient at dose level 3B
experienced a grade 4 neutropenia which Investigators reported as
probably related to study medication. The patient's complete blood
count presented trough values of grade 2 leukocytopenia, grade 1
anemia and grade 1 thrombocytopenia. Two concomitant medications
were initiated around the same time that treatment with Aileron
peptide-1 was started, both of which have been suspected to be
associated with the occurrence of neutropenia. There was no
association between the patient's neutropenia and drug exposure,
the patient's last complete blood count showed an improvement to
grade 3 neutropenia, no treatment was administered for neutropenia
and no infectious complications were reported.]
[0499] 4 formal Safety Review Meetings with Investigators confirm
no DLTs. For DL1, 2 and 3A there was an unanimous decision to
escalate at double the dose. For DL3B, there was an unanimous
decision to escalate via Fibonacci in DL4B. New Dose may be 0.53
mg/Kg instead of 0.64 mg/Kg.
[0500] Hematologic and non-hematologic adverse events were
generally consistent with our preclinical toxicology profile:
[0501] No genotoxicity [0502] No immunogenicity [0503] No relevant
findings in cardiovascular safety [0504] No relevant findings
suggestive of GI toxicity [0505] No Myelosuppression as the
dose-limiting toxicity
Biomarker Assessments
[0506] In the dose escalation phase, we used several exploratory
biomarkers to confirm Aileron peptide-1's pharmacological or
on-target biological activity, aid patient recruitment and help
inform dose selection.
[0507] Pharmacodynamic biomarkers were received on MDMX, MDM2, p21,
p53, apoptosis and MIC-1. The first biomarker for which we received
data is MIC-1. MIC-1 is a secreted p53-regulated cytokine that is
readily measured in blood if p53 is activated, and can serve as a
biomarker for p53 activation. Under normal conditions, p53
expression remains low, resulting in corresponding negligible
levels of MIC-1. However, when WT p53 activation occurs in response
to a tumor, this also leads to increased levels of MIC-1. We
measured MIC-1 one hour before initial infusion and again 24 hours
after initial infusion. In patients at dose levels ranging from
dose level 1 to dose level 4A, we observed a statistically
significant dose dependent response in the increase of MIC-1. See
FIG. 13.
[0508] Further, mononuclear blood cells from 4 patients confirmed
that Aileron peptide-1 penetrates cell membrane and activates
p53-signalling. We measured the amount of intracellular p53 and p21
in mononuclear blood cells from 4 patients at: (a) end of infusion
of Aileron peptide-1, (b) 1 hour after the end of infusion of
Aileron peptide-1 and (c) 4 hour after the end of infusion of
Aileron peptide-1. As seen in FIG. 14, a 1.8 times increase in the
levels of intracellular p53 and about 3 times increase in the
levels of intracellular p21 was observed.
[0509] Thus, we conclude that Aileron peptide-1 is penetrating the
cell membrane, localizing within the nucleus and releasing WT p53.
An increase of at least eight fold in MIC-1 levels from baseline
serves as guidance for the minimum dose needed for p53
re-activation.
[0510] Overall, at least two independent biomarker studies support
Aileron peptide-1-mediated activation of intracellular p53
signaling: (i) MIC-1 serum-protein (as measured by ELISA):
dose-response relationship, and (ii) p53 and p21 increase (as
measured by flow-cytometry) in blood cells.
Efficacy
[0511] Objective tumor response is an endpoint for efficacy in
trials. Patients in the 28-day cycle group are measured at baseline
and again after two cycles of therapy, or approximately within 56
days following initial dosing. Patients in the 21-day cycle group
are measured at baseline and again after three cycles of therapy,
or approximately within 63 days following initial dosing. RECIST
criteria definitions are as follows: [0512] Stable Disease, or SD:
Neither sufficient shrinkage to qualify for partial response nor
sufficient increase to qualify for progression, taking as reference
the smallest sum diameters while on study. [0513] Partial Response,
or PR: At least a 30% decrease in the sum of the diameters of
target lesions, taking as reference the baseline sum diameters.
[0514] Complete Response, or CR: Disappearance of all target
lesions. Any pathological lymph nodes, whether target or
non-target, must have reduction in short axis to less than 10
millimeters.
[0515] Tests show that patients who have completed at least two
cycles of treatment, some patients have stable disease. Aileron
peptide-1 has shown a stable disease rate. See FIG. 15.
[0516] Table 15 below shows exemplary patients who were treated
with Aileron peptide-1. These patients encompassed a range of solid
tumors with either wild type or mutant p53. As seen in Table 16,
after 2/3 cycle treatment each of the patient 4, 5, 7, 8, 10, 11
and 15 have stable disease, while only patients 2, 6 and 12 showed
a progressive disease. After completing 3/4 treatment cycles,
patient 11 continued to show stable disease. As used here, stable
disease refers to the situation where there is neither a sufficient
shrinkage of the tumor to qualify for partial response nor
sufficient increase to qualify for progression, taking as reference
the smallest sum diameters while on study.
TABLE-US-00022 TABLE 15 Patient information Tumor Tumor Patient P53
NGS Tissue(s) Size Burden No. 1.degree. Cancer Histopath 2.degree.
Cancer WT Tested Organ mm mm 1 Salivary gland squamous cell UNK
liver Lymph node 30 55 Right Hepatic 25 Lobe Posterior 2
liposarcoma liposarcoma Yes retroperitoneal: soft tissue 48 48 mass
3 Gall bladder adenocarcinoma Yes Gall bladder liner 16 36
endoperitoneal 20 4 Submandibular Adenoid cystic Yes adenoid
oropharynx 43 65 adenocarcinoma Carcinoma lung 22 5 liposarcoma
Liposarcoma Yes liposarcoma Retroperitoneal 175 301 retroperitoneal
126 6 GIST (sarcoma) GASTROINTESTINAL UNK N/A MESENTRIC 25 135
STROMAL TUMOR MASS lung 20 lung 16 liver 84 7 NSCLC invasive
Brain/head UNK N/A lung 10 71 andenocarcinoma Lymph node 20 lung 20
Lymph node 21 8 liposarcoma Basal Cell Yes tumor Stomach 147 443
resection- Stomach 127 retroperitoneal Bone 155 lymph node Liver 14
9 prostate Adenocarcinoma UNK N/A Lymph node 48 86 liver 15 Lymph
node 23 10 NSCLS Stage IV Yes right lung Lymph node 15 99
adenocarcinoma nodule Lymph node 21 the left lung Lymph node 21
Lung 25 Lung 17 11 ovarian met. ov cancer No Tumor Liver 17 64
stage iv ca with resection- Liver 30 liver mets uterus Other 17 12
endometrial ENDOMETRIAL No Liver 13 78 SEROUS Liver 17 CARCINOMA
Lymph node 18 Lymph node 20 13 breast Carcinoma osteosarcoma Yes
Tumor lung 64 181 resection- lung 117 osteosarcoma 14
leiomyosarcoma Unknown UNK Lung 54 195 Liver 12 Liver 99 Liver 30
15 leiomyosarcoma Uterine Yes Lymph node 44 76 Lymph node 32 16
lymphona b-cell lymphoma- Lymph node 300 follicular (CT & PET)
17 thymoma
TABLE-US-00023 TABLE 16 Patient response after treatment with
Aileron Peptide -1 for 2/3 cycle Tumor Tumor % change Patient Size
Burder from New No. Organ mm mm bsln Lesions? Target Non-Target
Overall 1 Lymph node Off Study: Right Hepatic Lobe Non- Posterior
compliance 2 soft tissue mass 62 62 29% No Progression Not Done
Progressive Disease 3 liner Off Study: endoperitoneal CDP 4
oropharynx 45 69 6% Yes Stable Progressive Stable lung 24 Disease
Disease Disease 5 retroperitoneal 186 336 11.2% NO Stable Not done
Stable retroperitoneal 150 Disease Disease 6 MESENTRIC 29 159 17.8%
Yes Stable Non-complete Progressive MASS (SUPERIOR Disease
Response/Non- Disease lung 11 HEPATIC progression lung 19 LOBE UP
liver 100 TO 1.5 CM) 7 lung 13 77 8% No Stable Non-complete Stable
Lymph node 20 Disease Response/Non- Disease lung 20 progression
Lymph node 24 8 Stomach 157 518 16.9% No Stable Progressive Stable
Stomach 145 Disease Disease Disease Bone 180 Liver 36 9 Lymph node
53 122 41.86% No Progressive Non-Complete Off Study: liver 35
Disease Response/Non- Objective Lymph node 34 Progression Disease
Progression 10 Lymph node 21 116 17.20% No Stable Not done Stable
Lymph node 24 Disease Disease Lymph node 24 Lung 28 Lung 19 11
Liver 17 71 10.90% No Stable Not done Stable Liver 24 Disease
Disease Other 30 12 Liver 15 100 28.21% Yes (LIVER Progression Not
all Progressive Liver 19 LESION) Evaluated or Disease Lymph node 32
Not Assessable Lymph node 32 13 lung -- -- -- lung -- 14 Lung 69
233 19.48% Yes (left Not entered Not entered yet Not entered Liver
19 subhepatic yet yet Liver 105 mass) Liver 40 15 Lymph node 44 77
1.30% No Stable Non-complete Stable Lymph node 33 Disease
Response/Non- Disease progression
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190269753A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190269753A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References