U.S. patent application number 13/914254 was filed with the patent office on 2013-10-10 for targeting vector-phospholipid conjugates.
The applicant listed for this patent is BRACCO SUISSE SA. Invention is credited to PHILIPPE BUSSAT, SAMIR CHERKAOUI, HONG (HELEN) FAN, BERNARD LAMY, PALANIAPPA NANJAPPAN, RADHAKRISHNA K. PILLAI, SIBYLLE POCHON, BO SONG, ROLF E. SWENSON.
Application Number | 20130266519 13/914254 |
Document ID | / |
Family ID | 39543102 |
Filed Date | 2013-10-10 |
United States Patent
Application |
20130266519 |
Kind Code |
A1 |
BUSSAT; PHILIPPE ; et
al. |
October 10, 2013 |
TARGETING VECTOR-PHOSPHOLIPID CONJUGATES
Abstract
Peptide vectors having high KDR binding affinity and processes
for making such vectors are provided. The peptide vectors may be
conjugated to phospholipids and included in ultrasound contrast
agent compositions. Such ultrasound contrast agents are
particularly useful in therapeutic and diagnostic methods, such as
in imaging KDR-containing tissue and in the evaluation and
treatment of angiogenic processes associated with neoplastic
conditions. The present invention also provides processes for the
large scale production of highly pure dimeric and monomeric peptide
phospholipid conjugates as well as precursor materials used to form
the conjugates. The present invention further provides processes
for the large scale production of highly pure peptide phospholipid
conjugates which contain very low levels of TFA.
Inventors: |
BUSSAT; PHILIPPE; (FEIGERES,
FR) ; CHERKAOUI; SAMIR; (FEIGERES, FR) ; FAN;
HONG (HELEN); (SHANGHAI, CN) ; LAMY; BERNARD;
(SAINT-JULIEN-EN-GENEVOIS, FR) ; NANJAPPAN;
PALANIAPPA; (PRINCETON, NJ) ; PILLAI; RADHAKRISHNA
K.; (CRANBURY, NJ) ; POCHON; SIBYLLE;
(TROINEX, CH) ; SONG; BO; (PRINCETON, NJ) ;
SWENSON; ROLF E.; (PRINCETON, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BRACCO SUISSE SA |
MANNO |
|
CH |
|
|
Family ID: |
39543102 |
Appl. No.: |
13/914254 |
Filed: |
June 10, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13160666 |
Jun 15, 2011 |
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13914254 |
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11954130 |
Dec 11, 2007 |
7985402 |
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13160666 |
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11608395 |
Dec 8, 2006 |
7794693 |
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11954130 |
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10661156 |
Sep 11, 2003 |
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13160666 |
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10382082 |
Mar 3, 2003 |
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10661156 |
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PCT/US03/06731 |
Mar 3, 2003 |
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13160666 |
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60833342 |
Jul 25, 2006 |
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60749240 |
Dec 9, 2005 |
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60440411 |
Jan 15, 2003 |
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60360851 |
Mar 1, 2002 |
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60440411 |
Jan 15, 2003 |
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60360851 |
Mar 1, 2002 |
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Current U.S.
Class: |
424/9.52 ;
530/324 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 14/00 20130101; Y02A 50/58 20180101; A61K 49/227 20130101;
C07K 14/52 20130101 |
Class at
Publication: |
424/9.52 ;
530/324 |
International
Class: |
A61K 49/22 20060101
A61K049/22; C07K 14/00 20060101 C07K014/00 |
Claims
1. An ultrasound contrast agent composition comprising one or more
peptide monomers selected from the group consisting of:
TABLE-US-00017 (SEQ. ID NO. 6)
Ac-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2; and (SEQ. ID NO. 7)
Ac-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH2.
2. The composition of claim 1, wherein the contrast agent comprises
a gas filled microvesicle.
3. The composition of claim 2, wherein said gas comprises a
fluorinated gas.
4. The composition of claim 2, further comprising a
phospholipid.
5. The composition of claim 4 further comprising two or more
components selected from the group consisting of: DSPC, DPPG, DPPA,
DSPA, DPPE, DPPG, DSPE-PEG1000, DSPE-PEG2000, palmitic acid and
stearic acid.
6. The composition of claim 5, wherein the gas the gas comprises
C.sub.3F.sub.8, C.sub.4F.sub.10 or SF.sub.6, optionally in
admixture with air, nitrogen, oxygen or carbon dioxide.
7. The composition of claim 5, further comprising a component
selected from the group consisting of: sugars, polysaccharides and
polyols.
8. (canceled)
9. A peptide-phospholipid conjugate comprising a peptide monomer
selected from the group consisting of
Ac-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2 (SEQ. ID NO. 6); and
Ac-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH2 (SEQ. ID NO. 7).
10. The peptide-phospholipid conjugate of claim 9, wherein the
phospholipid is selected from the group consisting of:
phosphatidylethanolamines and modified
phospatidylethanolamines.
11. The peptide-phospholipid conjugate of claim 10, wherein the
phospholipid is DSPE-PEG2000.
12. An ultrasound contrast agent composition comprising a conjugate
of claim 9.
13. The composition of claim 12, wherein the contrast agent
comprises a gas-filled microvesicle.
14. The composition of claim 13, wherein the gas filled
microvesicle comprises a phospholipid.
15. The composition of claim 14, wherein the gas-filled
microvesicle further comprises two or more components selected form
the group consisting of: DSPC, DPPG, DPPA, DSPA, DPPE, DPPG,
DSPE-PEG1000, DSPE-PEG2000, palmitic acid and stearic acid.
16-18. (canceled)
19. The composition of claim 15, further comprising a component
selected from the group consisting of: sugars, polysaccharides and
polyols.
20. (canceled)
21. The composition of claim 13, wherein said gas comprises a
fluorinated gas.
22. The composition of claim 21, wherein the gas comprises
C.sub.3F.sub.8, C.sub.4F.sub.10 or SF.sub.6, optionally in
admixture with air, nitrogen, oxygen or carbon dioxide.
23-24. (canceled)
25. A method for detecting or imaging tumor tissue containing KDR
in a mammal comprising administering an effective amount of a
composition of any one of claim 5 or 15 to the mammal and imaging
the mammal.
26. A KDR-binding peptide monomer selected from the group
consisting of: TABLE-US-00018 (SEQ. ID NO. 6)
Ac-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2; and (SEQ. ID NO. 7)
Ac-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH2.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 11,608,395, filed Dec. 8, 2006 which claims
priority to and benefit of U.S. Provisional Application No.
60/833,342, filed Jul. 25, 2006 and U.S. Provisional Application
No. 60/749,240, filed Dec. 9, 2005, and is a continuation-in-part
of U.S. application Ser. No. 10/661,156, filed Sep. 11, 2003, which
is a continuation-in-part of U.S. application Ser. No. 10/382,082,
filed Mar. 3, 2003, and a continuation in-part of International
Application No. PCT/US03/06731, filed Mar. 3, 2003, both of which
claim priority to and benefit of U.S. Provisional Application No.
60/440,411, filed Jan. 15, 2003; and U.S. Provisional Application
No. 60/360,851, filed Mar. 1, 2002, the contents of each which are
hereby incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to targeting
vector-phospholipid conjugates and particularly targeting
peptide-phospholipid conjugates, which are useful in therapeutic
and diagnostic compositions and methods of preparation of the same.
The invention includes targeted ultrasound contrast agents, and
particularly targeted microbubbles which include such targeting
vector-phospholipid conjugates.
BACKGROUND OF THE INVENTION
[0003] Angiogenesis, the formation of new blood vessels, occurs not
only during embryonic development and normal tissue growth and
repair, but is also involved in the female reproductive cycle,
establishment and maintenance of pregnancy, and repair of wounds
and fractures. In addition to angiogenesis that occurs in the
normal individual, angiogenic events are involved in a number of
pathological processes, notably tumor growth and metastasis, and
other conditions in which blood vessel proliferation is increased,
such as diabetic retinopathy, psoriasis and arthropathies. In
addition, angiogenesis is important in the transition of a tumor
from hyperplastic to neoplastic growth. Consequently, inhibition of
angiogenesis has become an active cancer therapy research
field.
[0004] Tumor-induced angiogenesis is thought to depend on the
production of pro-angiogenic growth factors by the tumor cells,
which overcome other forces that tend to keep existing vessels
quiescent and stable. The best characterized of these
pro-angiogenic agents or growth factors is vascular endothelial
growth factor (VEGF) (Cohen et al., FASEB J., 13: 9-22 (1999)).
VEGF is produced naturally by a variety of cell types in response
to hypoxia and some other stimuli. Many tumors also produce large
amounts of VEGF, and/or induce nearby stromal cells to make VEGF
(Fukumura et al., Cell, 94: 715-725 (1998)). VEGF, also referred to
as VEGF-A, is synthesized as five different splice isoforms of 121,
145, 165, 189, and 206 amino acids. VEGF.sub.121 and VEGF.sub.165
are the main forms produced, particularly in tumors (see Cohen et
al. 1999, supra). VEGF.sub.121 lacks a basic domain encoded by
exons 6 and 7 of the VEGF gene and does not bind to heparin or
extracellular matrix, unlike VEGF.sub.165. Each of the references
cited in this paragraph is incorporated by reference in its
entirety.
[0005] VEGF family members act primarily by binding to receptor
tyrosine kinases. In general, receptor tyrosine kinases are
glycoproteins having an extracellular domain capable of binding one
or more specific growth factors, a transmembrane domain (usually an
alpha helix), a juxtamembrane domain (where the receptor may be
regulated, e.g., by phosphorylation), a tyrosine kinase domain (the
catalytic component of the receptor), and a carboxy-terminal tail,
which in many receptors is involved in recognition and binding of
the substrates for the tyrosine kinase. There are three endothelial
cell-specific receptor tyrosine kinases known to bind VEGF: VEGFR-1
(Flt-1), VEGFR-2 (KDR or Flk-1), and VEGFR-3 (Flt4). Flt-1 and KDR
(also known as VEGFR-2 or Flk-1, which are used interchangeably
herein) have been identified as the primary high affinity VEGF
receptors. While Flt-1 has higher affinity for VEGF, KDR displays
more abundant endothelial cell expression (Bikfalvi et al., J.
Cell. Physiol., 149: 50-59 (1991)). Moreover, KDR is thought to
dominate the angiogenic response and is therefore of greater
therapeutic and diagnostic interest (see Cohen et al. 1999, supra).
Expression of KDR is highly upregulated in angiogenic vessels,
especially in tumors that induce a strong angiogenic response
(Veikkola et al., Cancer Res., 60: 203-212 (2000)). The critical
role of KDR in angiogenesis is highlighted by the complete lack of
vascular development in homozygous KDR knockout mouse embryos
(Folkman et al., Cancer Medicine, 5th Edition (B.C. Decker Inc.;
Ontario, Canada, 2000) pp. 132-152).
[0006] KDR (kinase domain region) is made up of 1336 amino acids in
its mature form. The glycosylated form of KDR migrates on an
SDS-PAGE gel with an apparent molecular weight of about 205 kDa.
KDR contains seven immunoglobulin-like domains in its extracellular
domain, of which the first three are the most important in VEGF
binding (Cohen et al. 1999, supra). VEGF itself is a homodimer
capable of binding to two KDR molecules simultaneously. The result
is that two KDR molecules become dimerized upon binding and
autophosphorylate, becoming much more active. The increased kinase
activity in turn initiates a signaling pathway that mediates the
KDR-specific biological effects of VEGF.
[0007] Thus, not only is the VEGF binding activity of KDR in vivo
critical to angiogenesis, but the ability to detect KDR
upregulation on endothelial cells or to detect VEGF/KDR binding
complexes would be extremely beneficial in detecting or monitoring
angiogenesis.
[0008] It is well known that gas filled ultrasound contrast agents
are exceptionally efficient ultrasound reflectors for echography.
Such ultrasound contrast agents include, for example, gas-filled
microvesicles such as gas-filled microbubbles and gas filled
microballoons. Gas filled microbubbles are particularly preferred
ultrasound contrast agents. (In this disclosure the term of
"microbubble" specifically designates a gaseous bubble surrounded
or stabilized by phospholipids). For instance injecting into the
bloodstream of living bodies suspensions of air- or gas-filled
microbubbles in a carrier liquid will strongly reinforce ultrasonic
echography imaging, thus aiding in the visualization of internal
anatomical structures. Imaging of vessels and internal organs can
strongly help in medical diagnosis, for instance for the detection
of neoplastic, cardiovascular and other diseases.
[0009] For both diagnostic and therapeutic purposes it would be
particularly beneficial to incorporate into gas filled ultrasound
contrast agents, targeting vector compositions which exhibit high
binding affinity for a desired target (such as, for example, KDR or
the VEGF/KDR complex). For example, targeting vector-phospholipid
conjugates and particularly targeting peptide-phospholipid
conjugates may be used to prepare targeted, gas filled ultrasound
contrast agents. In addition, it would be particularly beneficial
to have methods for large scale production of highly purified forms
of such targeting vector-phospholipid conjugates. Such compositions
and methods would allow for production of compositions for use in
diagnostic or therapeutic applications such as, for example,
precise targeting of reporter moieties, tumoricidal agents or
angiogenesis inhibitors to the target site.
SUMMARY OF THE INVENTION
[0010] The present invention provides targeting vector-phospholipid
conjugates and particularly targeting peptide-phospholipid
conjugates which are useful in the preparation of gas filled
ultrasound contrast agents. In a preferred embodiment the targeting
peptide-phospholipid conjugates include targeting peptides which
exhibit high KDR binding affinity and thus are useful components of
contrast agents for imaging of angiogenesis processes.
[0011] The present invention also provides monomeric and dimeric
peptide phospholipid conjugates (also referred to herein as
lipopeptides) which are useful in preparing gas filled ultrasound
contrast agents, and particularly in preparing ultrasound contrast
agents which target KDR and may be used for imaging of angiogenesis
processes.
[0012] The present invention also provides methods and processes
for the large scale production of highly pure monomeric and dimeric
peptide phospholipid conjugates, particularly monomeric and dimeric
peptide phospholipids conjugates having high KDR binding
affinity.
[0013] The present invention also provides methods and processes
for the large scale production of highly pure dimeric peptide
phospholipid conjugates having minimal levels of trifluoroacetic
acid (TFA).
[0014] The present invention also provides methods for synthesizing
monomeric peptides in high purity and the construction of peptide
phospholipid conjuages from multiple peptide sub-units.
[0015] The present invention also provides monomeric peptides which
bind KDR or the VEGF/KDR complex with high affinity, as well as
methods of synthesizing and using such monomeric peptides.
[0016] The present invention also provides targeted ultrasound
contrast agents prepared from such targeting vector-phospholipid
conjugates. Such targeted ultrasound contrast agents are useful for
imaging target-bearing tissue. In a preferred embodiment, the
targeted ultrasound contrast agents are targeted microbubbles and
the targeting vector-phospholipid conjugates include targeting
peptides which exhibit high KDR binding affinity and thus are
useful components of contrast agents for imaging KDR-bearing tissue
and particularly for imaging of tumors and angiogenesis processes.
Methods of preparing and using such targeted ultrasound contrast
agents are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 illustrates a method for the production of a
monomeric peptide phospholipid conjugate (1) from a linear peptide
monomer (2).
[0018] FIG. 2 illustrates a monomeric peptide phospholipid
conjugate (1) including a peptide with high binding affinity for
KDR.
[0019] FIG. 3 illustrates a method for the production of a
precursor dimer peptide (16) from peptide monomers.
[0020] FIG. 4 illustrates a method for the conjugation of the
precursor dimer peptide shown in FIG. 1 to DSPE-PEG2000-NH.sub.2 to
form a dimeric peptide phospholipid conjugate (11) containing
peptides which bind with high affinity to KDR.
[0021] FIG. 5 illustrates a dimeric peptide-phospholipid conjugate
(11) containing peptides which bind with high affinity to KDR.
[0022] FIG. 6 illustrates a method for the production of dimer
peptide-phospholipid conjugates (such as (21)) having minimal
levels of TFA.
[0023] FIG. 7 illustrates another method for the production of
dimer peptide-phospholipid conjugates (such as (21)) having minimal
levels of TFA.
[0024] FIG. 8 illustrates another method for the production of
dimer peptide-phospholipid conjugates having minimal levels of
TFA.
[0025] FIG. 9 illustrates another representative monomeric peptide
(32) having a high binding affinity for KDR.
[0026] FIG. 10 illustrates another monomeric peptide-phospholipid
conjugate (31) which includes the monomeric peptide shown in FIG.
9.
[0027] FIGS. 11A-C show images obtained by using the dimer
peptide-phospholipid conjugate (11) (shown in FIG. 52) in a
contrast agent at: 1) baseline (FIG. 11A); 2) after 25 minutes
(FIG. 11B); and 3) after subtraction of the baseline and free
circulating bubbles (FIG. 11C).
[0028] FIGS. 12A-C show images obtained by using the monomeric
phospholipid peptide conjugate (1) (shown in FIG. 2) in a contrast
agent at baseline (FIG. 12A); after 25 minutes (FIG. 12B); and
after subtraction of the baseline and free circulating bubbles
(FIG. 12C).
DETAILED DESCRIPTION
[0029] Applicants have unexpectedly discovered peptide phospholipid
conjugates, which are useful in producing targeted ultrasound
contrast agents and which have exceptional KDR binding efficiency.
These compounds include monomeric peptide phospholipid conjugates
having a linear peptide monomer which binds with high affinity to
KDRand dimeric peptide phospholipid conjugates having two distinct
monomer subunits, each binding to KDR. In addition, highly
efficient methods for large scale production of purified forms of
these conjugates and precursor materials have been discovered. Such
methods include the production of dimeric peptide phospholipid
conjugates having minimimal levels of TFA.
[0030] The phospholipid may be selected from the group consisting
of: phosphatidylethanolamines and modified
phosphatidylethanolamines. Particularly preferred phospholipids
include phosphatidylethanolamines modified by linking a hydrophilic
polymer thereto. Examples of modified phosphatidylethanolamines are
phosphatidylethanolamines (PE) modified with polyethylenglycol
(PEG), in brief "PE-PEGs", i.e. phosphatidylethanolamines where the
hydrophilic ethanolamine moiety is linked to a PEG molecule of
variable molecular weight (e.g. from 300 to 5000 daltons), such as
DPPE-PEG, DSPE-PEG, DMPE-PEG or DAPE-PEG. DSPE-PEG2000,
DSPE-PEG3400, DPPE-PEG2000 and DPPE-PEG3400 are preferred, with
DSPE-PEG2000 particularly preferred. Note that a salt form of the
phospholipid may be used, such as, for example, the trimethyl
ammonium salt, the tetramethylammonium salt, the triethylammonium
salt, sodium salt, etc.
[0031] These compounds may be incorporated into gas fillled
ultrasound contrast agents, such as, for example, gas filled
microbubbles to form contrast agents that provide excellent imaging
of target-bearing tissue. In a preferred embodiment, targeting
vector-phospholipid conjugates which include targeting peptides
which bind with high affinity to KDR are incorporated into targeted
microbubbles. As shown herein, such targeted microbubbles
selectively localize at KDR-bearing tissue, permitting imaging of
such tissue, and, in particular imaging of tumors and angiogenic
processes, including those processes associated with neoplastic
development.
Monomer Conjugates
[0032] Generally
[0033] Table 1 provides a description for the identification labels
shown in FIGS. 1, 2, 9 and 10.
TABLE-US-00001 TABLE 1 1 Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-
PEG2000-NH-Glut)-NH.sub.2 (SEQ ID NO. 1) 2
Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH.sub.2 (SEQ ID NO. 2) 3 mono-NHS
ester of glutaryl-peptide monomer (2)
Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(NHS- Glut)-NH.sub.2 (SEQ ID NO. 3)
4 DSPE-PEG2000-NH.sub.2 phospholipid 1,2-distearoyl-sn-glycero-3-
phosphoethanolaminocarbonyloxy-(PEG2000)- amine 31
Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE- PEG2000-NH-Glut)-NH.sub.2
(SEQ ID NO. 4) 32 Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK-NH.sub.2 (SEQ ID
NO. 5)
[0034] As shown if FIGS. 1 and 2 the monomeric peptide phospholipid
conjugate (1)
N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-trypto-
phyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-L-serinyl-L-methionyl-L-al-
anyl-L-aspartyl-L-glutaminyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L1-phe-
nylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-{N6-[1,2-d]-
stearoyl-sn-glycero-3-phosphoethanolaminocarbonyloxy-(PEG2000)-aminoglutar-
yl]}-L-lysinamide, is a phospholipid conjugate. This conjugate is
also referred to as
Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH.sub.2
(SEQ ID NO. 1) and
Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Ar-
g-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys(DSPE-PEG2000-NH-Glut)-NH.sub-
.2. It comprises a 29 amino acid linear peptide monomer (2)
N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-t
tophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-L-serinyl-L-methionyl-L-
-alanyl-L-aspartyl-L-glutaminyl-L-leucyl-L1-arginyl-L-histidyl-L-alanyl-L--
phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-L-lysina-
mide, also referred to as Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH.sub.2
(SEQ ID NO. 2) and
Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Ar-
g-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH.sub.2. This novel
peptide monomer binds with high affinity to KDR. It should be
understood that analogs and derivatives of the monomeric peptide
phospholipid conjugate (1) and the linear peptide monomer (2) are
intended to be included within the scope of the present
invention.
[0035] FIG. 10 provides the structure of another monmeric peptide
phospholipid conjugate (31),
N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosy-
l-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl--
L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalany-
l-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-{N6-[1,2-d]stearoyl--
sn-glycero-3-phosphoethanolaminocarbonyloxy-(PEG2000)-aminoglutaryl]}-L-ly-
sine-amide, a phospholipid conjugate. This conjugate is also
referred to as
Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH.sub.2 (SEQ
ID NO. 4) and
Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Ar-
g-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys(DSPE-PEG2000-NH-Glut)-NH.sub-
.2. As shown in FIG. 9, the conjugate comprises a 28 amino acid
linear peptide monomer (32),
N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosy-
l-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl--
L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalany-
l-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide,
which is also referred to as
Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK-NH.sub.2 (SEQ ID NO. 5) and
Ac-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-Hi-
s-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH.sub.2. As shown in
co-pending application, U.S. application Ser. No. 10/661,156, filed
Sep. 11, 2003, this peptide monomer binds with high affinity to
KDR. It should be understood that analogs and derivatives of the
monomeric peptide phospholipid conjugate and the linear peptide
monomer are intended to be included within the scope of the present
invention.
[0036] Additional monomeric peptide phospholip conjugates according
to the present invention include
Ac-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2 (SEQ. ID NO. 6) and
Ac-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH2 (SEQ. ID NO. 7). These peptide
monomers bind with high affinity to KDR. It should be understood
that analogs and derivatives of these monomeric peptide monomers
and peptide phospholipids conjugates comprising these monomers are
intended to be included within the scope of the present
invention.
[0037] As shown in the Examples, ultrasound contrast agents such as
gas filled microbubbles formulated with the monomeric peptide
phospholipid conjugates (1) and (31) displayed high KDR binding
which was confirmed using echographic examination of VX2 tumors in
rabbits.
[0038] Methods of Preparation of Monomer Peptide-Phospholipid
Conjugates
[0039] Ideally, to facilitate production of a monomeric peptide
phospholipid conjugate, the linear peptide monomer should be
prepared in bulk. Then conjugation of the purified linear peptide
monomer to the phospholipid, such as, for example, a pegylated
phospholipid in salt form, e.g., DSPE-PEG2000-NH.sub.2 phospholipid
ammonium salt via the linker disuccinimidyl glutarate (DSG), may be
used to provide monomeric peptide phospholipid conjugates.
[0040] In preparing monomeric peptide phospholipid conjugates,
methods according to the present invention provide at least the
following advantages: increased yield of peptide synthesis; reduced
extent of racemization; avoidance of previously observed piperidine
amide formation during synthesis, efficient purification of peptide
monomers, development of a procedure for conjugation of peptide
monomers on larger scale; and development of purification protocols
that would allow the ready separation of the monomeric peptide
phospholipid conjugates from the starting DSPE-PEG2000-NH.sub.2
phospholipid ammonium salt.
[0041] With respect to the methods described below (including the
Examples section of this specification), it should be understood
that while such methods may specifically reference the use of
linear peptide monomers (2) and (32) to form monomeric peptide
phospholipids conjugates (1) and (31), these methods also may be
used with peptide monomers Ac-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2
(SEQ. ID NO. 6) and Ac-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH2 (SEQ. ID
NO. 7), as well as analogs and derivatives of any of the above. It
also should be understood that the numerical values referred to in
the synthesis of monomeric peptide phospholipid conjugates are
representative.
[0042] Linear peptide monomers may be prepared by SPPS. The
sequence of the linear peptide monomers may be constructed as a
C-terminal carboxamide on Pal-Peg-PS-resin (substitution level: 0.2
mmol/g). Peptide synthesis may be accomplished using Fmoc chemistry
on a SONATA.RTM./Pilot Peptide Synthesizer. Problems previously
observed with this process have been racemization, incomplete
couplings and piperidine amide formation, each of which contribute
to suboptimal yield and purity. A dramatic decrease in the
formation of the piperidine amide may be attained by the use of 25%
piperidine in DMF containing HOBt (0.1M) as the reagent for Fmoc
removal. Racemization may be considerably reduced by using DIC/HOBt
as the activator for most couplings; a 3 h coupling time using a
four-fold excess of pre-activated Fmoc-amino acid with an
intervening wash with anhydrous DMF (6.times.). N.sup..alpha.-Fmoc
amino acids may be dissolved just before their coupling turn and
pre-activated with DIC/HOBt in DMF for 4 min and transferred to the
reaction vessel. This may be accomplished on the Sonata instrument
by loading the solid Fmoc-amino acids into the amino acid vessels
of the instrument and then programming the instrument to add DMF,
HOBt/DMF and DIC/DMF sequentially with bubbling of the
solution.
[0043] To optimize the yield, the problem of aggregation of the
resin during the synthesis of longer peptides, which can be
devastating even when optimal coupling reagents are employed, may
be addressed. To reduce aggregation during peptide assembly the
strategy of using pseudoproline dipeptides to incorporate X-Thr or
X-Ser as dipeptides instead of sequential couplings of X and Thr or
X and Ser, may be employed. For linear peptide monomers sequential
couplings of Leu.sup.11-Ser.sup.12 and Leu.sup.22-Ser.sup.23 may be
replaced by the single coupling of the pseudoproline dipeptide,
Fmoc-Leu-Ser(.PSI..sup.Me,Mepro)-OH. Additional optimization may be
accomplished by reducing the number of couplings by using
Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, in lieu of serial coupling
of Fmoc-Gly-OH. Activation of -Gly-Gly-OH segments may lead to
cyclization of the activated acid function with the distal amide
function to produce an inactive diketopiperazine; this may reduce
coupling yields in a time dependant manner. This problem may be
avoided by addition of Fmoc-Gly.sub.n-OH (n=2, 3) to the reaction
vessel and sequential addition of HOBt and DIC; the activated
Fmoc-Gly.sub.n-OH may be intercepted by the resin-bound amino group
before appreciable cyclization to the diketopiperazine takes place.
With these improvements, the synthesis of linear peptide monomers
may be completed on the Sonata Peptide Synthesizer on a 10 mmol
synthesis scale.
[0044] After chain elongation, the Fmoc may be removed from the
N-terminus. The peptide and the free amino group may be acetylated.
Then the peptide sequence may be cleaved from the resin and
deprotected using "Reagent B" (TFA:water:phenol:triisopropylsilane,
88:5:5:2, v/v/w/v) for 4 h. After the cleavage reaction the crude
peptide may be isolated as a solid by evaporation of the volatiles,
trituration of the residue with diethyl ether and washing of the
solid thus obtained using the same solvent. In another variation
the peptide may be precipitated from the reaction mixture by
addition of diethyl ether to the reaction mixture, collecting the
solid thus formed and washing with the same solvent.
[0045] Linear peptide monomers may be purified as described below.
Again, the numerical references are representative. Crude linear
peptide monomers (0.5 g) may be dissolved in CH.sub.3CN (40 mL/g)
and this solution may be diluted to a final volume of 100 mL with
water. The solution may then be filtered. The filtered solution may
be loaded onto the preparative HPLC column (Waters, XTerra.RTM.
Prep MS C18, 10.mu., 300 .ANG., 50.times.250 mm) equilibrated with
10% CH.sub.3CN in water (0.1% TFA). After loading, the composition
of the eluent may then be ramped to 20% CH.sub.3CN-water (0.1% TFA)
over 1 min, and a linear gradient may be initiated at a rate of
0.6%/min of CH.sub.3CN (0.1% TFA) into water (0.1% TFA) and run for
50 min. Eluted fractions may be checked for purity on an analytical
reversed phase C18 column (Waters XTerra MS-C18, 10.mu., 120 .ANG.,
4.6.times.50 mm) and fractions containing the product in >95%
purity may be combined and freeze-dried. For each purification of
0.5 g of crude peptide 0.12 g (24%) of linear peptide monomer may
be consistently isolated and will provide the peptide in the same
yield and purity.
[0046] Synthesis of monomeric peptide phospholipid conjugates may
be performed as described below. The numerical references are again
representative. The last step in the synthesis may be the
conjugation of the phospholipid, such as, for example, a pegylated
phospholipid such as DSPE-PEG2000-NH.sub.2 phospholipid ammonium
salt to a linear peptide monomer. The PEG2000 moiety of
DSPE-PEG2000-NH.sub.2 phospholipid ammonium salt (4) is nominally
comprised of 45 ethylene glycol units. It should be understood,
however, that this material is a distribution of PEG containing
species whose centroid is the nominal compound containing 45
ethylenoxy units. The conjugation of a linear peptide monomer with
DSPE-PEG2000-NH.sub.2 phospholipid ammonium salt may be
accomplished by preparation of the glutaric acid monoamide mono NHS
ester of a linear peptide monomer and reaction of this with the
free amino group of the phospholipid ammonium salt. Thus a linear
peptide monomer may be reacted with DSG (4 eq.) in DMF in the
presence of DIEA (5 eq.) for 30 min. The reaction mixture may be
diluted with ethyl acetate, which may result in precipitation of
the peptide glutaric acid monoamide mono-NHS ester. The supernatant
containing un-reacted DSG may be decanted and the intermediate
peptide mono-NHS ester may be washed several times with ethyl
acetate to remove traces of DSG. Mass spectral data confirms the
formation of the peptide mono-NHS ester as a clean product. The
solid mono-NHS ester may be dissolved in DMF and reacted with
DSPE-PEG2000-NH.sub.2 phospholipid ammonium salt (0.9 eq.) in the
presence of DIEA (4 eq.) for 24 h. The linear peptide monomer
glutaric acid monoamide mono-NHS ester may be used in excess to
maximize the consumption of the phospholipid ammonium salt because
free phospholipid ammonium salt may complicate the isolation of
monomeric peptide phospholipid conjugates in highly pure form.
[0047] The reaction mixture may be diluted with a 1:1 mixture of
water (0.1% TFA) and CH.sub.3CN--CH.sub.3OH (1:1, v/v) (0.1% TFA)
(.about.100 mL), applied to a reversed phase C2 column
(Kromasil.RTM. Prep C2, 10.mu., 300 .ANG., 50.times.250 mm, flow
rate 100 mL/min) and the column may be eluted with a 3:1 mixture of
water (0.1% TFA) and CH.sub.3CN--CH.sub.3OH (1:1, v/v) (0.1% TFA)
to remove hydrophilic impurities. Then the product may be eluted
using a gradient of CH.sub.3CN--CH.sub.3OH (1:1) (0.1% TFA) into
water (0.1% TFA) (see Experimental Section for details). The
collected fractions may be analyzed by reversed phase HPLC using an
ELS detector which allows the detection of the desired product and
the often difficult-to-separate DSPE-PEG2000-NH.sub.2 phospholipid
which has very little UV absorbance. This indicates the clear
separation of the monomeric peptide phospholipid conjugates and
DSPE-PEG2000-NH.sub.2 phospholipid. The pure product-containing
fractions may be collected, concentrated on a rotary evaporator (to
reduce the content of methanol) and freeze-dried to provide
monomeric peptide phospholipid conjugates as a colorless solid. In
order to prepare the required quantity of the monomeric peptide
phospholipid conjugates, several runs may be conducted employing
0.5 g to 1.0 g of linear peptide monomer. In all cases the target
monomeric peptide phospholipid conjugates may be were isolated in
high yield and purity (e.g., 57-60% yield and >99% purity).
Dimer Conjugate
[0048] Generally
[0049] Table 2 provides a description for the identification labels
shown in FIGS. 3, 4 and 5.
TABLE-US-00002 TABLE 2 11 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-
VCWEDSWGGEVCFRYDP-GGGK[-Adoa-Adoa-Glut-
K(DSPE-PEG2000-NH-Glut)]-NH.sub.2 cyclic (2-12) disulfide}-NH.sub.2
cyclic (6-13) disulfide 12
Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH.sub.2 cyclic (6-13)
disulfide 13 Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH.sub.2 cyclic
(2-12) disulfide 14 mono-NHS ester of glutaryl-peptide 12
Ac-AGPTWCEDDWYYCWLFGTGGGK[NHS-Glut-K(ivDde)]- NH.sub.2 cyclic
(6-13) disulfide 15 ivDde-bearing dimer
Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-
VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut- K(ivDde)]-NH.sub.2 cyclic
(2-12) disulfide}-NH.sub.2 cyclic (6-13) disulfide 16
Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-
VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)- NH.sub.2 cyclic (2-12)
disulfide]-NH.sub.2 cyclic (6-13) disulfide 17 Mono-NHS ester of
glutaryl-peptide 16 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-
VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut-K(NHS- Glut)]-NH.sub.2 cyclic
(2-12) disulfide}-NH.sub.2 cyclic (6-13) disulfide 18
DSPE-PEG2000-NH.sub.2 phospholipid
[0050] As shown in those figures the dimeric peptide phospholipid
conjugate (11)
Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glut-
amyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-t-
ryptophyl-1-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-
-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-sery-
l-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl--
L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(diste-
arylphosphoethanolaminocarbonoxy-PEG2000-amino-8-amino-3,6-dioxaoctanoyl-8-
-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl) amide cyclic (2-12)
disulfide]-amide cyclic (6-13) disulfide, consists of two monomeric
peptide chains which bind KDR: a 21 amino acid cyclic disulfide
peptide monomer (13)
Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tr-
yptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-argi-
nyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(8-amino-3,6-
-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl)amide cyclic (2-12)
disulfide, and a 22 amino acid cyclic disulfide peptide monomer
(12)
Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glut-
amyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-t-
ryptophyl-L-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-
-lysinamide cyclic 6-13 disulfide tethered by a glutaryl linker. It
should be understood that analogs and derivatives of the dimeric
peptide phospholipid conjugate (11) and the cyclic disulfide
peptide monomers (12) and (13) are intended to be included within
the scope of the present invention.
[0051] Ultrasound contrast agents (e.g. gas filled microbubbles)
formulated with the dimeric peptide phospholipid conjugate (11)
displayed high KDR binding which was confirmed using echographic
examination of VX2 tumors in rabbits.
[0052] Methods of Preparation of Dimer-Phospholipid Conjugates
[0053] To accomplish synthesis of the dimeric peptide phospholipid
conjugate (11), the monomers used for this purpose optimally should
be prepared in bulk. Then the monomers may be tethered to each
other using di-succinimidyl glutarate as a linker to form the
precursor dimer peptide (16),
Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl--
L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystin-
yl-L-tryptophyl-L-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-gl-
ycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl--
L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenyla-
lanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl-
(8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl)
amide cyclic (2-12) disulfide]-amide cyclic (6-13) disulfide. Then
conjugation of the purified precursor dimer peptide (16) to a
DSPE-PEG2000-NH.sub.2 phospholipid ammonium salt (18) again via
disuccinimidyl glutarate may be used in order to provide the target
dimeric peptide phospholipid conjugate (11).
[0054] In preparing dimeric peptide phospholipid conjugate (11),
methods according to the present invention provide at least the
following advantages: increased yield of automated chain elongation
of the peptide sequences; reduced extent of racemization
encountered during synthesis; avoidance of previously observed
piperidine amide formation during synthesis of peptide monomer
(13); cyclization of linear di-cysteine containing peptide
precursors of (12) and (13) using procedures amenable to multigram
scale yet allowing efficient and practical sample handling;
efficient purification of monomer peptides (12) and (13); maximized
yield and purity of precursor dimer peptide (16); development of a
procedure for conjugation of the precursor dimer peptide (16) on
larger scale; and development of purification protocols that would
allow the ready separation of the target dimeric peptide
phospholipid conjugate (11) from phospholipid ammonium salt
(18).
[0055] The dimeric peptide phospholipid conjugate (11) may be
prepared by automated synthesis of the peptide monomers (12),
Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Th-
r-Gly-Gly-Gly-Lys(ivDde)-NH.sub.2 cyclic (6-13) disulfide, and
(13),
Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gl-
y-Gly-Gly-Lys(Adoa-Adoa)-NH.sub.2 cyclic (2-12) disulfide, their
efficient coupling using disuccinimidyl glutarate (DSG) to give an
ivDde-protected dimer, its deprotection and subsequent coupling to
DSPE-PEG2000-NH.sub.2, also via a glutaryl linkage. Using
procedures according to the present invention, monomer peptides may
be synthesized on a 10 mmol scale without complication and after
HPLC purification may be obtained in about 20% yield and >95%
purity. Such methods allow dimer formation reactions and the
subsequent conjugation to the phospholipid component providing
formation of dimeric peptide phospholipid conjugate (11) to be
carried out on a gram scale. The precursor dimer peptide (16) may
be obtained from the monomer peptides routinely in about 32% yield
and >95% purity. The dimeric peptide phospholipid conjugate (11)
may be produced from the precursor dimer peptide (16) in 57-60%
yield and >99% purity.
[0056] Dimeric peptide phospholipid conjugates may be prepared as
described below. It should be appreciated that the numerical values
referred to in this representative description of the synthesis of
dimeric peptide phospholipid conjugates are representative.
[0057] Described below is a representative method for the solid
phase synthesis and disulfide cyclization of a peptide monomer (12)
Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Th-
r-Gly-Gly-Gly-Lys(ivDde)-NH.sub.2 cyclic (6-13) disulfide, and a
peptide monomer (13),
Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gl-
y-Gly-Gly-Lys(Adoa-Adoa)-NH.sub.2 cyclic (2-12) disulfide.
[0058] The peptides may be constructed as their C-terminal
carboxamides on Pal-Peg-PS-resin (substitution level: 0.2 mmol/g).
Chain elongation may be accomplished using Fmoc chemistry employing
optimized deprotection and coupling protocols on a
SONATA.RTM./Pilot Peptide Synthesizer on a 10 mmol synthesis scale.
The optimized synthesis of the peptides by automated SPSS may be
developed by study of peptide impurities and the effect of changes
of particular elements of the protocols on the overall yield and
purity of the peptides obtained.
[0059] Analysis of the impurities obtained from nonoptimized
syntheses of the monomer peptides indicates that the major problems
are racemization, incomplete couplings and piperidine amide
formation (presumably via an intermediate aspartimide or
glutarimide intermediate), each of which contributes to suboptimal
yield and purity. A dramatic decrease in formation of the
piperidine amide may be attained by the use of 25% piperidine in
DMF containing HOBt (0.1M) as the reagent for fmoc removal.
Racemization may be considerably reduced by using DIC/HOBt as the
activator for most couplings; and a 3 h coupling time using a
four-fold excess of pre-activated Fmoc-amino acid with an
intervening wash with anhydrous DMF (6.times.). N-.sup..alpha.Fmoc
amino acids may be dissolved just before their coupling turn and
pre-activated with DIC/HOBt in DMF for 4 min and transferred to the
reaction vessel. This may be accomplished on the Sonata instrument
by loading the solid Fmoc-amino acids into the amino acid vessels
of the instrument and then programming the instrument to add DMF,
HOBt/DMF and DIC/DMF sequentially with bubbling of the solution
after each addition.
[0060] To optimize the yield, the problem of aggregation of the
resin during the synthesis of longer peptides, which can be
devastating even when optimal coupling reagents are employed, may
be addressed. To reduce aggregation during peptide assembly the
strategy of using pseudoproline dipeptides to incorporate X-Thr or
X-Ser (X refers to the n-1 amino acid of the sequence) as
dipeptides instead of sequential couplings of X and Thr or X and
Ser, may be employed. Thus, for the monomer (12),
Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Th-
r-Gly-Gly-Gly-Lys(ivDde)-NH.sub.2 cyclic (6-13) disulfide,
sequential coupling of suitably protected Thr and Gly (shown in
bold above) may be replaced by the single coupling of the
pseudoproline dipeptide, Fmoc-Gly-Thr(.PSI..sup.Me,Mepro)-OH.
Similarly, in the synthesis of the monomer (13),
Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gl-
y-Gly-Gly-Lys(Adoa-Adoa)-NH.sub.2 cyclic (2-12) disulfide, the
pseudoproline dipeptide, Fmoc-Asp(OtBu)-Ser(.PSI..sup.Me,Mepro)-OH
may be employed to replace the sequential coupling of suitably
protected Ser and Asp (shown in bold font above). Further
optimization may be accomplished by reducing the number of
couplings by using Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, in lieu
of serial coupling of Fmoc-Gly-OH. Activation of -Gly-Gly-OH
segments can lead to cyclization of the activated acid function
with the distal amide function to produce an inactive
diketopiperazine; this may reduce coupling yields in a time
dependant manner. This problem may be avoided by addition of
Fmoc-Gly.sub.n-OH (n=2, 3) to the reaction vessel and sequential
addition of HOBt and DIC; the activated Fmoc-Gly.sub.n-OH may be
intercepted by the resin-bound amino group before appreciable
cyclization to the diketopiperazine takes place. After chain
elongation is completed the N-terminal Fmoc protecting group may be
removed from each of the peptides and the free amino group may be
acetylated.
[0061] The pseudo-orthogonally protected derivative,
Fmoc-Lys(ivDde)-OH may be used to enable the selective unmasking of
the .epsilon.-amine of the C-terminal lysine of the monomer and
dimer peptides and their subsequent functionalization, which also
may be optimized. The ivDde group on the .epsilon.-amine of the
C-terminal lysine of each of the peptide monomers may be removed
using 10% hydrazine in DMF. Then Fmoc-Adoa, for monomer (13) or
Lys(ivDde) for monomer (12) may be appended to the exposed lysine
.epsilon.-amino group using 4 equivalents of the Fmoc amino acid
and 4 equivalents each of DIC and HOBt in DMF for 10 h. After
completion of the synthesis, the peptide sequence may be cleaved
from the resin and deprotected using "Reagent B"
(TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) for 4 h.
After the cleavage reaction was complete the peptide may be
precipitated, washed with diethyl ether and dried.
[0062] The following procedures for cyclization of the linear
di-cysteine containing peptides may be used to provide optimal
scale-up of monomer peptides. Generally the aerial oxidation of
linear di-cysteine peptides may be carried out at ca 0.5-5 mg/mL
(for the disclosed peptide monomers .about.0.18-1.8 mM in peptide,
.about.0.36-3.6 mM in cysteine thiol). In order to work at
significantly higher concentrations DMSO-assisted cyclization of
di-cysteine peptides allows the cyclization of .about.10 g of the
linear peptides in good yields in as little as .about.50 mL of
solution. Therefore the crude linear di-cysteine peptides may be
cyclized in 95% DMSO-H.sub.2O (5 mL/g) at pH 8.5 at ambient
temperature. The progress of the cyclization may be routinely
followed by mass spectroscopy and HPLC. Although cyclization may be
essentially complete in .about.36 h, the reaction mixture may be
generally stirred for up to 48 h. The cyclic disulfide peptides may
be precipitated from the reaction mixture by dilution with
CH.sub.3CN and the resulting off-white crude solid peptides may be
collected by filtration. This is a convenient method for removing
DMSO from the crude cyclic peptide.
[0063] Purification and isolation of monomer peptide (12),
Ac-AGPTWC*EDDWYYC*WLFGTGGGK [K(ivDde)]-NH.sub.2 may be accomplished
as described below. Note that as used herein the designation "C*"
refers to a cysteine residue that contributes to a disulfide bond.
Attempts to dissolve 0.5 g of the crude peptide in up to 300 mL of
30% CH.sub.3CN in water (0.1% TFA) have been unsuccessful.
Therefore, as an alternative, the crude peptide, (0.5 g) may be
dissolved in DMSO (5 mL/g) and this solution may be diluted to a
final volume of 100 mL with 20% CH.sub.3CN-water. The solution may
be filtered. The filtered solution may be loaded onto the
preparative HPLC column (Waters, XTerra.RTM. Prep MS C18, 10.mu.,
300 .ANG., 50.times.250 mm) equilibrated with 10% CH.sub.3CN (0.1%
TFA) in water (0.1% TFA), and the column may be eluted with 10%
CH.sub.3CN (0.1% TFA) in water (0.1% TFA) to wash DMSO from the
column. The composition of the eluent then may be ramped to 35%
CH.sub.3CN-water (0.1% TFA) over 1 min, and a linear gradient may
be initiated at a rate of 0.5%/min of CH.sub.3CN (0.1% TFA) into
water (0.1% TFA) and run for 50 min. Eluted fractions may be
checked for purity on an analytical reversed phase C18 column
(Waters XTerra MS-C18, 10.mu., 120 .ANG., 4.6.times.50 mm) and
fractions containing the product in >95% purity may be combined
and freeze-dried. For each purification of 0.5 g of crude peptide
0.1 g (20%) for (12), Ac-AGPTWC*EDDWYYC*WLFGTGGGK
[K(ivDde)]-NH.sub.2 may be isolated. Repeat purifications have been
found to provide the peptide consistently in the same yield and
purity.
[0064] The peptide monomer (13),
Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH.sub.2 may be purified and
isolated as described for peptide monomer (12) except that the
subject peptide may be dissolved in 20% CH.sub.3CN (0.1% TFA) in
0.1% aqueous TFA (0.5 g peptide/100 mL) instead of a
DMSO-containing diluent. The resulting solution of crude peptide
may be loaded onto the preparative HPLC column (Waters, XTerra.RTM.
Prep MS C18, 10 g, 300 .ANG., 50.times.250 mm, flow rate 100
mL/min) equilibrated with 10% CH.sub.3CN in water (0.1% TFA). The
column may be eluted with 10% CH.sub.3CN (0.1% TFA)/water (0.1%
TFA) at 100 mL/min for 5 min. Then the composition of the eluent
may be ramped to 30% CH.sub.3CN (0.1% TFA)/water (0.1% TFA) over 1
min and a linear gradient rate of 0.5%/min of CH.sub.3CN (0.1% TFA)
into water (0.1% TFA) may be initiated, and maintained until the
desired peptide is completely eluted from the column.
Product-containing fractions may be analyzed on a Waters XTerra
analytical reversed phase C-18 column (10.mu., 120 .ANG.) and
fractions containing the product in >95% purity may be pooled
and freeze-dried to afford the cyclic disulfide peptide monomer
(13) (0.12 g, 24% yield) in >95% purity. The 10 g of crude
peptide monomer may be purified serially in this manner.
[0065] Described below is a representative method for preparing the
precursor dimer peptide (16),
Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH-
.sub.2 cyclic (2-12) disulfide]-NH.sub.2 cyclic (6-13) disulfide.
The preparation of the precursor dimer peptide may be accomplished
by the tethering of the monomer peptides in a two step procedure.
First, Ac-AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]-NH.sub.2 (12) may be
reacted with disuccinimidyl glutarate (DSG, 5 eq.) in DMF in the
presence of DIEA (5 eq.) for 30 min. The reaction mixture may be
diluted with ethyl acetate, which results in precipitation of the
glutaric acid monoamide mono-NHS ester of the peptide. The
supernatant, containing unreacted DSG, may be decanted and the
mono-NHS ester may be washed several times with ethyl acetate to
remove traces of DSG. Mass spectral data confirms the formation of
the mono-NHS ester as a clean product. This may be redissolved in
DMF and reacted with monomer peptide
Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH.sub.2 (13) in the presence
of DIEA (5 eq). HPLC and MS results indicate the formation of the
ivDde-bearing dimer, as a single major product. The ivDde
protecting group on the .epsilon.-amine of Lys of the dimer may be
removed by stirring the reaction mixture with hydrazine (10%) in
DMF for 20 min. The solution then may be acidified with TFA and
diluted with 10% CH.sub.3CN (0.1% TFA)-water (0.1% TFA), applied to
a preparative reversed phase C18 HPLC column and purified by a
gradient elution of acetonitrile (0.1% TFA) into 0.1% aqueous TFA.
In order to provide the needed quantity of the precursor dimer
peptide, the reaction may be conducted employing from 0.5 g to as
much as 1 g of each of the monomer peptides. In every case the
required precursor dimer peptide may be isolated in .about.32%
yield and >95% purity confirming the reproducibility and
scalability of the procedures.
[0066] The final step in the synthesis may be the conjugation of
DSPE-PEG2000-NH.sub.2 phospholipid ammonium salt (18) to the
precursor dimer peptide. As mentioned previously, the PEG2000
moiety of DSPE-PEG2000-NH.sub.2 is nominally comprised of 45
ethylene glycol units. It should be understood, however, that this
material is a distribution of PEG containing species whose centroid
is the nominal compound containing 45 ethylenoxy units.
[0067] Conjugation of the DSPE-PEG2000-NH.sub.2 to the precursor
dimer peptide may be accomplished by preparation of a glutaric acid
monoamide mono NHS ester of the precursor dimer and reaction of
this with the free amino group of the phospholipid ammonium salt.
Thus the ivDde bearing precursor dimer peptide (16) may be reacted
with DSG (4 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min.
As in the preparation of the precursor dimer peptide the solution
may be diluted with ethyl acetate to precipitate the glutaric acid
monoamide mono-NHS ester of the dimer (17), as a solid. The
supernatant may be decanted to remove the un-reacted DSG. The solid
glutaric acid monoamide mono-NHS ester of the dimer peptide (17)
may then be washed several times with ethyl acetate to remove
traces of DSG. Mass spectral results confirm the formation of the
glutaric acid monoamide mono-NHS ester of the peptide dimer as a
clean product.
[0068] The dimer glutaric acid monoamide mono-NHS ester (17) may be
dissolved in DMF-CH.sub.2Cl.sub.2 (8:2) and reacted with
DSPE-PEG2000-NH.sub.2 phospholipid ammonium salt (0.9 eq.) in the
presence of DIEA (4 eq.) for 24 h. The NHS ester (17) may be used
in excess to maximize the consumption of the phospholipid ammonium
salt because any free phospholipid may complicate the purification
and isolation of the final product. The reaction mixture may be
diluted with water (0.1% TFA)-CH.sub.3CN--CH.sub.3OH (1:1) (0.1%
TFA) (.about.100 mL), applied to a reversed phase C4 column
(Kromasil.RTM. Prep C4, 10.mu., 300 .ANG., 50.times.250 mm, flow
rate 100 mL/min) and the column may be eluted with water (0.1%
TFA)-CH.sub.3CN--CH.sub.3OH (1:1) (0.1% TFA) solvent mixture to
remove hydrophilic impurities. Then the product may be eluted using
a gradient of CH.sub.3CN--CH.sub.3OH (1:1) (0.1% TFA) into water
(0.1% TFA). The collected fractions may be analyzed by reversed
phase HPLC using an ELS detector which allows the detection of the
desired product and the often difficult to separate
DSPE-PEG2000-NH.sub.2 phospholipid ammonium salt which has no
strong UV chromophore. This indicates the clear separation of
dimeric peptide phospholipid conjugate and DSPE-PEG2000-NH.sub.2
phospholipid ammonium salt. The pure product-containing fractions
may be collected, concentrated on a rotary evaporator (to reduce
the content of methanol) and freeze-dried to provide the dimer
peptide phospholipid conjugate as a colorless solid.
[0069] In order to prepare the required quantity of the dimer
peptide phospholipid conjugate, several runs may be conducted
employing 0.5 g to 1.0 g of the precursor dimer peptide. In all
cases the target dimer peptide phospholipid conjugate may be
isolated in 57-60% yield and in >99% purity. The bulk quantity
of dimer peptide phospholipid conjugate, obtained from the serial
runs described above may be obtained by dissolution of the product
from the individual runs in t-butanol-acetonitrile-water (1:1:3)
followed by lyophilization. The procedure of Ellman for
quantitative estimation of free thiol may be applied to the bulk
sample of the dimeric peptide phospholipid conjugate; free thiol,
if present will be below the limit of detection. Amino acid
composition analysis gives results within the acceptable limits,
supporting the assigned structure of the peptide derivative.
MALDI-TOF mass spectral analysis also supports the presumed
structure of the dimeric peptide phospholipid conjugate.
Methods of Preparation of Dimer-Phospholipid Conjugates Having Low
or Negligible Levels of TFA
[0070] The present invention also provides methods for producing
dimeric peptide-phospholipid conjugates having very low levels of
TFA. While certain methods provide for the synthesis and
purification of such conjugates on a gram scale, formation of a
lyso-version of the conjugates has been observed upon storage of
lyophilized material at 5.degree. C. or upon storage of aqueous
solutions of the conjugates. It is believed that the lyso-compound
is formed by TFA-promoted acid hydrolysis of one of the
phospholipid fatty acid esters in dimer peptide-phospholipid
conjugates.
[0071] To obtain the phospholipid peptide as a stable material
bearing a pharmaceutically acceptable counterion, highly efficient
methods for obtaining dimer peptide-phospholipid conjugates were
discovered which convert the TFA salts of the dimer
peptide-phospholipid conjugate, or any suitable precursor(s), to
analogous pharmaceutical acetate salt(s). Representative
embodiments of these methods are provided below.
[0072] Table 3 provides a description for the identification labels
shown in FIGS. 6, 7 and 8.
TABLE-US-00003 TABLE 3 21 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-
VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut-K(DSPE-
PEG2000-NH-Glut)]-NH.sub.2 cyclic (2-12) disulfide}- NH.sub.2
cyclic (6-13) disulfide 22
Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH.sub.2 cyclic (2-12)
disulfide .cndot.nTFA 23
Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH.sub.2 cyclic (2-12)
disulfide .cndot.xHOAc 24 mono-NHS ester of glutaryl-peptide 23
Ac-AGPTWCEDDWYYCWLFGTGGGK[NHS-Glut-K(ivDde)]- NH.sub.2 cyclic
(2-12) disulfide 25 Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH.sub.2
cyclic (2-12) disulfide .cndot.yTFA 26
Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH.sub.2 cyclic (2-12)
disulfide .cndot.zHOAc 27 Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-
VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)-NH.sub.2 cyclic (2-12)
disulfide[-NH.sub.2 cyclic (6-13) disulfide .cndot.X HOAc 28
Mono-NHS ester of glutaryl-peptide 27 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-
VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut- K(NHS-Glut)]-NH.sub.2 cyclic
(2-12) disulfide}-NH.sub.2 cyclic (6-13) disulfide 29
DSPE-PEG2000-NH.sub.2 Where m, n, x, y, z are variable depending on
lyophilization conditions.
[0073] Referring now to FIGS. 6 and 7, in certain embodiments
monomer peptide components of heterodimer peptide (27), namely TFA
salts compounds (22) and (25), are subjected to ion exchange
chromatography on the macroporous sulfonic acid cation exchange
resin AG MP-50 using a step gradient of ammonium acetate to convert
them to their acetate salts. Then the two peptide monomer acetates
(23) and (26) may be tethered through a glutaryl linker to form the
dimer (27) as an acetate salt. Purification of the crude dimer
acetate salt of (27), by C-18 preparative HPLC using a linear
gradient method employing CH.sub.3CN/H.sub.2O each containing 10 mM
NH.sub.4OAc provides the pure dimer acetate (27). Conjugation of
this dimer to DSPE-PEG2000-NH.sub.2 (29) and final purification of
the crude mixture by C-3 preparative HPLC using
CH.sub.3CN/H.sub.2O/NH.sub.4OAc provides compound (21) as the
acetate salt.
[0074] More specifically, compounds (22), (25) and (27) all bear
side-chain carboxylic acid and amino groups. AG MP-50, a
macroporous cation-exchange resin, may be used to allow full
penetration of the resin by the peptides and to exploit the
immobilization of the peptides via their basic (amino and guanidine
groups). TFA salts of the peptides may be adsorbed to an AG MP-50
column (sulfonic acid form) and the column may be washed with water
and then eluted with a step gradient of NH.sub.4OAc in 0 or 30%
CH.sub.3CN/H.sub.2O, depending on the solubility of the peptides.
The peptides may be eluted at about 600 mM NH.sub.4OAc and the
acetate form of the peptides then may be obtained in pure form.
Both IC fluorine analysis and CE TFA counter-ion analysis
consistently show very low TFA content of the peptides.
[0075] Preferred methods also include
redissolution/relyophilization of the final peptides several times
to remove residual NH.sub.4OAc. Otherwise, residual traces of
NH.sub.4OAc present in the peptides may give rise to free ammonia
in the presence of DIEA. This may result in the formation of
unwanted peptide-Glut-amide as a major product in subsequent
preparation of (27) from the monomers (23) and (26) or final
phospholipid-peptide conjugate (21) from the acetate salt of
(27).
[0076] Referring now to FIG. 7, another embodiment provides the
conversion of the TFA salt of dimer (27) to its analogous acetate
salt by ion exchange chromatography on the macroporous sulfonic
acid cation exchange resin AG MP-50. This dimer acetate then may be
conjugated with DSPE-PEG2000-NH.sub.2 followed by purification of
the crude material by C-3 preparative column using
CH.sub.3CN/H.sub.2O/NH.sub.4OAc to give the final compound (21) as
an acetate salt.
[0077] While the methods described above and in FIGS. 6 and 7
provide excellent results, the second approach has the advantage of
requiring fewer steps. Additional details are provided below in the
Examples section.
[0078] Turning to FIG. 8, another embodiment provides methods for
providing dimeric conjugates having minimal amounts of TFA
utilizing the size differential between the phospholipid-peptide
conjugate (21) and TFA ions. In this embodiment 21.cndot.nTFA
adduct may be eluted down a size exclusion column in the presence
of ammonium bicarbonate buffer. The crude 21.cndot.nTFA initially
may be freed of the lyso-compound by preparative HPLC on a Zorbax
C-3 column using a linear gradient of acetonitrile into water. Both
phases may be buffered with 10 mM ammonium acetate. This provides
separation of the lyso-compound as indicated by analytical
HPLC.
[0079] To further reduce the amount of TFA, the material may be
applied to a Sephadex G-25 column and eluted with aqueous ammonium
bicarbonate solution. The eluate may be monitored by HPLC.
Product-containing fractions may be pooled and lyophilized to
afford the desired material (21) essentially free of TFA and with
high recovery rates. Additional detail is provided below in the
Examples section.
[0080] Both the monomeric and dimeric peptide phospholipid
conjugates described herein may be incorporated into ultrasound
contrast agents such as, for example, gas filled microvesicles.
Such gas filled microvesicles include, for example, gas filled
microbubbles, gas filled microballoons, gas filled microcapsules,
etc. In a preferred embodiment, the peptide phospholipid conjugates
may be incorporated into ultrasound contrast agents comprising gas
filled microbubbles. Methods of preparation of gas filled
microbubbles from phospholipids and phospholipid conjugates are
known to those skilled in the art. For example, microbubbles
according to the present invention can be prepared by methods
described in any one of the following patents: EP 554213, WO
04/069284, U.S. Pat. No. 5,413,774, U.S. Pat. No. 5,578,292, EP
744962, EP 682530, U.S. Pat. No. 5,556,610, U.S. Pat. No.
5,846,518, U.S. Pat. No. 6,183,725, EP 474833, U.S. Pat. No.
5,271,928, U.S. Pat. No. 5,380,519, U.S. Pat. No. 5,531,980, U.S.
Pat. No. 5,567,414, U.S. Pat. No. 5,658,551, U.S. Pat. No.
5,643,553, U.S. Pat. No. 5,911,972, U.S. Pat. No. 6,110,443, U.S.
Pat. No. 6,136,293, EP 619743, U.S. Pat. No. 5,445,813, U.S. Pat.
No. 5,597,549, U.S. Pat. No. 5,686,060, U.S. Pat. No. 6,187,288,
and U.S. Pat. No. 5,908,610, which are incorporated by reference
herein in their entirety. The methods disclosed in WO 04/069284 are
particularly preferred.
[0081] Suitable phospholipids include esters of glycerol with one
or two molecules of fatty acids (the same or different) and
phosphoric acid, wherein the phosphoric acid residue is in turn
bonded to a hydrophilic group, such as choline, serine, inositol,
glycerol, ethanolamine, and the like groups. Fatty acids present in
the phospholipids are in general long chain aliphatic acids,
typically containing from 12 to 24 carbon atoms, preferably from 14
to 22, that may be saturated or may contain one or more
unsaturations. Examples of suitable fatty acids are lauric acid,
myristic acid, palmitic acid, stearic acid, arachidic acid, behenic
acid, oleic acid, linoleic acid, and linolenic acid. Mono esters of
phospholipids are known in the art as the "lyso" forms of the
phospholipid.
[0082] Further examples of phospholipids are phosphatidic acids,
i.e., the diesters of glycerol-phosphoric acid with fatty acids,
sphingomyelins, i.e., those phosphatidylcholine analogs where the
residue of glycerol diester with fatty acids is replaced by a
ceramide chain, cardiolipins, i.e. the esters of
1,3-diphosphatidylglycerol with a fatty acid, gangliosides,
cerebrosides, etc.
[0083] As used herein, the term phospholipids includes either
naturally occurring, semisynthetic or synthetically prepared
products that can be employed either singularly or as mixtures.
[0084] Examples of naturally occurring phospholipids are natural
lecithins (phosphatidylcholine (PC) derivatives) such as,
typically, soya bean or egg yolk lecithins. Examples of
semisynthetic phospholipids are the partially or fully hydrogenated
derivatives of the naturally occurring lecithins.
[0085] Examples of synthetic phospholipids are e.g.,
dilauryloyl-phosphatidylcholine ("DLPC"),
dimyristoylphosphatidylcholine ("DMPC"),
dipalmitoyl-phosphatidylcholine ("DPPC"),
diarachidoylphosphatidylcholine ("DAPC"),
distearoyl-phosphatidylcholine ("DSPC"),
1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"),
1-palmitoyl-2-myristoylphosphatidylcholine ("PMPC"),
1-palmitoyl-2-stearoylphosphatidylcholine ("PSPC"),
1-stearoyl-2-palmitoyl-phosphatidylcholine ("SPPC"),
dioleoylphosphatidylycholine ("DOPC"), 1,2
Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),
dilauryloyl-phosphatidylglycerol ("DLPG") and its alkali metal
salts, diarachidoylphosphatidylglycerol ("DAPG") and its alkali
metal salts, dimyristoylphosphatidylglycerol ("DMPG") and its
alkali metal salts, dipalmitoyl-phosphatidylglycerol ("DPPG") and
its alkali metal salts, distearolyphosphatidylglycerol ("DSPG") and
its alkali metal salts, dioleoylphosphatidylglycerol ("DOPG") and
its alkali metal salts, dimyristoyl phosphatidic acid ("DMPA") and
its alkali metal salts, dipalmitoyl phosphatidic acid ("DPPA") and
its alkali metal salts, distearoyl phosphatidic acid ("DSPA"),
diarachidoyl phosphatidic acid ("DAPA") and its alkali metal salts,
dimyristoyl phosphatidyl-ethanolamin-e ("DMPE"), dipalmitoyl
phosphatidylethanolamine ("DPPE"), distearoyl
phosphatidyl-ethanolamine ("D SPE"), dimyristoyl phosphatidylserine
("DMPS"), diarachidoyl phosphatidylserine ("DAPS"), dipalmitoyl
phosphatidylserine ("DPPS"), distearoylphosphatidylserine ("DSPS"),
dioleoylphosphatidylserine ("DOPS"), dipalmitoyl sphingomyelin
("DPSP"), and distearoyl sphingomyelin ("DS SP").
[0086] Suitable phospholipids further include phospholipids
modified by linking a hydrophilic polymer thereto. Examples of
modified phospholipids are phosphatidylethanolamines (PE) modified
with polyethylenglycol (PEG), in brief "PE-PEGs", i.e.
phosphatidylethanolamines where the hydrophilic ethanolamine moiety
is linked to a PEG molecule of variable molecular weight (e.g. from
300 to 5000 daltons), such as DPPE-PEG, DSPE-PEG, DMPE-PEG or
DAPE-PEG (where DAPE is
1,2-diarachidoyl-sn-glycero-3-phosphoethanolamine). The
compositions also may contain other amphiphilic compounds
including, for instance, fatty acids, such as palmitic acid,
stearic acid, arachidonic acid or oleic acid; sterols, such as
cholesterol, or esters of sterols with fatty acids or with sugar
acids; glycerol or glycerol esters including glycerol tripalmitate,
glycerol distearate, glycerol tristearate, glycerol dimyristate,
glycerol trimyristate, glycerol dilaurate, glycerol trilaurate,
glycerol dipalmitate; tertiary or quaternary alkyl-ammonium salts,
such as 1,2-distearoyl-3-trimethylammonium-propane (DSTAP),
1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), and mixtures
or combinations thereof.
[0087] Preferably, the formulation comprises at least one component
bearing an overall net charge, such as, for instance, phosphatidic
acid, PE-PEG, palmitic acid, stearic acid, Ethyl-DSPC or DSTAP,
preferably in a molar amount of less than about 50%. Particularly
preferred formulations may include mixtures of two or more of the
following components: DSPC, DPPG, DPPA, DSPE-PEG1000, DSPE-PEG2000,
palmitic acid and stearic acid. Some preferred phospholipids and
formulations are set forth in the examples Any of the gases
disclosed herein or known to the skilled artisan may be employed;
however, inert gases, such as SF.sub.6 or perfluorocarbons like
CF.sub.4, C.sub.3F.sub.8 and C.sub.4F.sub.10, are preferred,
optionally in admixture with other gases such as air, nitrogen,
oxygen or carbon dioxide
[0088] The preferred microbubble suspensions of the present
invention may be prepared from phospholipids using known processes
such as a freeze-drying or spray-drying solutions of the crude
phospholipids in a suitable solvent or using the processes set
forth in EP 554213; WO 04/069284; U.S. Pat. No. 5,413,774; U.S.
Pat. No. 5,578,292; EP 744962; EP 682530; U.S. Pat. No. 5,556,610;
U.S. Pat. No. 5,846,518; U.S. Pat. No. 6,183,725; EP 474833; U.S.
Pat. No. 5,271,928; U.S. Pat. No. 5,380,519; U.S. Pat. No.
5,531,980; U.S. Pat. No. 5,567,414; U.S. Pat. No. 5,658,551; U.S.
Pat. No. 5,643,553; U.S. Pat. No. 5,911,972; U.S. Pat. No.
6,110,443; U.S. Pat. No. 6,136,293; EP 619743; U.S. Pat. No.
5,445,813; U.S. Pat. No. 5,597,549; U.S. Pat. No. 5,686,060; U.S.
Pat. No. 6,187,288; and U.S. Pat. No. 5,908,610, which are
incorporated by reference herein in their entirety. Preferably, as
disclosed in International patent application WO 04/069284, a
microemulsion can be prepared which contains the phospholipids (e.g
DSPC and/or DSPA) in admixture with a lyoprotecting agent (such as,
for instance, carbohydrates, sugar alcohols, polyglycols and
mixtures thereof, as indicated in detail hereinafter) and
optionally other amphiphilic materials (such as stearic acid),
dispersed in an emulsion of water and of a water immiscible organic
solvent. Preferred organic solvents are those having solubility in
water of 1.0 g/l or lower, preferably lower about 0.01 g/l, and
include, for instance, pentane, hexane, heptane, octane, nonane,
decane, 1-pentene, 2-pentene, 1-octene, cyclopentane, cyclohexane,
cyclooctane, 1-methyl-cyclohexane, benzene, toluene, ethylbenzene,
1,2-dimethylbenzene, 1,3-dimethylbenzene, di-butyl ether and
di-isopropylketone, chloroform, carbon tetrachloride,
2-chloro-1-(difluoromethoxy)-1,1,2-trifluoroethane (enflurane),
2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane (isoflurane),
tetrachloro-1,1-difluoroethane, perfluoropentane, perfluorohexane,
perfluoroheptane, perfluorononane, perfluorobenzene,
perfluorodecalin, methylperfluorobutylether,
methylperfluoroisobutylether, ethylperfluorobutylether,
ethylperfluoroisobutylether and mixtures thereof. The
peptide-phospholipid conjugate of the invention can be admixed
together with the phospholipid forming the microvesicle's envelope,
in the microemulsion. Preferably, an aqueous suspension of the
peptide-phospholipid conjugate and of a PE-PEG (e.g. DSPE-PEG2000)
is first prepared, which is then admixed together with an
aqueous-organic emulsion comprising the phospholipid and the
lyoprotecting agent. Preferably said mixing is effected under
heating, e.g. form about 40.degree. C. to 80.degree. C.
[0089] Prior to formation of the suspension of microbubbles by
dispersion in an aqueous carrier, the freeze dried or spray dried
phospholipid powders are contacted with air or another gas. When
contacted with the aqueous carrier the powdered phospholipids whose
structure has been disrupted will form lamellarized or laminarized
segments that will stabilize the microbubbles of the gas dispersed
therein. This method permits production of suspensions of
microbubbles that are stable even when stored for prolonged periods
and are obtained by simple dissolution of the dried laminarized
phospholipids (which have been stored under a desired gas) without
shaking or any violent agitation.
[0090] Alternatively, microbubbles can be prepared by suspending a
gas into an aqueous solution at high agitation speed, as disclosed
e.g. in WO 97/29783. A further process for preparing microbubbles
is disclosed in WO 2004/069284, herein incorporated by reference,
which comprises preparing an emulsion of an organic solvent in an
aqueous medium in the presence of a phospholipid and subsequently
lyophilizing said emulsion, after optional washing and/or
filtration steps. Some preferred preparation methods are disclosed
in the examples.
[0091] The formulation for the preparation of the gas-filled
microbubbles may advantageously further comprise a lyophilization
additive, such as an agent with cryoprotective and/or lyoprotective
effect and/or a bulking agent, for example an amino-acid such as
glycine; a carbohydrate, e.g. a sugar such as sucrose, mannitol,
maltose, trehalose, glucose, lactose or a cyclodextrin, or a
polysaccharide such as dextran; or a polyglycol such as
polyethylene glycol (e.g. PEG-4000).
[0092] Any of these ultrasound compositions should also be, as far
as possible, isotonic with blood. Hence, before injection, small
amounts of isotonic agents may be added to any of above ultrasound
contrast agent suspensions. The isotonic agents are physiological
solutions commonly used in medicine and they comprise aqueous
saline solution (0.9% NaCl), 2.6% glycerol solution, 5% dextrose
solution, etc. Additionally, the ultrasound compositions may
include standard pharmaceutically acceptable additives, including,
for example, emulsifying agents, viscosity modifiers,
cryoprotectants, lyoprotectants, bulking agents etc.
[0093] Any biocompatible gas may be used in the ultrasound contrast
agents of the invention. The term "gas" as used herein includes any
substances (including mixtures) substantially in gaseous form at
the normal human body temperature. The gas may thus include, for
example, air, nitrogen, oxygen, CO.sub.2, argon, xenon or krypton,
fluorinated gases (including for example, perfluorocarbons,
SF.sub.6, SeF.sub.6) a low molecular weight hydrocarbon (e.g.,
containing from 1 to 7 carbon atoms), for example, an alkane such
as methane, ethane, a propane, a butane or a pentane, a cycloalkane
such as cyclopropane, cyclobutane or cyclopentene, an alkene such
as ethylene, propene, propadiene or a butene, or an alkyne such as
acetylene or propyne and/or mixtures thereof. However, fluorinated
gases are preferred. Fluorinated gases include materials that
contain at least one fluorine atom such as SF.sub.6, freons
(organic compounds containing one or more carbon atoms and
fluorine, i.e., CF.sub.4, C.sub.2F.sub.6, C.sub.3F.sub.8,
C.sub.4F.sub.8, C.sub.4F.sub.10, CBrF.sub.3, CCl.sub.2F.sub.2,
C.sub.2ClF.sub.5, and CBrClF.sub.2) and perfluorocarbons. The term
perfluorocarbon refers to compounds containing only carbon and
fluorine atoms and includes, in particular, saturated, unsaturated,
and cyclic perfluorocarbons. The saturated perfluorocarbons, which
are usually preferred, have the formula C.sub.nF.sub.n+2, where n
is from 1 to 12, preferably from 2 to 10, most preferably from 3 to
8 and even more preferably from 3 to 6. Suitable perfluorocarbons
include, for example, CF.sub.4, C.sub.2F.sub.6,
C.sub.3F.sub.8C.sub.4F.sub.8, C.sub.4F.sub.10, C.sub.5F.sub.12,
C.sub.6F.sub.2, C.sub.7F.sub.14, C.sub.8F.sub.18, and
C.sub.9F.sub.20. Most preferably the gas or gas mixture comprises
SF.sub.6 or a perfluorocarbon selected from the group consisting of
C.sub.3F.sub.8C.sub.4F.sub.8, C.sub.4F.sub.10, C.sub.5F.sub.2,
C.sub.6F.sub.12, C.sub.7F.sub.14, C.sub.8F.sub.18, with
C.sub.4F.sub.10 being particularly preferred. See also WO 97/29783,
WO 98/53857, WO 98/18498, WO 98/18495, WO 98/18496, WO 98/18497, WO
98/18501, WO 98/05364, WO 98/17324. In a preferred embodiment the
gas comprises C.sub.4F.sub.10 or SF.sub.6, optionally in admixture
with air, nitrogen, oxygen or carbon dioxide.
[0094] In certain circumstances it may be desirable to include a
precursor to a gaseous substance (e.g., a material that is capable
of being converted to a gas in vivo, often referred to as a "gas
precursor"). Preferably the gas precursor and the gas it produces
are physiologically acceptable. The gas precursor may be
pH-activated, photo-activated, temperature activated, etc. For
example, certain perfluorocarbons may be used as temperature
activated gas precursors. These perfluorocarbons, such as
perfluoropentane, have a liquid/gas phase transition temperature
above room temperature (or the temperature at which the agents are
produced and/or stored) but below body temperature; thus they
undergo a phase shift and are converted to a gas within the human
body.
[0095] As discussed above, the gas can comprise a mixture of gases.
The following combinations are particularly preferred gas mixtures:
a mixture of gases (A) and (B) in which, at least one of the gases
(B), present in an amount of between 0.5-41% by vol., has a
molecular weight greater than 80 daltons and is a fluorinated gas
and (A) is selected from the group consisting of air, oxygen,
nitrogen, carbon dioxide and mixtures thereof, the balance of the
mixture being gas A.
[0096] Unless it contains a hyperpolarized gas, known to require
special storage conditions, the lyophilized product may be stored
and transported without need of temperature control of its
environment and in particular it may be supplied to hospitals and
physicians for on site formulation into a ready-to-use
administrable suspension without requiring such users to have
special storage facilities. Preferably in such a case it can be
supplied in the form of a two-component kit, which can include two
separate containers or a dual-chamber container. In the former case
preferably the container is a conventional septum-sealed vial,
wherein the vial containing the lyophilized residue of step b) is
sealed with a septum through which the carrier liquid may be
injected using an optionally prefilled syringe. In such a case the
syringe used as the container of the second component is also used
then for injecting the contrast agent. In the latter case,
preferably the dual-chamber container is a dual-chamber syringe and
once the lyophilizate has been reconstituted and then suitably
mixed or gently shaken, the container can be used directly for
injecting the contrast agent. In both cases means for directing or
permitting application of sufficient bubble forming energy into the
contents of the container are provided. However, as noted above, in
the stabilised contrast agents according to the invention the size
of the gas microbubbles is substantially independent of the amount
of agitation energy applied to the reconstituted dried product.
Accordingly, no more than gentle hand shaking is generally required
to give reproducible products with consistent microbubble size.
[0097] It can be appreciated by one of ordinary skilled in the art
that other two-chamber reconstitution systems capable of combining
the dried powder with the aqueous solution in a sterile manner are
also within the scope of the present invention. In such systems, it
is particularly advantageous if the aqueous phase can be interposed
between the water-insoluble gas and the environment, to increase
shelf life of the product. Where a material necessary for forming
the contrast agent is not already present in the container (e.g. a
targeting ligand to be linked to the phospholipid during
reconstitution), it can be packaged with the other components of
the kit, preferably in a form or container adapted to facilitate
ready combination with the other components of the kit.
[0098] No specific containers, vial or connection systems are
required; the present invention may use conventional containers,
vials and adapters. The only requirement is a good seal between the
stopper and the container. The quality of the seal, therefore,
becomes a matter of primary concern; any degradation of seal
integrity could allow undesirable substances to enter the vial. In
addition to assuring sterility, vacuum retention is essential for
products stoppered at ambient or reduced pressures to assure safe
and proper reconstitution. The stopper may be a compound or
multicomponent formulation based on an elastomer, such as
poly(isobutylene) or butyl rubber.
[0099] In ultrasound applications the contrast agents formed by
phospholipid stabilized microbubbles can be administered, for
example, in doses such that the amount of phospholipid injected is
in the range 0.1 to 200 .mu.g/kg body weight, preferably from about
0.1 to 30 .mu.g/kg.
[0100] Ultrasound imaging techniques that can be used in accordance
with the present invention include known techniques, such as color
Doppler, power Doppler, Doppler amplitude, stimulated acoustic
imaging, and two- or three-dimensional imaging techniques. Imaging
may be done in harmonic (resonant frequency) or fundamental modes,
with the second harmonic preferred.
[0101] The ultrasound contrast agents of the present invention may
further be used in a variety of therapeutic imaging methods. The
term therapeutic imaging includes within its meaning any method for
the treatment of a disease in a patient which comprises the use of
a contrast imaging agent (e.g. for the delivery of a therapeutic
agent to a selected receptor or tissue), and which is capable of
exerting or is responsible to exert a biological effect in vitro
and/or in vivo. Therapeutic imaging may advantageously be
associated with the controlled localized destruction of the
gas-filled microvesicles, e.g. by means of an ultrasound burst at
high acoustic pressure (typically higher than the one generally
employed in non-destructive diagnostic imaging methods). This
controlled destruction may be used, for instance, for the treatment
of blood clots (a technique also known as sonothrombolysis),
optionally in combination with the localized release of a suitable
therapeutic agent. Alternatively, said therapeutic imaging may
include the delivery of a therapeutic agent into cells, as a result
of a transient membrane permeabilization at the cellular level
induced by the localized burst of the microvesicles. This technique
can be used, for instance, for an effective delivery of genetic
material into the cells; optionally, a drug can be locally
delivered in combination with genetic material, thus allowing a
combined pharmaceutical/genetic therapy of the patient (e.g. in
case of tumor treatment).
[0102] The term "therapeutic agent" includes within its meaning any
substance, composition or particle which may be used in any
therapeutic application, such as in methods for the treatment of a
disease in a patient, as well as any substance which is capable of
exerting or responsible to exert a biological effect in vitro
and/or in vivo. Therapeutic agents thus include any compound or
material capable of being used in the treatment (including
diagnosis, prevention, alleviation, pain relief or cure) of any
pathological status in a patient (including malady, affliction,
disease lesion or injury). Examples of therapeutic agents are
drugs, pharmaceuticals, bioactive agents, cytotoxic agents,
chemotherapy agents, radiotherapeutic agents, proteins, natural or
synthetic peptides, including oligopeptides and polypeptides,
vitamins, steroids and genetic material, including nucleosides,
nucleotides, oligonucleotides, polynucleotides and plasmids.
[0103] Materials and Analytical Methods
[0104] Solvents for reactions, chromatographic purification and
HPLC analyses were E. Merck Omni grade solvents from VWR
Corporation (West Chester, Pa.). N-Methylpyrrolidinone (NMP) and
N,N-dimethylformamide (DMF) were obtained from Pharmco Products
Inc. (Brookfield, Conn.), and were peptide synthesis grade or low
water/amine-free Biotech grade quality. Piperidine (sequencing
grade, redistilled 99+%) and trifluoroacetic acid (TFA)
(spectrophotometric grade or sequencing grade) were obtained from
Sigma-Aldrich Corporation (Milwaukee, Wis.) or from the Fluka
Chemical Division of Sigma-Alrich Corporation.
N,N'-Diisopropylcarbodiimide (DIC), phenol (99%),
N,N-diisopropylethylamine (DIEA) and triisopropylsilane (TIS) were
purchased from Sigma-Aldrich Corporation. Fmoc-protected amino
acids, pseudoproline dipeptides,
Fmoc-Asp(O-tBu)-Ser(.PSI..sup.Me,Mepro)-OH and
Fmoc-Gly-Thr(.PSI..sup.Me,Mepro)-OH and N-hydroxybenzotriazole
(HOBt) were obtained from Novabiochem (San Diego, Calif.).
Fmoc-8-amino-3,6-dioxaoctanoic acid (Adoa) was obtained from NeoMPS
Corp (San Diego, Calif.) or Suven Life Sciences (Hyderabad, India).
Disuccinimidyl glutarate (DSG) and
1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[amino
(polyethyleneglycol)2000] ammonium salt, [DSPE-PEG2000-NH.sub.2]
were obtained from Pierce Chemical Co. (Rockford, Ill.) and
Avanti.RTM. Polar Lipids (Alabaster, Ala.), respectively.
Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH were prepared in-house from
the corresponding triglycine or diglycine by the reaction with
Fmoc-OSu. An AG MP-50 ion-exchange resin was obtained from Bio-Rad
(Hercules, Calif.).
[0105] Analytical HPLC data were generally obtained using a
Shimadzu LC-10AT VP dual pump gradient system employing a Waters
XTerra MS-C18 4.6.times.50 mm column, (particle size: 5.mu.; 120
.ANG. pore size) and gradient or isocratic elution systems using
water (0.1% TFA) as eluent A and CH.sub.3CN (0.1% TFA) or
CH.sub.3CN--CH.sub.3OH (1:1, v/v) (0.1% TFA) as eluent B. Detection
of compounds was accomplished using UV at 220 and 254 nm. The
purity of the phospholipid-PEG-peptide derivatives was determined
on a YMC C-4 (5 .mu.M, 300 .ANG., 4.6.times.250 mm) column or on a
Zorbax 300 SB-C3 (3.5 .mu.M; 300 .ANG., 3.times.150 mm) column
using a SEDEX 55 Light Scattering Detector (LSD) and with a UV
detector.
[0106] Preparative HPLC was conducted on a Shimadzu LC-8A dual pump
gradient system equipped with a SPD-10AV UV detector fitted with a
preparative flow cell. Generally the solution containing the crude
peptide was loaded onto a reversed phase C18, C4 or C3 column,
depending on the compound characteristics, using a third pump
attached to the preparative Shimadzu LC-8A dual pump gradient
system. After the solution of the crude product mixture was applied
to the preparative HPLC column the reaction solvents and solvents
employed as diluents, such as DMF or DMSO, were eluted from the
column at low organic phase composition. Then the desired product
was eluted using a gradient elution of eluent B into eluent A.
Product-containing fractions were combined based on their purity as
determined by analytical HPLC and mass spectral analysis. The
combined fractions were freeze-dried to provide the desired
product.
[0107] Amino acid composition analyses were performed at the Keck
Biotechnology Resource Laboratory at Yale University, New Haven,
Conn. Mass spectral data were obtained from MScan Inc. (606
Brandywine Parkway, West Chester Pa. 19380) or obtained in-house on
an Agilent LC-MSD 1100 Mass Spectrometer. For the purposes of
fraction selection and characterization of the products mass
spectral values were usually obtained using API-ES in negative ion
mode. Generally the molecular weight of the target peptides was
.about.3000; the mass spectra usually exhibited doubly or triply
negatively charged ion mass values rather than [M-H].sup.-. These
were generally employed for selection of fractions for collection
and combination to obtain the pure peptide during HPLC
purification. In some cases fractions exhibited dominant peaks
attributable to [M-2H]/2+57 or [M-2H]/2+114 in the mass spectrum.
These peaks are due to the formation of adducts of one or two
molecules of trifluoroacetic acid per molecule of the peptide.
After careful collection of fractions by comparing MS results and
HPLC purities and freeze-drying process, a small amount of the
isolated fluffy solid was dissolved in water (0.5 mg/mL) and
treated with a drop of aqueous N-methyl-D-glucamine (.about.0.5 M).
This solution was analyzed by HPLC and MS for final purity results
of the purified peptide. Peptide solutions in the presence of
N-methyl-D-glucamine did not exhibit [M-2H]/2+57 or [M-2H]/2+114
mass value peaks in the mass spectrum, instead the expected
[M-2H]/2 or [M-3H]/3 peaks were observed.
[0108] The following non-limiting Examples provide additional
detail on efficient processes used for obtaining large quantities
of highly purified forms of the monomeric and dimeric peptide
phospholipid conjugates. These non-limiting Examples also describe
the preparation of representative targeted microbubbles which
include these monomeric and dimeric peptide phospholipid
conjugates. These Examples also describe the use of such targeted
microbubbles in static binding tests on KDR-transfected cells and
dynamic binding tests on rh VEGF-R2/Fc chimeric protein. The
Examples further describe the evaluation of ultrasound contrast
agents containing KDR binding lipopeptides in a rabbit VX2 tumor
model.
EXAMPLES
[0109] Examples 1-2 below refer to the monomeric peptide
phospholipid conjugate shown in FIG. 2. A process for synthesizing
this compound is shown in FIG. 1. Although these Examples refer
more specifically to the process for synthesizing the compound
shown in FIG. 2, similar processes may used to prepare the
monomeric peptide phospholipid conjugate shown in FIG. 10 and the
linear peptide monomer (32) shown in FIG. 9 as well as other
monomer peptide-phospholipid conjugates including, for example,
those formed using peptide monomers
Ac-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2 (SEQ. ID NO. 6) and
Ac-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH2 (SEQ. ID NO. 7), as well as
analogs and derivatives of any of the above. Additionally,
co-pending U.S. application Ser. No. 10/661,156, filed Sep. 11,
2003, sets forth methods for the preparation of the peptide
monomers and is incorporated by reference herein in its
entirety.
Example 1
TABLE-US-00004 [0110] Solid Phase Synthesis (SPPS) and Purification
of Linear Peptide Monomer (2) Ac-
RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH.sub.2, (SEQ ID NO. 2)
Ac-Arg-Ala-Gln-Asp-
Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Se-
r-Gly-Gly- Gly-Gly-Gly-Lys-NH.sub.2,;
N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-
tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-a-
lanyl-L-aspartyl-L-
glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L--
seryl-glycyl-glycyl- glycyl-glycyl-glycyl-L-lysinamide
[0111] The linear peptide monomer (2) was synthesized by an
established automated protocol on a SONATA/Pilot Peptide
Synthesizer using Fmoc-Pal-Peg-PS resin (0.2 mmol/g),
Fmoc-protected amino acids and DIC-mediated HOBt ester activation
in DMF. The peptide sequence was synthesized in stepwise fashion by
SPPS methods on the Fmoc-Pal-Peg-PS resin, typically on a 10 mmol
scale. The amino acid couplings were carried out with a 4-fold
excess each of amino acid and the DIC-HOBt reagent pair in DMF.
[0112] In a typical coupling of an amino acid, 5 mL of dry DMF per
gram of resin was used. The total volume of DMF, calculated on the
basis of resin used, was allocated among amino acid, HOBt and DIC
for solution preparation. For example, for the synthesis involving
50 g (10 mmol scale) of resin, the calculated volume of 250 mL of
DMF was distributed among amino acid (150 mL), HOBt (50 mL) and DIC
(50 mL). The amino acid vessel on the Sonata Pilot Peptide
Synthesizer was charged with the solid dry amino acid (4-fold
excess with respect to the resin). At inception of the coupling
step, the software of the instrument was employed to deliver
successively the chosen volume of DMF (for dilution of the amino
acid) and HOBt (4 eq.) in DMF and DIC (4 eq.) in DMF and mixing by
nitrogen bubbling was initiated and conducted for 4 min. This
served to pre-activate the amino acid and to insure complete
dissolution of all components of the mixture. After activation, the
software mediated the transfer of the solution of the activated
Fmoc-amino acid to the reaction vessel containing the resin. After
transfer was complete the vessel was agitated for 3 h with
recurrent nitrogen bubbling. After the 3 h coupling time, the resin
was washed thoroughly with DMF (5 mL/g, 6.times.) and the cleavage
of the Fmoc-group was performed with 25% piperidine in DMF (5 mL/g)
containing HOBt (0.1M) (2.times.10 min). The resin was thoroughly
washed with DMF (5 mL/g, 6.times.) to assure complete removal of
piperidine from the resin in preparation for the ensuing amino acid
coupling. In the case of Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH,
the pre-activation in the amino acid bottle was not conducted in
order to minimize the formation of diketopiperazine during the
activation time as discussed in the text. Therefore, in these two
cases, the solutions of amino acid, HOBt and DIC were added to the
reaction vessel sequentially and the coupling process was conducted
with `in situ` activation.
[0113] After chain elongation was completed, the Fmoc group of the
N-terminal amino acid was removed in the standard manner followed
by the standard wash with DMF (vide supra). The N-terminal amino
acid was then capped by treatment with freshly prepared acetylation
mixture (0.5M acetic anhydride, 0.125M DIEA and 0.015M HOBt in
DMF/6 mL/g of resin), 2.times.20 min. After completion of the
peptide synthesis, the resin was treated with the cleavage
cocktail, `Reagent B` (TFA:water:phenol:triisopropylsilane,
88:5:5:2, v/v/w/v) (10 mL/g of resin) for 4 h. The volatiles were
removed and the paste thus obtained was triturated with ether to
provide a solid which was washed with ether (3.times.) with
intervening centrifugation (to compact the suspended solids in
order to allow decantation of the supernatant) and then dried under
vacuum to provide the required peptide as an off-white solid. A 10
mmol scale synthesis of the linear peptide monomer (2) gave 33.82 g
(103% of theory) of the crude peptide. The greater than theoretical
yield was most likely due to moisture and residual solvents.
TABLE-US-00005 Purification of the Linear Peptide Monomer (2) Ac-
RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH.sub.2 (SEQ ID NO. 2);
Ac-Arg-Ala-Gln-Asp-
Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Se-
r-Gly-Gly- Gly-Gly-Gly-Lys-NH.sub.2;
N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-
tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-a-
lanyl-L-aspartyl-L-
glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L--
seryl-glycyl-glycyl- glycyl-glycyl-glycyl-L-lysinamide
[0114] A .about.0.5 g portion of the crude linear peptide monomer
(2) was dissolved in a minimum amount of CH.sub.3CN (.about.20 mL).
The volume of the solution was adjusted to .about.100 mL with water
and employing a third pump the solution was loaded onto a reversed
phase C18 preparative column (Waters, XTerra.RTM. Prep MS C18,
10.mu., 300 .ANG., 50.times.250 mm, flow rate 100 mL/min) which had
been pre-equilibrated with 10% CH.sub.3CN in water (0.1% TFA). The
column was not eluted with the equilibrating eluent during
application of the sample solution. After the sample solution was
applied to the column, the composition of the eluent was ramped to
20% CH.sub.3CN-water (0.1% TFA) over 1 min, and a linear gradient
at a rate of 0.6%/min of CH.sub.3CN (0.1% TFA) into water (0.1%
TFA) was initiated and maintained for 50 min. Fractions (15 mL)
were manually collected using UV at 220 nm as an indicator of
product elution. The collected fractions were analyzed on a Waters
XTerra analytical reversed phase C-18 column (5.mu. particle, 120
.ANG. pore) and product-containing fractions of >95% purity were
pooled and freeze-dried to afford the corresponding pure linear
peptide monomer (2). Typically the purification of 0.5 g of crude
(2) afforded 0.12 g (24% yield) of the desired product (>95%
purity).
Example 2
TABLE-US-00006 [0115] Preparation of Monomeric Peptide Phospholipid
Conjugate (1) Ac-
RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH.sub.2 (SEQ
ID NO. 1);
Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-
-Arg-His-
Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-(DSPE-PEG2000-NH-Glut)-NH.sub.2;
N-acetyl-L-
arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspar-
tyl-L-isoleucyl-L-
glutamyl-L-leucy1-1-serinyl-L-methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L-
-leucyl-L-arginyl-
L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl--
glycyl-glycyl-L- lysinamide
[0116] The monomeric peptide phospholipid conjugate (1),
Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH.sub.2(SEQ
ID NO. 1), was prepared by conjugation of (3), the glutaric acid
monoamide mono-NHS ester of peptide monomer (2), with
DSPE-PEG2000-NH.sub.2 phospholipid ammonium salt (4).
[0117] A round-bottomed flask equipped with magnetic stir bar and
septum cap was charged sequentially with anhydrous
dimethylformamide (7.5 mL), disuccinimidyl glutarate (DSG, 0.25 g,
0.75 mmol) and diisopropylethylamine (0.10 g, 0.78 mmol) with
stirring. Solid linear peptide monomer (2) (0.5 g, 0.152 mmol) was
added portionwise to the above solution over a period of 2 min;
then the solution was stirred for 30 min at ambient temperature.
The reaction mixture was diluted to .about.50 mL with anhydrous
ethyl acetate; this resulted in precipitation of the intermediate
mono-NHS ester (3), the glutaric acid monoamide mono-NHS ester of
peptide monomer (2). The solution was centrifuged to bring down
mono-NHS ester (3)) as a colorless solid. The supernatant
containing excess DSG was decanted from the compacted solid
mono-NHS ester (3) which was again dispersed in ethyl acetate,
centrifuged and washed twice more to remove the remaining traces of
DSG. The solid intermediate mono-NHS ester (3) thus obtained was
dissolved in anhydrous DMF (10.0 mL); diisopropylethylamine (0.10
g, 0.78 mmol) was added; and the mixture was stirred.
[0118] Meanwhile, DSPE-PEG2000-NH.sub.2 phospholipid ammonium salt
(4) (0.38 g, 0.14 mmol, 0.9 eq.) was suspended in dry
dichloromethane (2 mL) in a separate flask and trifluoroacetic acid
(2 drops) was added to protonate the phosphodiester oxygen
facilitating solubilization of phospholipid ammonium salt in
dichloromethane. The clear solution was then evaporated on a rotary
evaporator to remove the volatiles and dried further under
vacuum.
[0119] The solid phospholipid ammonium salt (4) was dissolved in
DMF (5 mL) and transferred to the stirred solution of mono-NHS
ester (3) and the resulting mixture was stirred for 24 h at ambient
temperature. The reaction mixture was diluted to 100 mL with a 1:1
mixture of CH.sub.3OH and CH.sub.3CN-water (1:1, v/v) and the
insolubles were filtered. Half of the filtered solution was loaded
onto a reversed phase C2 preparative column (Kromasil.RTM. Prep C2,
10.mu., 300 .ANG., 50.times.250 mm) which had been pre-equilibrated
with 3:1 (v/v) mixture of water (0.1% TFA) and
CH.sub.3OH--CH.sub.3CN (1:1, v/v, 0.1% TFA) at a flow rate of 100
mL/min. Note that the column was not eluted with the equilibrating
eluent during loading of the sample. After the sample solution was
loaded the column was washed with the equilibration eluent until
the plug of DMF was eluted. The composition of the eluent was
ramped to 70% CH.sub.3OH--CH.sub.3CN (1:1, 0.1% TFA) over 9 min and
a linear gradient of 0.75%/min of CH.sub.3OH--CH.sub.3CN (1:1, 0.1%
TFA) into water (0.1% TFA) was initiated and run for 40 min.
Fractions (15 mL) were collected using UV (220 nm) as an indicator
of product elution. Fractions were checked for purity on an
analytical HPLC system (column: YMC C-4, 5.mu., 300 .ANG.,
4.6.times.250 mm) using UV at 220 nm and an evaporative light
scattering detector (ELSD). The latter detector (ELSD) was employed
to detect DSPE-PEG2000-NH.sub.2 phospholipid ammonium salt (4)
which has very little UV absorbance at 220 nm. Product-containing
fractions of >98% purity, and devoid of DSPE-PEG2000-NH.sub.2
phospholipid ammonium salt (4) were combined and concentrated on a
rotary evaporator to reduce the content of CH.sub.3OH. The
concentrated solution was then diluted with 10% CH.sub.3CN in water
until a faint flocculent precipitate formed. The resulting solution
was freeze-dried to provide monomeric peptide phospholipid
conjugate (1) as a colorless solid. The second portion of crude
monomeric peptide phospholipid conjugate (1) was purified as
described above. The combined yield of the target monomeric peptide
phospholipid conjugate (1) was 0.40 g (47% yield).
[0120] Examples 3-5 below refer to the dimeric peptide phospholipid
conjugate shown in FIG. 5. Representative methods of synthesizing
the dimeric conjugate are shown in FIGS. 3, 4, 6, 7 and 8.
Example 3
TABLE-US-00007 [0121] Solid Phase Synthesis (SPPS), Cyclization and
Purification of Monomer Peptides (12) Ac-
AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH.sub.2 and (13) Ac-
VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH.sub.2
[0122] The linear peptides were synthesized by an established
automated protocol on a SONATA.RTM./Pilot Peptide Synthesizer using
Fmoc-Pal-Peg-PS resin (0.2 mmol/g), Fmoc-protected amino acids and
DCI-mediated HOBt ester activation in DMF. The peptide sequence on
the Fmoc-Pal-Peg-PS resin was synthesized in stepwise fashion by
SPPS methods typically on a 10 mmol scale. The amino acid coupling
was carried out with a 4-fold excess each of amino acid and
DIC-HOBt reagent in DMF.
[0123] In a typical coupling of an amino acid in the sequence, 5 mL
of dry DMF per gram of resin was used. The total volume of DMF,
calculated on the basis of resin used, was allocated among amino
acid, HOBt and DIC for solution preparation. For example, for the
synthesis involving 50 g of resin, the calculated volume of 250 mL
of DMF was distributed among amino acid (150 mL), HOBt (50 mL) and
DIC (50 mL). The amino acid vessel on the Sonata.RTM. Pilot Peptide
Synthesizer was charged with the solid dry amino acid (4-fold
excess with respect to the resin). At inception of the coupling
step, the chosen volume of DMF and HOBt (4 eq.) in DMF and DIC (4
eq.) in DMF were delivered successively and after each delivery
mixing by nitrogen bubbling was conducted. After the last reagent
was delivered mixing by nitrogen bubbling was initiated and
conducted for 4 min. This served to preactivate the amino acid and
to insure complete dissolution of all components of the
mixture.
[0124] After activation, the solution of the activated Fmoc-amino
acid was transferred to the reaction vessel containing the resin.
After transfer was complete the vessel was agitated for 3 h with
recurrent nitrogen bubbling. After the 3 h coupling time, the resin
was washed thoroughly with DMF (5 mL/g, 6.times.) and the cleavage
of the Fmoc-group was performed with 25% piperidine in DMF (5 mL/g)
containing HOBt (0.1M) (2.times.10 min). The resin was thoroughly
washed with DMF (5 mL/g, 6.times.) to assure complete removal of
piperidine from the resin in preparation for the ensuing amino acid
coupling. In the case of Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH,
the pre-activation in the amino acid bottle was not conducted in
order to minimize the formation of diketopiperazine during the
activation time as discussed in the text. Therefore, in these two
cases, the solution of the amino acid, HOBt and DIC were added to
the reaction vessel sequentially and the coupling process was
conducted with `in situ` activation. After chain elongation was
completed, the fmoc group of the N-terminal amino acid was removed
in the standard manner followed by the standard wash with DMF (vide
supra). The N-terminal amino acid was then capped by treatment with
freshly prepared acetylation mixture (0.5M acetic anhydride, 0.125M
DIEA and 0.015M HOBt in DMF-6 mL/g of resin), 2.times.20 min.
[0125] Functionalization of the .epsilon.-amino group of C-terminal
Lysine moieties of the monomer peptides (with Fmoc-Adoa or with
Fmoc-Lys(ivDde) as required) was accomplished by first removing the
ivDde group of the .epsilon.-amino group with freshly prepared 10%
hydrazine in DMF (5 mL/g of resin-2.times.10 min). For appending of
Fmoc-Adoa or Fmoc-Lys(ivDde) the coupling time was increased to 10
h. After completion of the peptide synthesis, the resin was treated
with the cleavage cocktail, `Reagent B`
(TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) (10 mL/g
of resin) for 4 h. After evaporation of the volatiles under vacuum,
the paste was triturated with ether to provide a solid which was
collected by filtration washed with diethyl ether and dried. A 10
mmol scale synthesis of (12),
Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH.sub.2 gave 30 g (103% of
theory) of the crude peptide. In the case of (13)
Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH.sub.2, a 10 mmol scale
synthesis gave 28 g (107% of theory) of crude peptide. The greater
than theoretical yields are most likely due to moisture and
residual solvents.
[0126] Cyclization of the Linear Di-Cysteine Peptides to Cyclic
Disulfide Peptides
[0127] Cyclic disulfide peptides were prepared from the
corresponding linear di-cysteine peptides by DMSO-assisted
oxidation using DMSO/water (95/5, v/v). The crude linear peptide
was dissolved in the solvent mixture (5 mL/g) in a wide mouth
beaker, and the pH of the solution was adjusted to 8.5 by the
addition of solid N-methyl-D-glucamine in portions. The resulting
mixture was stirred for 36 h at ambient temperature. The solution
was then diluted with acetonitrile (50 mL/g) and the mixture was
stirred for 2 min. The solid cyclic disulfide peptide was collected
by filtration, washed with diethyl ether and dried.
[0128] Purification of Monomer Peptides
TABLE-US-00008 Peptide Monomer (12)
Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH.sub.2; Ac-Ala-
Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gl-
y-Gly- Lys[Lys(ivDde)]-NH.sub.2 cyclic (6-13) disulfide
[0129] A .about.0.5 g portion of the crude cyclic disulfide peptide
monomer (12) was dissolved in a minimum amount of DMSO (.about.3
mL). The volume of the solution was adjusted to .about.100 mL with
20% CH.sub.3CN-water and employing a third pump, the solution was
loaded onto a reversed phase C18 preparative column (Waters,
XTerra.RTM. Prep MS C18, 10.mu., 300 .ANG., 50.times.250 mm, flow
rate 100 mL/min), which had been pre-equilibrated with 10%
CH.sub.3CN in water (0.1% TFA). During application of the sample
solution to the column the flow of the equilibrating eluent from
the preparative HPLC system was stopped. After the sample solution
was applied to the column, the flow of equilibrating eluent from
the gradient HPLC system was reinitiated and the column was eluted
with 10% CH.sub.3CN-water (0.1% TFA) until the DMSO was eluted.
Then the eluent composition was ramped to 35% CH.sub.3CN-water
(0.1% TFA) over 1 min after which a linear gradient at a rate of
0.5%/min CH.sub.3CN (0.1% TFA) into water (0.1% TFA) was initiated
and maintained for 50 min. Fractions (15 mL) were manually
collected using UV at 220 nm as an indicator of product elution.
The collected fractions were analyzed on a Waters XTerra analytical
reversed phase C-18 column (5.mu. particle, 120 .ANG. pore) and
product-containing fractions of >95% purity were pooled and
freeze-dried to afford the corresponding cyclic disulfide peptide
monomer (12). Typically the purification of 0.5 g of crude peptide
monomer (12) afforded 0.1 g (20% yield) of the desired product
(>95% purity).
TABLE-US-00009 Peptide Monomer (13)
Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH.sub.2; Ac-Val-
Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gl-
y- Lys(Adoa-Adoa)-NH.sub.2 cyclic (2-12) disulfide
[0130] Following the procedure employed for the HPLC purification
of peptide monomer (2), the crude cyclic disulfide peptide monomer
(13) Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH.sub.2 (0.5 g)
dissolved in 20% CH.sub.3CN-water mixture (100 mL) was loaded onto
a reversed phase C18 preparative column (Waters, XTerra.RTM. Prep
MS C18, 50.times.250 mm, 10.mu. particle, 300 .ANG. pore, flow rate
100 mL/min), which had been pre-equilibrated with 10% CH.sub.3CN
(0.1% TFA) in water (0.1% TFA). During application of the sample
solution to the column the flow of the equilibrating eluent from
the preparative HPLC system was stopped. After the sample solution
was applied to the column, the flow of equilibrating eluent from
the gradient HPLC system was reinitiated and the column was eluted
with 10% CH.sub.3CN-water (0.1% TFA) for 5 min. Then the eluent
composition was ramped to 30% CH.sub.3CN (0.1% TFA)-water (0.1%
TFA) over 1 min and a linear gradient elution at a rate of 0.5%/min
of CH.sub.3CN (0.1% TFA) into water (0.1% TFA) was initiated and
maintained for 50 min. Fractions (15 mL) were manually collected
using UV at 220 nm as an indicator of product elution. The
fractions were analyzed on a Waters XTerra analytical reversed
phase C-18 column (4.6 mm i.d..times.50 mm, 5.mu., particle, 120
.ANG. pore) and product-containing fractions of >95% purity were
pooled and freeze-dried to afford the corresponding cyclic
disulfide peptide monomer (13). Typically the purification of 0.5 g
of crude peptide monomer (3) afforded 0.12 g (24% yield) of the
desired product (>95% purity).
Example 4
TABLE-US-00010 [0131] Preparation and Purification of Precursor
Dimer Peptide (16) Ac-
AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-
Glut-K)]-NH.sub.2 cyclic (2-12) disulfide]-NH.sub.2 cyclic (6-13)
disulfide; Ac-Ala-Gly-Pro-Thr-Trp-
Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys[Ac-Val-
-Cys-Trp-
Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(-A-
doa-Adoa- Glut-Lys)]-NH.sub.2 cyclic (2-12) disulfide ]-NH.sub.2
cyclic (6-13) disulfide
[0132] As shown in FIG. 3, disuccinimidyl glutarate (DSG, 0.28 g,
0.86 mmol) was dissolved in stirred anhydrous dimethylformamide
(2.0 mL) and diisopropylethylamine (0.11 g, 0.85 mmol) was added in
one portion. Then solid peptide monomer (12)
Ac-AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]-NH.sub.2 (0.50 g, 0.17 mmol)
was added in portions to the stirred solution of DSG over a period
of two min. After stirring for 30 min at room temperature, the
solution was diluted with anhydrous ethyl acetate to .about.50 mL,
(this served to precipitate intermediate mono-NHS ester (14)). The
entire mixture was centrifuged and the supernatant was decanted
leaving intermediate mono-NHS ester (14) as a colorless solid. The
solid was resuspended with ethyl acetate; the solution containing
the suspended solid mono-NHS ester (14) was centrifuged to separate
the solid and the supernatant was again decanted. This washing
process was repeated twice to remove completely the excess DSG.
[0133] The solid mono-NHS ester (14) was dissolved in stirred
anhydrous dimethylformamide (2.0 mL) and diisopropylethylamine
(0.11 g, 0.85 mmol) was added. Then solid peptide monomer (13),
Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH.sub.2, (0.50 g, 0.19 mmol,
1.12 eq.) was added in portions to the stirred solution over a
three min. period and the resulting mixture was stirred for 18 h.
The reaction was monitored by mass spectrometry; after the complete
consumption of the peptide monomer glutaric acid monoamide mono-NHS
ester (14) was confirmed, neat hydrazine (0.1 mL) was added to
remove the ivDde protecting group of the ivDde-bearing dimer (15)
and the mixture was stirred for 20 min at room temperature.
[0134] The solution was then acidified by dropwise addition of TFA
and the mixture was diluted to 100 mL with 10% CH.sub.3CN (0.1%
TFA) in water (0.1% TFA). The solution was filtered to remove
particulates and half of the clarified solution was loaded onto a
reversed phase C18 preparative column (Waters, XTerra.RTM. Prep MS
C18, 10.mu., 50.times.250 mm, flow rate 100 mL/min)
pre-equilibrated with 10% CH.sub.3CN in water (0.1% TFA). During
application of the sample solution to the column the flow of the
equilibrating eluent from the preparative HPLC system was stopped.
After the sample solution was applied to the column, the flow of
equilibrating eluent from the gradient HPLC system was reinitiated
and the column was eluted with 10% CH.sub.3CN-water (0.1% TFA) in
order to flush DMF from the column. After elution of the DMF plug
was completed the eluent composition was increased to 20%
CH.sub.3CN over one min. and the elution was continued with a
linear gradient rate of 0.6%/min of CH.sub.3CN (0.1% TFA) into
water (0.1% TFA). Fractions (15 mL) were collected using UV (220
nm) as an indicator of product elution. The fractions were analyzed
on a reversed phased C18 column (Waters MS C18, 4.6 mm
i.d..times.50 mm, 5.mu., particle, 120 .ANG. pore) and the
product-containing fractions of >95% purity were pooled and
freeze-dried to provide precursor dimer peptide (16) as a
colorless, fluffy solid. The remaining crude precursor dimer
peptide (16) was purified in the same manner. From 0.5 g each of
monomer peptides (12) and (13), 320 mg (overall yield 33%) of the
desired dimer (16) was obtained (>95% purity).
Example 5
[0135] Preparation of KDR-Binding Dimeric Peptide Phospholipid
Conjugate (11)
Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-
-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystiny-
l-L-tryptophyl-1-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-gly-
cyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-
-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylal-
anyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(-
distearylphosphoethanolaminocarbonoxy-PEG2000-amino-8-amino-3,6-dioxaoctan-
oyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl) amide cyclic (2-12)
disulfide]-amide cyclic (6-13) disulfide; Ac-AGPTWCEDDWYYCWLFGTGGGK
{Ac-VCWEDSWGGEVCFRYDP-GGGK[-Adoa-Adoa-Glut-K(DSPE-PEG2000-NH-Glut)]-NH.su-
b.2 cyclic (2-12) disulfide}-NH.sub.2 cyclic (6-13) disulfide;
Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Th-
r-Gly-Gly-Gly-Lys{Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-A-
rg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys[-Adoa-Adoa-Glut-Lys(DSPE-PEG2000-NH-Glut)-]-
-NH.sub.2 cyclic (2-12) disulfide}-NH.sub.2 cyclic (6-13)
disulfide.
[0136] The KDR-binding dimer (11) may be prepared by conjugation of
precursor dimer peptide (16),
Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH-
.sub.2 cyclic (2-12) disulfide]-NH.sub.2 cyclic (6-13) disulfide,
with DSPE-PEG2000-NH.sub.2 phospholipid ammonium salt (18) as shown
in FIG. 4.
[0137] Solid precursor dimer peptide (16) (0.5 g, 0.092 mmol) was
added portionwise to a solution of disuccinimidyl glutarate (DSG,
0.15 g, 0.46 mmol), and diisopropylethylamine (0.06 g, 0.47 mmol)
in anhydrous DMF (3.0 mL) with stirring over a period of 3 min.
Then the solution was stirred at ambient temperature for 30 min.
The reaction mixture was diluted to .about.50 mL with anhydrous
ethyl acetate; this resulted in precipitation of the dimer glutaric
acid monoamide mono-NHS ester (17), the glutaric acid monoamide
mono-NHS ester of the precursor dimer peptide (16). The solution
was centrifuged to pellet 6 (m/z, neg. ion, 1887.3 (M-3H)/3, 1415.1
(M-4H)/4, 1131.9 (M-5H)/5) as a colorless solid. The supernatant
ethyl acetate layer containing excess DSG was decanted from the
compacted solid dimer glutaric acid monoamide mono-NHS ester (17)
which was again resuspended in ethyl acetate, centrifuged and
washed twice more to remove the remaining traces of DSG. The solid
intermediate glutaric acid monoamide mono-NHS ester dimer
derivative (17) thus obtained was dissolved in anhydrous
DMF/CH.sub.2Cl.sub.2 (8:2, v/v) (3.0 mL); diisopropylethylamine
(0.06 g, 0.47 mmol) was added and the solution was stirred.
[0138] Meanwhile, DSPE-PEG2000-NH.sub.2 phospholipid ammonium salt
(18) (0.235 g, 0.084 mmol, 0.9 eq.) was suspended in dry
dichloromethane (2 mL) in a separate flask and TFA (2 drops) was
added to protonate the phosphodiester oxygen, facilitating
solubilization of phospholipid ammonium salt (18) in
dichloromethane. The clear solution was concentrated to remove the
volatiles and dried further under vacuum.
[0139] The solid phospholipid ammonium salt (18) was dissolved in
DMF (2 mL) and transferred to the stirred solution of glutaric acid
monoamide mono-NHS ester dimer derivative (17) and the resulting
mixture was stirred for 24 h at ambient temperature. The reaction
mixture was diluted with a solution of 50% CH.sub.3OH, 25%
CH.sub.3CN and 25% water (1:1) to .about.100 mL and the insolubles
were filtered. Half of the filtered solution was loaded onto a
reverse phased C4 preparative column (Kromasil.RTM. Prep C4,
10.mu., 300 .ANG., 50.times.250 mm) which had been pre-equilibrated
with 1:1 mixture of CH.sub.3OH and CH.sub.3CN (1:1, 0.1% TFA) and
water (0.1% TFA) at a flow rate of 100 mL/min. During application
of the sample solution to the column the flow of the equilibrating
eluent from the preparative HPLC system was stopped. After the
sample solution was loaded the flow of the equilibrating eluent was
reinitiated and the column was washed until the plug of DMF was
eluted.
[0140] The composition of the eluent was then ramped to 70%
CH.sub.3OH--CH.sub.3CN (1:1, 0.1% TFA)-water (0.1% TFA) over 1 min
and a linear gradient of 0.75%/min of CH.sub.3OH--CH.sub.3CN (1:1,
0.1% TFA) into water (0.1% TFA) was initiated. The elution was
continued after reaching 100% B in order to achieve complete
elution of the product from the column. Fractions (15 mL) were
collected using UV (220 nm) as an indicator of product elution and
after the main product was eluted fraction collection was continued
for several minutes in order to insure elution of trace amounts of
starting phospholipid ammonium salt (18). Fractions were checked
for purity on an analytical HPLC system (column: YMC C4, 5 .mu.M,
300 .ANG., 4.6.times.250 mm) using UV at 220 nm and an evaporative
light scattering detector (ELSD). The latter detector is employed
to detect DSPE-PEG2000-NH.sub.2 which has a weak chromophore at 220
nm. Product-containing fractions of >98% purity, and devoid of
DSPE-PEG2000-NH.sub.2 phospholipid ammonium salt (8) were combined
and concentrated to reduce the content of CH.sub.3OH. The solution
was then diluted with 10% CH.sub.3CN in water until a faint
flocculent precipitate formed. The resulting solution was
freeze-dried to afford the dimeric peptide phospholipid conjugate
(11) as a colorless solid. The second portion of crude dimeric
peptide phospholipid conjugate (11) was purified as described
above. The combined yield of the target dimeric peptide
phospholipid conjugate (11) was 0.39 g (57% yield). The samples of
the dimeric peptide phospholipid conjugate (11) made from different
sample purification runs were pooled together, dissolved in
tert-butanol-acetonitrile-water mixture and re-lyophilized to
provide the dimeric peptide phospholipid conjugate (11) as a
colorless, fluffy solid which was further dried under vacuum.
[0141] Examples 6-8 below refer to the preparation of the dimer
peptide-phospholipid conjugate shown in FIG. 5, wherein the dimeric
conjugate contains very low levels of TFA. FIGS. 6-8 illustrate the
methods described in the Examples below.
Example 6
Preparation of Dimeric Conjugate Having Low TFA Levels Via the Use
of a Glutaryl Linker
Preparation of (23), (26) and Dimer Peptide (27) Acetate Salt by
Conversion of (22), (25) and Dimer Peptide 27.cndot.nTFA Salts to
Acetates by AG MP-50 Ion-Exchange Resin
[0142] For compound (23) an AG MP-50 ion-exchange resin (1.5 meq/mL
resin bed) was suspended in 20% of CH.sub.3CN/H.sub.2O. The
suspension was packed in a 3.times.30 cm glass column and the final
volume was 150 mL. The column was connected to a pump and a
conductivity meter. It was washed with 20% of CH.sub.3CN/H.sub.2O
at 17 mL/min flow rate until the conductivity was below 1 .mu.s/cm.
Compound (22) (210 mg) was dissolved in 20% of CH.sub.3CN/H.sub.2O
(80 mL) and the resulting solution was loaded to the column. The
column was washed again with the same eluent until its conductivity
was below 1 .mu.s/cm. A gradient of NH.sub.4OAc in 20% of
CH.sub.3CN/H.sub.2O was applied at 200 mM, 400 mM, 600 mM and 800
mM, 250 mL each. The compound came out at 600 mM NH.sub.4OAc. The
fractions were analyzed by HPLC and the ones containing the
compound were combined and lyophilized several times until the
weight of the material was constant. 176 mg of the pure material
(23) was obtained as a white fluffy solid. The yield was 83.8%.
[0143] Additional parameters and results were as follows: HPLC:
Ret. Time: 5.6 min; Assay >98% (area %); Column: Waters XTerra
MS-C18, 4.6.times.50 mm, 5.mu. particle, 120 .ANG. pore; Eluent: A:
H.sub.2O (0.1% TFA), B: CH.sub.3CN (0.1% TFA); Elution: Initial
condition: 15% B, linear gradient 15-50% B over 8 min; Flow rate: 3
mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES; Mode:
Negative ion; 1441.7 [M-2H]/2, 960.9 [M-3H]/3. CE analysis
(counter-ion % wt./wt.): TFA estimated to be 0.3%; acetate
1.1%.
[0144] For compound (26), following the same procedure for compound
(23), 300 mg of the peptide TFA salt (25) in 80 mL of water was
loaded at 17 mL/min. to a 150 mL of AG MP-50 column, which was
washed with H.sub.2O to conductivity of 1 .mu.s/cm. The column was
then washed with H.sub.2O again after loading, and the same step
gradient of aqueous NH.sub.4OAc into H.sub.2O as employed for the
ion exchange of compound (23) was applied. Lyophilization of the
combined fractions to a constant weight afforded 200 mg of the
acetate (26) as a white fluffy solid. The yield was 66.7%.
[0145] Additional parameters and results were as follows: HPLC:
Ret. Time: 5.6 min; Assay 97.0% (area %); Column: Waters XTerra
MS-C18, 4.6.times.50 mm, 5.mu. particle, 120 .ANG. pore; Eluent: A:
H.sub.2O (0.1% TFA), B: CH.sub.3CN (0.1% TFA); Elution: Initial
condition: 15% B, linear gradient 15-50% B over 8 min; Flow rate: 3
mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES; Mode:
Negative ion; 1336.9 [M-2H]/2, 890.8 [M-3H]/3; CE analysis
(counter-ion % wt./wt.): TFA estimated to be 0.4%; acetate 4.2%; IC
analysis (F %): 0.26.
[0146] For the dimer peptide (27) acetate salt, similar to the
procedure for compound (23), an AG MP-50 column (100 mL wet volume)
was washed with 30% CH.sub.3CN/H.sub.2O until the conductivity was
below 1 .mu.s/cm. Compound (27) as the TFA salt, (120 mg in 80 mL
of 30% of CH.sub.3CN/H.sub.2O) was loaded onto the column and the
column was washed with the same eluent until the conductivity was
stable at 1 .mu.s/cm. A step gradient of NH.sub.4OAc 30% of
CH.sub.3CN/H.sub.2O into 30% of CH.sub.3CN/H.sub.2O was run as for
compound (23) and the compound was eluted at ca 600 mM NH.sub.4OAc.
The combined fractions were lyophilized and then relyophilized
several times until the material displayed a constant weight to
provide 104 mg of the pure material (27) as an acetate salt. The
yield was 86.7%.
[0147] Additional parameters and results were as follows: HPLC:
Ret. time: 5.2 min; Assay >99% (area %); Column: Waters XTerra
MS-C18, 4.6.times.50 mm, 5.mu. particle, 120 .ANG. pore; Eluent: A:
H.sub.2O (0.1% TFA), B: CH.sub.3CN (0.1% TFA); Elution: Initial
condition: 20% B, linear gradient 20-60% B over 8 min; Flow rate: 3
mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES; Mode:
Negative ion; 1816.3 [M-3H]/3, 1362.0 [M-4H]/4, 1089.2 [M-5H]/5; CE
analysis (counter-ion % wt./wt.): TFA estimated to be 0.2%; acetate
0.15%.
Preparation and Purification of the Dimer Peptide (27) Acetate Salt
from Compound (23) and Compound (26)
[0148] To a solution of disuccinimidyl glutarate (18 mg, 0.055
mmol) in anhydrous DMF (0.1 mL) was added a solution of compound
(23) (61 mg, 0.021 mmol) in 0.2 mL of anhydrous DMF dropwise (pH 8,
neutralized by DIEA). The clear solution was stirred at RT for 0.5
h. HPLC and MS showed the completion of the reaction. Solvent was
removed in vacuo and EtOAc (8 mL) was added to precipitate the
intermediate (24). The mixture was centrifuged and decanted to
remove excess glutarate. This EtOAc washing was repeated 3 more
times and the resulting solid was dried using a stream of dry
nitrogen. It was then dissolved in 0.3 mL of anhydrous DMF.
Compound (26), (56 mg, 0.021 mmol) was added and the pH of the
solution was adjusted to 8 by addition of DIEA. The solution was
stirred for 16 h at room temperature after which by HPLC and MS
analysis indicated completion of the reaction. A 30 .mu.L aliquot
of NH.sub.2NH.sub.2 was added and the mixture was stirred for 5 min
to cleave the ivDde group. The reaction mixture was analyzed by
HPLC and MS, which indicated complete removal of the ivDde
group.
[0149] Before purification of the dimer peptide (27) acetate,
caution was taken to carefully wash the whole preparative HPLC
system including the column with TFA-free eluents,
CH.sub.3CN/H.sub.2O/10 mM NH.sub.4OAc. The crude reaction mixture
was then applied to a reverse phase C-18 preparative column
(Atlantis C-18, 5 .mu.m particle, 100 .ANG. pore, 30.times.150 mm,
flow rate 30 mL/min), pre-equilibrated with 15% B (A: 10 mM
NH.sub.4OAc in H.sub.2O; B: 10 mM NH.sub.4OAc in
CH.sub.3CN/H.sub.2O, 9/1, v/v). The column was washed with the same
eluent until the DMF plug was eluted. The eluent composition was
increased to 25% B over 2 min. and then ramped to 65% B over 40
min. The fractions were analyzed on an analytical reverse phase
C-18 column (Waters MS C-18, 4.6.times.50 mm, 5 .mu.m particle, 100
.ANG. pore, flow rate 3 mL/min) and the product-containing
fractions of >95% purity were pooled and freeze-dried to afford
25 mg of the dimer peptide (27) as its acetate salt as a fluffy
white solid. The yield was 21.8%.
[0150] Additional parameters and results were as follows: HPLC:
Ret. time: 5.2 min; Assay >99% (area %); Column: Waters XTerra
MS-C18, 4.6.times.50 mm, 5.mu. particle, 120 .ANG. pore; Eluent: A:
H.sub.2O (0.1% TFA), B: CH.sub.3CN (0.1% TFA); Elution: Initial
condition: 20% B, linear gradient 20-60% B over 8 min; Flow rate: 3
mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES; Mode:
Negative ion; [M-3H]/3, 1362.0 [M-4H]/4, 1089.2 [M-5H]/5; CE
analysis (counter-ion % wt./wt.): TFA estimated to be less than
0.2%; acetate 1.1%.
Example 7
FIG. 7
Preparation of Dimer Peptide-Phospholipid Conjugates Having Low/TFA
Levels Via Ion Exchange Resin
Preparation and Purification of the Phospholipid Peptide Conjugate
(21) as its Acetate Salt From Dimer Peptide (27) Acetate Salt
[0151] To a solution of disuccinimidyl glutarate-DSG (3.7 mg, 11.3
mmol) in anhydrous DMF (0.1 mL) was added a solution of neutralized
dimer peptide (27) acetate salt, (15 mg, 2.75 mmol) in anhydrous
DMF (0.2 mL), dropwise. The reaction solution was stirred at RT for
0.5 h. HPLC analysis with a Waters Xterra C-18 column and MS showed
the completion of the reaction. The solvent was evaporated and
EtOAc (8 mL) was added to precipitate the intermediate (28). The
vessel containing the precipitated intermediate (28) was
centrifuged and the liquid layer was decanted. This procedure was
repeated 3 times to remove the excess of DSG. The solid was dried
with a stream of dry nitrogen and then dissolved in 0.3 mL of
anhydrous DMF. DSPE-PEG2000-NH.sub.2 ammonium salt (29) (6.5 mg,
2.33 mmol) was added in solid form and the pH of the mixture was
adjusted to (28). The reaction mixture was stirred at RT for 16 h.
The mixture was analyzed by MS and HPLC with a Zorbax 300 SB-C3
column and this indicated that the reaction was complete.
[0152] To minimize the potential contamination of the product with
TFA, the crude reaction mixture was purified by preparative HPLC
equipped using a new Zorbax 300SB-C3 column (21.2.times.150 mm,
5.mu. particle) which had never been exposed to TFA. The HPLC
system was pre-washed by CH.sub.3CN/H.sub.2O/NH.sub.4OAc
extensively to remove traces of TFA. The reaction mixture was
loaded onto the column which was pre-equilibrated with 20% B (A: 10
mM NH.sub.4OAc in H.sub.2O; B: 10 mM NH.sub.4OAc in
CH.sub.3CN/H.sub.2O, 9/1 v/v) at a flow rate of 30 mL/min. The
column was eluted at 30 mL/min with the same eluent until the plug
of DMF was eluted. The eluent composition was then increased to 40%
B over 3 min and then ramped to 90% B over 50 min. The collected
fractions were analyzed on an analytical reverse phase C-3 column
(Zorbax 300SB-C3, 3.times.150 mm, 3.5 .mu.m particle, 300 .ANG.
pore, flow rate: 0.5 mL/min), where detection was accomplished
using UV at 220 nm and an evaporative light scattering detector
(ELSD). The fractions containing the pure product were pooled and
lyophilized. A 6.5 mg portion of the final product (21) acetate
salt was obtained. The yield was 33.0%.
[0153] Additional parameters and results were as follows: HPLC:
Ret. Time: 13.3 min; Assay >99% (area %); Column: Zorbax
300SB-C3, 3.times.150 mm, 3.5 .mu.m, 300 .ANG. pore; Eluent: A:
H.sub.2O (0.1% TFA), B: CH.sub.3CN/MeOH 1/1 (0.1% TFA); Elution:
Initial condition: 60% B, linear gradient 60-90% B over 3 min; Flow
rate: 0.5 mL/min; Detection: UV at 220 nm and ELSD; CE analysis
(counter-ion % wt./wt.): % wt. TFA: 0.3%; % wt acetate 0.4%.
Example 8
FIG. 8
Preparation of Dimeric Conjugate Having Low TFA Levels Via
Sequential Purification Using Zorbax C-3 RP Preparative HPLC and
Sephadex G-25 Gel Permeation Chromatography
[0154] Materials used and conditions for the analytical HPLC system
include the following: Column: Zorbax 300SB C-3; 3 mm
i.d..times.150 mm; 3.5 .mu.m particle; Eluent A: H.sub.2O (HPLC
Grade with 0.1% TFA by volume); Eluent B: CH.sub.3CN (0.1% TFA by
volume). Elution: Initial condition: 50% B then a linear gradient
of 50-90% B over 3 min, hold at 90% B for 11 min; Flow rate: 0.5
mL/min; Detection: UV at 220 nm. Ret. time: (Compound (21)): 6.77
min, Rt (lyso): 4.06 min.
Preparative HPLC Using Preparative Zorbax C-3 Column to Remove the
Lyso-Compound from (21)
[0155] The crude compound was loaded at a concentration of 30%
eluent B. Materials used and conditions include: Conditions:
Column: Waters Zorbax 300SB C-3; 21.2 mm i.d..times.150 mm; 3.5
.mu.m particle; Eluents: Eluent A: H.sub.2O(HPLC Grade with 10 mM
NH.sub.4OAc); Eluent B: CH.sub.3CN/H.sub.2O, 9/1 (final NH.sub.4OAc
concentration:10 mM).
[0156] The composition of the eluent was then changed to 45% B over
2 min, then the column was eluted with a linear gradient of 45-100%
B over 40 min; Flow rate: 30 mL/min; Detection: UV at 220 nm.
[0157] The crude compound (100 mg) was dissolved in 25 mL of a
solution of 30% B. The preparative HPLC system was equilibrated at
30% B. The compound was loaded on to the Zorbax C-3 column. The
mobile phase composition was ramped to 45% B over 2 min. A linear
gradient from 45-100% B over 40 min was used for the elution of
(21). The product eluted between 26.5-33 min.
[0158] The fractions that contained (21) were combined and
lyophilized to give a white fluffy solid. This was dissolved in
water-acetonitrile, then lyophilized again. This provided 70 mg
product devoid of the lyso-compound. The recovery was about 70%.
After chromatography was completed, the system was washed with 95%
B for 15 min at a flow rate of 30 mL/min. The column was then
washed with CH.sub.3CN/H.sub.2O (50/50, without TFA or buffer) for
30 min at a flow rate of 15 mL/min. The column was then stored at
room temperature for future use. Analytical HPLC confirmed the
absence of the lyso-compound in the isolated material. Further
analysis confirmed that no lyso-compound formed after 5 days at
room temperature. The material still contained significant amounts
(4.2 wt %) of TFA.
Removal of TFA from (21) by Gel Permeation Chromatography on
Sephadex G-25
[0159] A Sephadex G-25 column (100 g resin, bead size 20-80 .mu.m,
total gel volume 500 mL, column height: 27 cm) was equilibrated
with 4 L of 50 mM ammonium bicarbonate. Then (21) (70 mg) was
dissolved in 30 mL (final volume) of 60 mM ammonium bicarbonate in
10% aqueous acetonitrile. The solution was filtered and then loaded
on to the Sephadex G-25 column. The column was eluted with 50 mM
ammonium bicarbonate buffer with collection of 10 mL fractions. The
collected fractions were monitored by analytical HPLC (UV detection
at 220 nm). The results are provided in Table 4 below.
TABLE-US-00011 TABLE 4 Compound present (by Fraction # Volume (mL)
HPLC analysis of fraction) 1 10 No 3 10 No 6 10 No 9 10 No 12 10 No
15 10 No 18 10 No 19 10 No 20 10 Yes 21 10 Yes 24 10 Yes 27 10 Yes
28 10 Yes 29 10 No
[0160] Fractions 20-28 were pooled and lyophilized. The lyophilized
material obtained was re-dissolved in a small volume of water and
the solution was frozen and lyophilized to remove residual amounts
of ammonium bicarbonate. The final weight of the desired material
was 58 mg. The recovery was 83%.
[0161] To ascertain the effective removal of TFA, the sample was
subjected to CE analysis for TFA and acetate ions. The TFA is
clearly present in the starting material (4.2%) according to the
previous assay, while it is hardly detected (0.2%) after the gel
permeation procedure. No acetate ion was detected.
Analytical Data for (21) Obtained by Serial Zorbax C-3 Preparative
HPLC and Sephadex G-25 Gel Permeation Chromatography
[0162] Materials used and conditions for collecting analytical data
include: Fluorine analysis (IC by QTI): 751 ppm (0.15% TFA wt/wt);
Mass Spectrum: Method: MALDI-TOF; Mode: Positive Ion; Average
molecular weight detected was 8461 the typical PEG2000 mass
distribution curve was observed. HPLC: System A: Column: Zorbax
300SB C-3; 3 mm i.d..times.150 mm; 3.5 .mu.m particle; Eluent A:
Water (HPLC Grade with 0.1% TFA by volume); Eluent B: Acetonitrile
(0.1% TFA by volume). Initial condition: 50% B; Elution: linear
gradient of 50-90% B over 3 min, hold at 90% B for 11 min; Flow
rate: 0.5 mL/min; Detection: UV at 220 nm. Ret time: 6.77 min; Area
%: 99.6%. System B: Column: Zorbax 300SB C-3; 3 mm i.d..times.150
mm; 3.5 .mu.m particle; Eluent A: Water (HPLC Grade with 0.1% TFA
by volume); Eluent B: Acetonitrile (0.1% TFA by volume). Initial
condition: 50% B; Elution: linear gradient of 50-90% B over 3 min
then ramp to 100% B over 12 min. Flow rate: 0.5 mL/min; Detection:
LSD; Ret: time: 13.98 min. Area %: 99.3%.
[0163] Table 5 below provides definitions for the abbreviations
used and the sources of materials referred to in Examples 9-12.
TABLE-US-00012 TABLE 5 DSPA.Na (Genzyme) IUPAC:
1,2-Distearoyl-sn-glycero-3- phosphosphatidic acid, sodium salt
DPPG.Na (Genzyme) IUPAC: 1,2-Dipalmitoyl-sn-glycero-3-
phosphoglycerol, sodium salt DPPE (Genzyme) IUPAC:
1,2-Dipalmitoyl-sn-glycero-3- phosphoethanolamine DSPC
Distearoyl-glycero-phosphatidylcholine (Genzyme) IUPAC:
1,2-Distearoyl-sn-glycero-3-phosphocholine DSPG.Na (Genzyme) IUPAC:
1,2-Distearoyl-sn-glycero-3- phosphoglycerol, sodium salt
DSPE-PEG1000 Distearoyl-glycero-phosphoethanolamine-N-
methoxy(polyethylene glycol)1000 (Avanti Polar) DSPE-PEG2000
Distearoyl-glycero-phosphoethanolamine-N- methoxy(polyethylene
glycol)2000 (Avanti Polar) Stearate* Sodium Stearate (Fluka)
PEG4000 (polyethylene glycol) MW 4000 (Fluka) Mannitol (Fluka) *the
acid form, i.e., stearic acid, can also be used in any of the
microbubble preparations herein.
Example 9
Preparation of Targeted Microbubbles with DSPC/DPPG Envelope
Example 9A
[0164] 383 mg of a mixture of DSPC/DPPG/ and the dimeric peptide
phospholipid conjugate (11) shown in FIG. 5 (molar ratio
49.75/49.75/0.5, corresponding to 187.1, 176.4 and 19.8 mg of the
three components, respectively) and PEG-4000 (22.6 g) were
solubilized in 120 g of t-butyl alcohol at 60.degree. C., in a
water bath. The solution was filled in vials with 0.8 mL of
solution each. The samples were frozen at -45.degree. C. and
lyophilized. The air in the headspace was replaced with a mixture
of C.sub.4F.sub.10/Nitrogen (50/50) and vials capped and crimped.
The lyophilized samples were reconstituted with 5 mL of H.sub.2O
per vial.
Example 9B
[0165] Example 9A was repeated using a mixture of DSPC/DPPG/ and
the monomeric peptide phospholipid conjugate (31) shown in FIG. 10
(molar ratio 49.5/49.5/1, corresponding to 182.8, 172.3 and 28.2 mg
of the three components, respectively).
Example 10
Preparation of Targeted Microbubbles with DPPE/DPPG Envelope
Example 10A
[0166] An aqueous suspension of DSPE-PEG1000 (0.43 mg-0.24
.mu.mmole) and the monomeric peptide phospholipid conjugate (31)
shown in FIG. 10 (3.0 mg-0.5 .mu.mmole) was prepared in 500 .mu.L
of distilled water at 60.degree. C. to obtain a micellar
suspension.
[0167] Separately, DPPE (15.8 mg-22.8 .mu.mmoles) and DPPG (4.2
mg-5.7 .mu.mmoles) were dispersed in a solution of mannitol 10% in
distilled water (20 mL) at 70.degree. C. for 20 minutes. The
dispersion was then cooled to room temperature. Perfluoroheptane
(1.6 mL) was emulsified in the aqueous phase using a high speed
homogenizer (Polytron PT3000, probe diameter of 3 cm) for 1 minute
at 10500 rpm to obtain an emulsion.
[0168] The micellar suspension was added to the emulsion and the
resulting mixture was heated at 60.degree. C. for 1 hour under
stirring. After cooling to room temperature (1 hour), the obtained
emulsion was divided in 4 mL fractions in 50 mL round bottom
flasks. The emulsion was frozen at -45.degree. C. for 5 minutes and
freeze-dried at 0.2 mBar for 24 hours (Freeze-Drier Christ Beta
1-8K).
[0169] Before redispersion, the lyophilisate was exposed to an
atmosphere containing C4F10/nitrogen (50/50 by volume). The
lyophilized product was then dispersed in a volume of water twice
the initial one by gentle hand shaking
Example 10B
[0170] An aqueous suspension of DSPE-PEG1000 (0.5 mg-0.27
.mu.mmole) and dimeric peptidephospholipid conjugate (11) shown in
FIG. 5 (5.3 mg-0.63 .mu.mmole) was prepared in 500 .mu.L of
distilled water at 60.degree. C. to obtain a micellar
suspension.
[0171] Separately, DPPE (15.8 mg-22.8 .mu.mmoles) and DPPG (4.2
mg-5.7 .mu.mmoles) were dispersed in a solution of PEG4000 10% in
distilled water (20 mL) at 70.degree. C. for 20 minutes. The
dispersion was then cooled to room temperature. Perfluoroheptane
(1.6 mL) was emulsified in the aqueous phase using a high speed
homogenizer (Polytron PT3000, probe diameter of 3 cm) for 1 minute
at 10000 rpm to obtain an emulsion.
[0172] The micellar suspension was added to the emulsion and the
resulting mixture was heated at 80.degree. C. for 1 hour under
stirring. After cooling to room temperature (1 hour), the obtained
emulsion was washed once by centrifugation (200 g/10 min-Sigma
centrifuge 3K10) to eliminate the excess of phospholipid. The
separated pellet (containing emulsified microdroplets of solvent)
was recovered and re-suspended with the initial volume of a 10%
PEG4000 aqueous solution.
[0173] The obtained emulsion was sampled into DIN8R vials (1
mL/vial). Then vials were cooled at -50.degree. C. (Christ Epsilon
2-12DS Freeze Dryer) and freeze-dried at -25.degree. C. and 0.2
mBar for 12 hours with a final drying step at 30.degree. C. and 0.1
mBar for 7 hours. Vials were exposed to an atmosphere containing
C4F10/nitrogen (35/65 by volume) and sealed. The lyophilized
product was redispersed in a volume of water twice the initial one
by gentle hand shaking
Example 11
Preparation of Targeted Microbubbles with DSPC/DSPA Envelope
Example 11A
[0174] An aqueous suspension of DSPE-PEG1000 (2.5 mg-1.4 .mu.mmole)
and dimeric peptide conjugate (11) shown in FIG. 5 (7.0 mg-0.84
.mu.mmole) was prepared in 1 mL of distilled water at 60.degree. C.
to obtain a micellar suspension.
[0175] Separately, DSPC (16.3 mg-20.6 .mu.mmoles) and DSPA (3.7
mg-5.15 .mu.mmoles) were dissolved in cyclooctane (1.6 mL) at
80.degree. C. This organic phase was added to a PEG4000 10%
solution in water (20 mL) using a high speed homogenizer (Polytron
T3000, probe diameter of 3 cm) for 1 minute at 8000 rpm, to obtain
an emulsion.
[0176] The micellar suspension was mixed with the emulsion and the
resulting mixture was heated at 80.degree. C. for 1 hour under
agitation. After cooling to room temperature (1 hour), the obtained
emulsion was washed once by centrifugation (1500 g/10 min--Sigma
centrifuge 3K10) to eliminate the excess of the phospholipid. The
separated supernatant (containing emulsified microdroplets of
solvent) was recovered and re-suspended in twice the initial volume
of a 10% PEG 4000 aqueous solution.
[0177] The obtained suspension was sampled into DIN8R vials (1
mL/vial). Then vials were cooled to -50.degree. C. (Christ Epsilon
2-12DS Freeze Dryer) and freeze-dried at -25.degree. C. and 0.2
mbar for 12 hours, with a final drying step at 30.degree. C. and
0.1 mbar for 7 hours. Vials were exposed to an atmosphere
containing C.sub.4F.sub.10/Nitrogen (35/65 by volume) and sealed.
The lyophilized product was then dispersed in a volume of water
twice the initial one by gentle hand shaking
Example 11B
[0178] Example 11A was repeated, but using 0.7 mg of DSPE-PEG2000
(0.26 .mu.mmoles) and 1.6 mg of monomeric peptide-phospholipid
conjugate (1) shown in FIG. 2 (0.26 .mu.mmole) to prepare the
micellar suspension.
Example 11C
[0179] DSPC (16.3 mg-20.6 .mu.mmoles), DSPA (3.7 mg-5.15
.mu.mmoles) and monomeric peptide phospholipid conjugate (1) shown
in FIG. 1 (1.6 mg-0.26 .mu.mmole) were dissolved in cyclooctane
(1.6 mL) at 80.degree. C. This organic phase was emulsified in a
PEG4000 10% aqueous phase (20 mL) using a high speed homogenizer
(Polytron PT3000, probe diameter of 3 cm) for 1 minute at 8000 rpm
to obtain an emulsion.
[0180] The resulting emulsion was heated at 80.degree. C. for 1
hour under stirring. After cooling to room temperature (1 hour),
the obtained emulsion was diluted with 20 ml of a PEG4000 10%
aqueous solution.
[0181] The emulsion was sampled into DIN8R vials (1 mL/vial). Then
vials were cooled at -50.degree. C. (Christ Epsilon 2-12DS Freeze
Dryer) and freeze-dried at -25.degree. C. and 0.2 mBar for 12 hours
with a final drying step at 30.degree. C. and 0.1 mBar for 7 hours.
Vials were exposed to an atmosphere containing C4F10/nitrogen
(35/65 by volume) and sealed. The lyophilized product was
redispersed in a volume of water twice the initial one by gentle
hand shaking
Example 12
Preparation of Targeted Microbubbles with DSPC/Stearate
Envelope
Example 12A
[0182] An aqueous suspension of DSPE-PEG2000 (2.5 mg-0.9
.mu.mmoles) and the dimeric phospholipid conjugate (11) shown in
FIG. 5 (2.5 mg-0.3 .mu.mmoles) was prepared in 660 .mu.L of
distilled water at 60.degree. C. to obtain the micellar
suspension.
[0183] Separately, DSPC (18.2 mg-23.1 .mu.mmoles) and stearate (1.8
mg-5.8 .mu.mmoles) were dissolved in cyclooctane (1.6 mL) at
80.degree. C. This organic phase was added to a PEG4000 10%
solution in water (20 mL) using a high speed homogenizer (Polytron
T3000, probe diameter of 3 cm) for 1 minute at 9000 rpm, to obtain
an emulsion.
[0184] The micellar solution was mixed with the emulsion and the
resulting mixture was heated at 80.degree. C. for 1 hour under
agitation. After cooling to room temperature (1 hour), the obtained
emulsion was washed once by centrifugation (1500 g/10 min--Sigma
centrifuge 3K10) to eliminate the excess of phospholipids. The
separated supernatant (containing emulsified microdroplets of
solvent) was recovered and re-suspended with twice the initial
volume of a 10% PEG 4000 aqueous solution.
[0185] The obtained suspension was sampled into DIN8R vials (1
mL/vial). Then vials were cooled to -50.degree. C. (Christ Epsilon
2-12DS Freeze Dryer) and freeze-dried at -25.degree. C. and 0.2
mbar for 12 hours, with a final drying step at 30.degree. C. and
0.1 mbar for 7 hours. Vials were exposed to an atmosphere
containing C.sub.4F.sub.10/Nitrogen (35/65 by volume) and sealed.
The lyophilized product was dispersed in a volume of water twice
the initial one by gentle hand shaking
Example 12B
[0186] Example 12A was repeated by replacing the dimeric peptide
phospholipid conjugate (11) shown in FIG. 5 with the same relative
molar amount of the monomeric peptide phospholipid conjugate (1)
shown in FIG. 2.
Example 12C
[0187] Example 11C was repeated with DSPC (18.2 mg-23.1
.mu.mmoles), sodium stearate (1.8 mg-5.8 .mu.mmoles) and the
dimeric peptide phospholipid conjugate (11) shown in FIG. 5 (2.2
mg-0.26 .mu.mmole). The agitation speed for emulsification was
fixed to 9000 rpm. After cooling to room temperature (1 hour), the
obtained emulsion was washed once by centrifugation (1500 g/10
min-Sigma centrifuge 3K10) to eliminate the excess of the
phospholipid. The separated supernatant (containing emulsified
microdroplets of solvent) was recovered and re-suspended in twice
the initial volume of a 10% PEG 4000 aqueous solution.
Example 13
Static Binding Test on KDR-Transfected Cells
[0188] Plasmid Production and Purification
[0189] Full-length KDR was cloned into the pcDNA6 vector and the
plasmid was amplified in competent DH5.alpha. E. coli. Plasmid
amplification and purification was performed using E. coli JM 109
and a kit from Quiagen.
[0190] Transfection of 293H Cells on Thermanox.RTM. Coverslips
[0191] Cells were grown on poly-D-lysine-coated Thermanox.RTM.
circular coverslips in 24-well plate. Transfection was done as
recommended in the lipofectamine 2000 protocol (Invitrogen,
cat#11668-019) using 1 .mu.g of DNA (pc-DNA6-fKDR)/per coverslip
(1.3 cm2) in 0.1 mL. Transfection was done in serum-free media, the
transfection reagent mix was removed from cells after 2 hours and
replaced with regular serum-containing medium. Some of the
cell-coated coverslips were mock-transfected (with no DNA). The
next day, expression of the KDR receptor was assessed by
immunocytochemistry and the binding assay was performed.
[0192] Bubble Binding Assay
[0193] The transfected cells were incubated with KDR-targeted
microbubbles resuspended in 50% human plasma in PBS. For the
incubation with the transfected cells a small plastic cap was
filled with a suspension containing a 1.3.times.10.sup.8 bubbles
and the cap was covered with an inverted Thermanox.RTM. coverslip
so as to put the transfected cells in contact with the targeted
microbubbles. After 30 min of incubation at RT, the coverslip was
lifted with tweezers, rinsed three times in PBS and examined under
a microscope to assess binding of the targeted microbubbles.
[0194] Determination of the % of Surface Covered by
Microbubbles
[0195] Images were acquired with a digital camera DC300F (Leica)
and the percent of surface covered by bound microbubbles in the
imaged area was determined using the software QWin version 3.1
(Leica Microsystem AG, Basel, Switzerland). Pictures were taken of
each Thermanox.RTM.coverslip. For each preparation of Examples 9
and 10, the binding assay was repeated a minimum of two times thus
obtaining an average value of the surface covered. In the following
Tables 6 and 7, the binding activity of the microbubbles prepared
according to Examples 9 and 10 above are recorded.
[0196] As indicated by the Tables, the same peptide may show
different binding activities when included (as a lipopeptide) in
different phospholipid formulations forming the stabilizing
envelope of the microbubble. Microbubbles containing KDR binding
lipopeptides of the invention bind specifically to KDR-expressing
cells while they did not bind appreciably to mock transfected
cells.
Example 14
Dynamic Binding Test on Rh VEGF-R2/Fc Chimeric Protein
[0197] Preparation of Fc-VEGF-R2-Coated Coverslips
[0198] Glass coverslips (40 mm in diameter, Bioptechs Inc., Butler,
Pa., USA) were coated with recombinant human VEGF-R2/Fc Chimeric
protein (R&D Systems) according the following methodology.
[0199] A surface of dimensions 14.times.25 mm was delimited on the
glass coverslip using a special marker (Dako Pen) and 400 .mu.L of
Fc-VEGF-R2 solution at 4 .mu.g/mL in PBS was deposited on this
surface. After an overnight incubation at 4.degree. C., the
solution was aspirated, replaced by 0.5 mL of a solution of BSA 1%
w/v in PBS-0.05% Tween 80, pH 7.4 and incubated for 3 hours at RT.
Then the coverslip was washed three times with 5 ml of PBS-0.05%
Tween 80.
[0200] Binding Assay
[0201] Binding studies of targeted bubbles were carried out using a
parallel-plate flow chamber (FCS2, Bioptech Inc., Butler, Pa., USA)
with a chamber gasket of 0.25 mm in thickness, with a customized
adapter for upside-down chamber inversion. The coated coverslip was
inserted as a plate of the flow chamber. Microbubbles
(5.times.10.sup.6 bubbles/mL in 50% human plasma in PBS) were drawn
through the flow chamber using an adjustable infusion pump (Auto
Syringe.RTM. AS50 Infusion Pump, Baxter, Deerfield, Ill., USA) with
a 60 mL syringe (Terumo). The pump flow rate was adjusted to 1
mL/min to obtain the desired shear rate of about 114 s.sup.-1.
After 10 minutes, the flow was stopped and pictures were taken
randomly at different positions on the coverslip (on areas of about
0.025 mm.sup.2) using a 40.times. objective and a CCD monochrome
camera (F-View II, Soft Imaging Systems, Germany) connected to an
inverted Olympus IX 50 microscope.
[0202] The number of microbubbles on each picture was determined,
averaged with respect to the total number of pictures and the
obtained value was then divided by ten (to obtain the "slope", i.e.
the average amount of bound microbubbles per minute).
[0203] For each preparation of Examples 11 and 12, the binding
assay was repeated four times thus obtaining an average value of
the slope.
[0204] The slope represents the bubble binding rate on the target
substrate. For instance, a slope value of 8 indicates that an
average of eighty (80) microbubbles was bound on the coated
coverslip in ten minutes. A higher slope indicates a better
capacity of bubbles to bind to the target under flow
conditions.
[0205] In the following tables 8 and 9, the binding activity of the
microbubbles prepared according to Examples 11 and 12 above were
illustrated.
[0206] As inferable from the tables, the same peptide may show
different binding activities when included (as a
peptide-phospholipid conjugate or lipopeptide) in different
phospholipid formulations forming the stabilizing envelope of the
microbubble.
TABLE-US-00013 TABLE 6 KDR- Example KDR Mock Mock 9A 28.6% 0.4%
28.3% 9B 28.0% 0.3% 27.7%
TABLE-US-00014 TABLE 7 KDR- Example KDR Mock Mock 10A 23.6% 0.2%
23.5% 10B 28.0% 0.0% 28.0%
TABLE-US-00015 TABLE 8 Example Slope 11A 8.2 11B 8.1 11C 5.8
TABLE-US-00016 TABLE 9 Example Slope 12A 9.0 12B 8.0 12C 7.8
Example 15
In Vivo Evaluation of Ultrasound Contrast Agents Targeted to
KDR
[0207] The ability of ultrasound contrast agents containing KDR
binding lipopeptides of the invention to bind to KDR-expressing
tissue in vivo was assessed using a known model of angiogenesis:
the rabbit VX2 tumor model.
[0208] A known model of angiogenic tissue was used to examine the
ability of the KDR-targeted ultrasound microbubbles to localize to
and provide an image of angiogenic tissue. The VX2 rabbit carcinoma
was serially implanted in the dorsal muscle of New Zealand rabbits
(Charles River Laboratories, France) weighting 2.5/3 kg.
[0209] Preparation of Tumor Homogenate
[0210] Tumor was surgically removed, placed into McCoy's culture
medium containing 10% fetal calf serum, antibiotics, 1.5 mM
Glutamax I and cut into small pieces that were rinsed to remove
blood and debris. Then tumor pieces (3 to 5 cm.sup.3) were placed
in a 50 ml Falcon tube containing 5 mL of complete medium. The
tumor tissue was ground (Polytron) until no more solid pieces were
visible. The murky fluid was centrifuged for 5 minutes at 300 g and
the supernatant discarded. Seven mL of fresh medium was added per 5
mL of pellet.
[0211] Tumor Implantation
[0212] Rabbits received first 0.3 mL of Vetranquil (Acepromazine,
Sanofi, injected intramuscularly) and were then anesthetized with
an intramuscular injection of Ketaminol.RTM.5/Xylazine (Veterinaria
AG/Sigma) mixture (50/10 mg/mL, 0.7 mL/kg). One hundred microliters
of VX2 tumor homogenate was injected intramuscularly. Fifteen days
after implantation of VX2 tumors, animals were anesthetized again
with the same mixture, plus subcutaneous injection of 50% Urethane
(2 mL/kg, s.c.) (Sigma) for imaging experiments.
[0213] In Vivo Ultrasound Imaging
[0214] VX2 tumor imaging was performed using an ultrasound imaging
system ATL HDI 5000 apparatus equipped with a L7-4 linear probe.
B-mode pulse inversion at high acoustic power (MI=0.9) was used to
evaluate accumulation of targeted microbubbles on the KDR receptor
expressed on the endothelium of neovessels. The linear probe was
fixed on the skin directly over the implanted tumors.
[0215] After bubble injection (0.1 .mu.L/kg of gas) using the
preparations of either Example 16 or Example 17, insonation was
stopped allowing bubbles to accumulate for 25 minutes. Then,
insonation was reactivated at high acoustic power (MI 0.9)
destroying all the bubbles present in the tumor. The amount of free
circulating bubbles was then assessed by recording the signal
obtained after 20 sec accumulation without insonation.
Video frames from VX2 tumor imaging experiments were captured with
video-capture and analysed with Image-Pro Plus 2.0 software. The
image representing free circulating bubbles was subtracted from the
image obtained at 25 min, to provide an image representing bound
bubbles. Referring to FIG. 11 (which shows the results with the
preparation of Example 16) and FIG. 12 (which shows the results
with the preparation of Example 17), FIGS. 11A and 12A show an
image before bubble injection (baseline); FIGS. 11B and 12B show
retention of bubble contrast in the tumor 25 minutes post
injection; and FIGS. 11C and 12C show the result obtained after
subtraction of the baseline and free circulating bubbles and
represent bound microbubbles containing KDR lipopeptides according
to the present invention. Examples 15-17 and FIGS. 11 and 12
confirm that ultrasound contrast agents bearing such KDR binding
moieties localize to KDR expressing (and thus angiogenic) tissue in
animal models.
Example 16
[0216] Example 12A was repeated by replacing DSPE-PEG2000 with
DSPE-PEG1000 (2.7 mg, 1.54 .mu.mmol) and using 2.5 mg (0.31
.mu.mmol) of dimeric peptide phospholipid conjugate (11) shown in
FIG. 5.
Example 17
[0217] Example 16 was repeated by replacing the dimeric peptide
phospholipid conjugate with the same molar amount of monomeric
phospholipid conjugate (1) shown in FIG. 2.
Sequence CWU 1
1
7129PRTArtificial Sequencesynthetic peptide 1Arg Ala Gln Asp Trp
Tyr Tyr Asp Glu Ile Leu Ser Met Ala Asp Gln 1 5 10 15 Leu Arg His
Ala Pro Leu Ser Gly Gly Gly Gly Gly Lys 20 25 229PRTArtificial
Sequencesynthetic peptide 2Arg Ala Gln Asp Trp Tyr Tyr Asp Glu Ile
Leu Ser Met Ala Asp Gln 1 5 10 15 Leu Arg His Ala Phe Leu Ser Gly
Gly Gly Gly Gly Lys 20 25 329PRTArtificial Sequencesynthetic
peptide 3Arg Ala Gln Asp Trp Tyr Tyr Asp Glu Ile Leu Ser Met Ala
Asp Gln 1 5 10 15 Leu Arg His Ala Phe Leu Ser Gly Gly Gly Gly Gly
Lys 20 25 428PRTArtificial Sequencesynthetic peptide 4Ala Gln Asp
Trp Tyr Tyr Asp Glu Ile Leu Ser Met Ala Asp Gln Leu 1 5 10 15 Arg
His Ala Phe Leu Ser Gly Gly Gly Gly Gly Lys 20 25 528PRTArtificial
Sequencesynthetic peptide 5Ala Gln Asp Trp Tyr Tyr Asp Glu Ile Leu
Ser Met Ala Asp Gln Leu 1 5 10 15 Arg His Ala Phe Leu Ser Gly Gly
Gly Gly Gly Lys 20 25 629PRTArtificial Sequencesynthetic peptide
6Arg Ala Gln Asp Trp Tyr Tyr Asp Glu Ile Leu Ser Met Ala Asp Gln 1
5 10 15 Leu Arg His Ala Phe Leu Ser Gly Gly Gly Gly Gly Lys 20 25
729PRTArtificial Sequencesynthetic peptide 7Arg Ala Gln Asp Trp Tyr
Tyr Asp Glu Ile Leu Ser Met Ala Asp Gln 1 5 10 15 Leu Arg His Ala
Phe Leu Ser Gly Ser Ala Gly Ser Lys 20 25
* * * * *