U.S. patent application number 14/329803 was filed with the patent office on 2015-02-05 for etoposide and doxorubicin conjugates for drug delivery.
The applicant listed for this patent is Angiochem Inc.. Invention is credited to Christian Che, Michel DEMEULE, Reinhard Gabathuler, Gaoqiang Yang.
Application Number | 20150038429 14/329803 |
Document ID | / |
Family ID | 42106173 |
Filed Date | 2015-02-05 |
United States Patent
Application |
20150038429 |
Kind Code |
A1 |
DEMEULE; Michel ; et
al. |
February 5, 2015 |
ETOPOSIDE AND DOXORUBICIN CONJUGATES FOR DRUG DELIVERY
Abstract
The invention relates to improvements in the field of drug
delivery. More particularly, the invention relates to polypeptides
having a hydrolyzable covalent bond to a therapeutic agent that
includes, etoposide, etoposide 4'-dimethylglycine or doxorubicin.
These polypeptide conjugates can be used as vectors to transport
the podophyllotoxin derivative across the blood brain barrier (BBB)
or into particular cell types such as ovary, liver, lung, or
kidney. The invention also relates to pharmaceutical compositions
that include the compounds of the invention and to uses thereof in
methods of treatment.
Inventors: |
DEMEULE; Michel;
(Beaconsfield, CA) ; Che; Christian; (Longueuil,
CA) ; Gabathuler; Reinhard; (Verdun, CA) ;
Yang; Gaoqiang; (Montreal, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Angiochem Inc. |
Montreal |
|
CA |
|
|
Family ID: |
42106173 |
Appl. No.: |
14/329803 |
Filed: |
July 11, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13124022 |
Feb 8, 2012 |
8828925 |
|
|
PCT/CA09/01481 |
Oct 15, 2009 |
|
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14329803 |
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61171010 |
Apr 20, 2009 |
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61105654 |
Oct 15, 2008 |
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Current U.S.
Class: |
514/19.3 ;
530/322 |
Current CPC
Class: |
A61K 31/7048 20130101;
A61K 38/10 20130101; C07H 17/04 20130101; A61K 31/04 20130101; A61K
47/64 20170801; A61K 47/62 20170801; A61K 31/704 20130101; C07K
14/515 20130101; A61K 45/06 20130101; A61K 31/337 20130101; A61P
35/00 20180101; A61K 39/39558 20130101; A61K 31/04 20130101; A61K
2300/00 20130101; A61K 31/704 20130101; A61K 2300/00 20130101; A61K
31/7048 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/19.3 ;
530/322 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 31/337 20060101 A61K031/337; A61K 31/704 20060101
A61K031/704 |
Claims
1-43. (canceled)
44. A compound having the structure: ##STR00042## wherein Y is a
hydrolyzable linker; or a pharmaceutically acceptable salt
thereof.
45. The compound of claim 44, wherein Y has the structure:
##STR00043## wherein each G and G' is, independently, --C(O)--,
--C(O)O--, --OC(O)--, --S(O).sub.2O--, --OS(O).sub.2--,
--S(O).sub.2NH--, --NHS(O).sub.2--, or --OP(O)(OR.sub.11)O--; X is
-(optionally substituted aryl)-, --(CR.sub.12R.sub.13).sub.r,
--O{(CR.sub.12R.sub.13).sub.2O}.sub.n--,
--{(CR.sub.12R.sub.13).sub.2O(CR.sub.12R.sub.13).sub.2}.sub.n--, or
--(CR.sub.12R.sub.13).sub.oR.sub.14(CR.sub.12R.sub.13).sub.p--;
each n, o, and p is, independently, 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10; R.sub.11 is H or C.sub.1-6 alkyl; R.sub.12 and R.sub.13 are
each selected, independently, from H, OH, or C.sub.1-6 alkyl; and
R.sub.14 is O, NH, N(C.sub.1-6 alkyl), or -optionally substituted
aryl.
46. The compound of claim 45, wherein G is --C(O)--.
47. The compound of claim 45, wherein G' is --C(O)--.
48. The compound of claim 45, wherein X is
--(CR.sub.12R.sub.13).sub.n--.
49. The compound of claim 48, wherein n is 1, 2, 3, 4, 5, or 6.
50. The compound of claim 49, wherein n is 2 or 3.
51. The compound of claim 50, wherein n is 3.
52. A pharmaceutical composition comprising the compound claim 44
and a pharmaceutically acceptable carrier.
53. A method of treating a cancer, said method comprising
administering to a patient a therapeutically effective amount of
the compound of claim 44.
54. The method of claim 53, further comprising the administration
of a second therapeutic agent.
55. The method of claim 54, wherein said second therapeutic agent
is a polypeptide comprising the sequence of Angiopep-2 (SEQ ID
NO:97), and wherein said Angiopep-2 is conjugated to an anticancer
agent.
56. The method of claim 55, wherein said anticancer agent is
paclitaxel.
57. The method of claim 56, wherein said second therapeutic agent
is ANG1005, which has the following structure ##STR00044##
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/124,022, filed Feb. 8, 2012, which is the U.S. national
stage filing under 35 U.S.C. .sctn.371 of international application
PCT/CA2009/001481, filed Oct. 15, 2009, which claims benefit of the
filing date of U.S. Provisional Application No. 61/171,010, filed
Apr. 20, 2009, and U.S. Provisional Application No. 61/105,654,
filed Oct. 15, 2008, each of which is incorporated by
reference.
REFERENCE TO A SEQUENCE LISTING
[0002] This application contains a Sequence Listing in computer
readable form. The computer readable form is incorporated herein by
reference.
FIELD OF THE INVENTION
[0003] The invention relates to improvements in the field of drug
delivery. More particularly, the invention relates to polypeptides
having a hydrolyzable covalent bond to a therapeutic agent such as
a podophyllotoxin derivative (e.g., etoposide or an etoposide
derivative such as etoposide 4'-dimethylglycine) or to doxorubicin
or a doxorubicin derivative. These polypeptide conjugates can be
used as vectors to transport the therapeutic agent across the blood
brain barrier (BBB) or into particular cell types such as ovary,
liver, lung, or kidney. These conjugates can show improved
physicochemical (e.g., increased solubility) and pharmaceutical
properties (e.g., enhanced targeting that allows for subtherapeutic
doses or reduced toxicity that allows for supertherapeutic doses)
relative to the unconjugated therapeutic agent. The invention also
relates to pharmaceutical compositions that include the compounds
of the invention and to uses thereof in methods of treatment.
BACKGROUND OF THE INVENTION
[0004] Many therapeutic agents for such diseases have undesirable
side effects (e.g., chemotherapeutic agents) or, for reasons such
as in vivo stability, transport, or other pharmacokinetic
properties, are difficult to provide at a sufficiently high
concentration in the target tissue or for a sufficiently long
duration to allow maximal therapeutic effect in the target tissue.
Accordingly, there is a need for methods and compositions that
increase concentrations of therapeutic and diagnostic agents in
target organs or tissues such as the brain, ovary, liver, or
lung.
SUMMARY OF THE INVENTION
[0005] We have developed peptide-therapeutic conjugates, and
pharmaceutically acceptable salts thereof, where etoposide is
covalently attached to the Angiopep-2 polypeptide (SEQ ID NO:97) at
the 2'' hydroxyl via a hydrolyzable glutaric acid linker (e.g.,
Compound (1) shown in Scheme 1). A related peptide-therapeutic
conjugate has also been prepared in which etoposide
4'-dimethylglycine is used instead of etoposide, and improved
properties (e.g., solubility) are observed.
##STR00001##
[0006] Doxorubicin has also been covalently attached at the
14-hydroxyl to the Angiopep-2 polypeptide using a succinic acid
linker (e.g., the trihydrochloride salt of Compound (2) shown in
Scheme 2). Covalent attachment of the doxorubicin hydrochloride
salt can also afford improved properties (e.g., solubility).
##STR00002##
[0007] These conjugates can show improved properties relative to
the corresponding unconjugated therapeutic agent such as improved
physicochemical (e.g., increased solubility) and pharmaceutical
(e.g., enhanced targeting that allows for subtherapeutic doses or
reduced toxicity that allows for supertherapeutic doses)
properties. The solubility of the etoposide.sub.DMG and the
doxorubicin hydrochloride conjugates can also be useful in
adjusting dosing regimens. The invention therefore features these
compounds, as well as related compounds. Methods of making and
using these compounds are also provided.
[0008] Accordingly, in one aspect, the invention features a
compound, or a pharmaceutically acceptable salt thereof, that
includes an amino acid sequence substantially identical to an amino
acid sequence selected from the group consisting of SEQ ID
NOS:1-105 and 107-116, or a functional derivative thereof, where
the amino acid sequence includes a covalent bond from an amino acid
of the amino acid sequence to a podophyllotoxin derivative. In some
embodiments, the podophyllotoxin derivative is a compound having a
structure according to Formula (I):
##STR00003##
[0009] or a stereoisomer or pharmaceutically acceptable salt
thereof, where
[0010] each R.sub.1, R.sub.2, and R.sub.3 is selected,
independently, from H, optionally substituted C.sub.1-6 alkyl,
C(O)R.sub.8, P(O)(OR.sub.9)(OR.sub.10), S(O).sub.2(OR.sub.9), or a
hydrolyzable linker Y that comprises a covalent bond to an amino
acid of the polypeptide;
[0011] X is O or NR.sub.7;
[0012] each R.sub.4, R.sub.5, and R.sub.7 is selected,
independently, from H, optionally substituted C.sub.1-6 alkyl,
C(O)R.sub.8, or a hydrolyzable linker Y that comprises a covalent
bond to an amino acid of the polypeptide;
[0013] R.sub.6 is H, optionally substituted C.sub.1-6 alkyl,
optionally substituted aryl, optionally substituted heteroaryl,
[0014] R.sub.8 is selected from optionally substituted C.sub.1-6
alkyl or optionally substituted aryl;
[0015] each R.sub.9 and R.sub.10 is selected, independently, from
H, optionally substituted C.sub.1-6 alkyl, or optionally
substituted aryl; and
[0016] n is 1, 2, 3, 4, 5, 6, 7, or 8;
[0017] where one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
and R.sub.7 is Y and no more than one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, and R.sub.7 is Y.
[0018] In some embodiments, Y is --C(O)(CH.sub.2).sub.nC(O)-- and n
is 2, 3, or 4. In certain embodiments, n is 3.
[0019] In some embodiments, the pharmaceutically acceptable salt of
the compound is the mono-, di-, or tri-acid addition salt (e.g.,
the mono-, di-, or trihydrochloride salt).
[0020] In some embodiments, each compound of Formula (I) is
selected, independently, from:
##STR00004##
where each R.sub.2 is, independently, H, P(O)(OH).sub.2, or
C(O)CH.sub.2N(CH.sub.3).sub.2; each R.sub.6 is, independently,
CH.sub.3 or 2-thiophene; each Y is selected from
--C(O)(CH.sub.2).sub.nC(O)--;
--[C(O){OCH.sub.2CH.sub.2}.sub.nOC(O)]--;
--S(O).sub.2(CH.sub.2).sub.n S(O).sub.2--;
--[S(O).sub.2{OCH.sub.2CH.sub.2}.sub.nOS(O).sub.2]--;
--[{P(O)(OR.sub.9)}(CH.sub.2).sub.n{P(O)(OR.sub.9)}]--; and
--[{P(O)(OR.sub.9)}(OCH.sub.2CH.sub.2).sub.nO{P(O)(OR.sub.9)}]--;
each n is, independently, 1, 2, 3, 4, 5, or 6; and where each Y is
covalently bound to an amino acid. In some embodiments, each Y is
--C(O)(CH.sub.2).sub.nC(O)-- or
--[C(O){OCH.sub.2CH.sub.2}.sub.nOC(O)]-- and n is 2, 3, or 4. In
some embodiments, each R.sub.2 is C(O)CH.sub.2N(CH.sub.3).sub.2. In
some embodiments, each compound of Formula (I) is:
##STR00005##
[0021] In some embodiments, the compound of the invention has the
following structure
##STR00006##
where each (-(Formula(I)) group represents an optional covalent
bond between the indicated amino acid and a compound of Formula
(I), and where there is at least one covalent bond between an amino
acid of the polypeptide and said compound of Formula (I). In some
embodiments, two compounds of Formula (I) are attached to the amino
acid sequence. In other embodiments, the threonine at position 1
and the lysines at positions 10 and 15 of the polypeptide each
include a covalent bond to a compound having a structure according
to Formula (I).
[0022] In some embodiments, R.sub.2 is H or
--C(O)CH.sub.2N(CH.sub.3).sub.2 (i.e., C-linked
N,N-dimethylglycine). In other embodiments, each R.sub.2 is H. In
still other embodiments, each R.sub.2 is
--C(O)CH.sub.2N(CH.sub.3).sub.2.
[0023] In some embodiments, the optionally substituted C.sub.1-6
alkyl is selected, independently, from methyl, ethyl, n-propyl,
isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl,
tert-butyl, n-pentyl, neopentyl, n-hexyl, or sec-hexyl. In some
embodiments, the C.sub.1-6 alkyl is substituted with at least one
optionally substituted amino group (e.g., NH.sub.2 or
N(CH.sub.3).sub.2) at any carbon.
[0024] In some embodiments, the optionally substituted C.sub.3-10
cycloalkyl is selected, independently, from cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
[0025] In some embodiments, the optionally substituted aryl group
is selected, independently, from phenyl, naphthyl,
tetrahydronaphthyl, indanyl, or indenyl.
[0026] In some embodiments, the optionally substituted heterocyclyl
group is selected, independently, from azacyclopropanyl,
azacyclobutanyl, 1,3-diazatidinyl, pyrrolidinyl, piperidinyl,
piperazinyl, thiranyl, thietanyl, tetrahydrothiophenyl,
dithiolanyl, tetrahydrothiopyranyl, oxiranyl, oxetanyl,
tetrahydrofuranyl, tetrahydropyranyl, dioxanyl, oxathiolanyl,
morpholinyl, thiomorpholinyl, thioxanyl, and quinuclidinyl.
[0027] In some embodiments, the optionally substituted heterocyclyl
group is selected from pyrrolyl, pyrazolyl, imadazolyl, pyridinyl,
pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl,
pryyrolizinyl, indolyl, quinolinyl, isoquinolynyl, benzimidazolyl,
indazolyl, quinolizinyl, cinnolinyl, quinazolinyl, phthalazinyl,
napthyridinyl, quinoxalinyl, thiophenyl, thiepinyl, furanyl,
benzofuranyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl,
isoxazolyl, and oxadiazolyl.
[0028] In some embodiments, a substituted alkyl, cycloalkyl, aryl,
heterocyclyl, or heteroaryl is substituted with 1, 2, 3, 4, 5, or 6
substituents selected from: C.sub.1-6 alkyl; halogen; azido
(--N.sub.3), nitro (--NO.sub.2), cyano (--CN), acyloxy, acyl
(--C(O)R), (--OC(O)R), alkoxy (--OR), amido (--NRC(O)R' or
--C(O)NRR'), amino (--NRR'), aryl, carboxylic acid (--CO.sub.2H),
carboxylic ester (--CO.sub.2R), carbamoyl (--OC(O)NRR' or
--NRC(O)OR'), cycloalkyl, heterocyclyl, hydroxy (--OH), isocyano
(--NC), phosphate (--P(O)(OR)(OR')), sulfonate (--SO.sub.2OR), or
sulfonyl (--SO.sub.2R), where each R or R' is selected,
independently, from H, C.sub.1-6 alkyl, cycloalkyl, heterocyclyl,
aryl, or heteroaryl, as defined herein. In some embodiments, these
substituents are not further substituted. In other embodiments, the
substituents may themselves be further substituted with 1, 2, 3, 4,
5, or 6 substituent groups.
[0029] In some embodiments, R.sub.4 is Y. In other embodiments,
R.sub.5 is Y.
[0030] In other embodiments, the amino acid sequence is covalently
bonded to additional podophyllotoxin derivatives through a second,
third, fourth, or fifth amino acid of said amino acid sequence. In
some embodiments, the podophyllotoxin derivative is a compound of
Formula (I).
[0031] In certain embodiments, the compound of Formula I has the
structure:
##STR00007##
where Y is --C(O)(CH.sub.2)--C(O)-- or
--[C(O){OCH.sub.2CH.sub.2}--OC(O)]-- and n is 2, 3, or 4.
[0032] In other embodiments, the compound of Formula (I) has the
following structure:
##STR00008##
where Y is --C(O)(CH.sub.2).sub.nC(O)-- or
--[C(O){OCH.sub.2CH.sub.2}.sub.nOC(O)]-- and n is 2, 3, or 4.
[0033] In still other embodiments, the compound of Formula (I) has
the following structure:
##STR00009##
where Y is --C(O)(CH.sub.2)--C(O)-- or
--[C(O){OCH.sub.2CH.sub.2}--OC(O)]-- and n is 2, 3, or 4.
[0034] In other embodiments, each compound of Formula (I) is
selected, independently, from:
##STR00010##
where each R.sub.8A and R.sub.8B is, independently, H or optionally
substituted C.sub.1-6 alkyl, or R.sub.8A and R.sub.8B combine to
form an optionally substituted 3-7 membered ring. In some
embodiments, each R.sub.8A and R.sub.8B is optionally substituted
C.sub.1-6 alkyl. In other embodiments, each compound of Formula (I)
has the following structure:
##STR00011##
[0035] In further embodiments, the compound the following
structure:
##STR00012##
[0036] In particular embodiments, the compound has the
structure:
##STR00013##
or a pharmaceutically acceptable salt thereof (e.g., the
trihydrochloride salt), where in Compound (I), etoposide refers to
etoposide 4'-dimethylglycine.
[0037] In certain embodiments, each amino acid that is covalently
bonded to the hydrolyzable linker Y is attached via an amino-, a
guanidino-, a hydroxyl-, a phenol-, or a thiol functional group of
said amino acid. In some embodiments, the amino acid that is
covalently bonded to the hydrolyzable linker Y is lysine, tyrosine,
serine, threonine, or arginine.
[0038] In a second aspect, the invention features a compound, or a
pharmaceutically acceptable salt thereof, that includes an amino
acid sequence substantially identical to an amino acid sequence
selected from the group consisting of SEQ ID NOS:1-105 and 107-116,
or a functional derivative thereof, wherein said amino acid
sequence includes a covalent bond from an amino acid of said amino
acid sequence to a doxorubicin derivative, and wherein said
doxorubicin derivative is a compound having a structure according
to Formula (II):
##STR00014##
or a stereoisomer or a pharmaceutically acceptable salt thereof,
wherein
[0039] each X.sub.1, X.sub.2, X.sub.3, X.sub.4, and X.sub.5 is
selected, independently, from a covalent bond, O, or NR.sub.25;
[0040] each R.sub.17, R.sub.18, R.sub.19, R.sub.20, R.sub.20,
R.sub.21, R.sub.22, R.sub.23, R.sub.24, and R.sub.25 is selected,
independently, from H, optionally substituted C.sub.1-6 alkyl,
optionally substituted C.sub.2-6 alkenyl, optionally substituted
C.sub.2-6 alkynyl, optionally substituted cycloalkyl, optionally
substituted heterocyclyl, or is a hydrolyzable linker Y; and
[0041] wherein one and only one of R.sub.17, R.sub.18, R.sub.19,
R.sub.20, R.sub.20, R.sub.21, R.sub.22, R.sub.23, R.sub.24, and
R.sub.25 is Y.
[0042] In some embodiments, the pharmaceutically acceptable salt of
the compound is the mono-, di-, or tri-acid addition salt (e.g.,
the mono-, di-, or trihydrochloride salt).
[0043] In certain embodiments, the compound of Formula (II) has the
following structure:
##STR00015##
where X.sub.2R.sub.18 is H or NH.sub.2; X.sub.3R.sub.19 is H or OH;
X.sub.4R.sub.20 is H or optionally substituted C.sub.1-3 alkyl; and
Y is a hydrolyzable linker as described herein. In further
embodiments, the compound of Formula (II) has the following
structure:
##STR00016##
or a pharmaceutically acceptable salt thereof.
[0044] In further embodiments, the compound of Formula (II) has the
following structure:
##STR00017##
[0045] In certain embodiments, the compound has the following
structure:
##STR00018##
[0046] wherein each (-(Formula(II)) represents an optional covalent
bond between the indicated amino acid and a compound of Formula
(II), and wherein there is at least one covalent bond between an
amino acid of the polypeptide and said compound of Formula (II). In
some embodiments, the threonine at position 1 and the lysines at
positions 10 and 15 of the polypeptide each comprise a covalent
bond to a compound having a structure according to Formula
(II).
[0047] In some embodiments, Y is --C(O)(CH.sub.2).sub.nC(O)--, and
n is 2, 3, or 4. In certain embodiments, n is 2. In other
embodiments, the amino acid sequence is covalently bonded to a
compound having a structure according to Formula (II) through a
second, third, fourth, or fifth amino acid of the amino acid
sequence. In another embodiment, each amino acid covalently bonded
to said hydrolyzable linker Y is attached via an amino-, a
guanidino-, a hydroxyl-, a phenol-, or a thiol functional group of
said amino acid. In certain embodiments, the amino acid is lysine
or threonine.
[0048] In some embodiments, the compound of Formula (II) has the
following structure:
##STR00019##
or a pharmaceutically acceptable salt thereof.
[0049] In particular embodiments, the compound has the
structure:
##STR00020##
or a pharmaceutically acceptable salt thereof (e.g., the
trihydrochloride salt).
[0050] In another aspect, the invention features the following
compound,
##STR00021##
("etoposide 4'-dimethylglycine" or "etoposide.sub.DMG"), or any
stereoisomer, or any pharmaceutically acceptable salt or solvent
thereof.
[0051] In any of the above aspects, the amino acid sequence may be
substantially identical to any of the sequences set forth in Table
1, or a fragment thereof or a pharmaceutically acceptable salt
thereof. In certain embodiments, the amino acid sequence has a
sequence of Angiopep-1 (SEQ ID NO:67), Angiopep-2 (SEQ ID NO:97),
Angiopep-3 (SEQ ID NO:107), Angiopep-4a (SEQ ID NO:108),
Angiopep-4b (SEQ ID NO:109), Angiopep-5 (SEQ ID NO:110), Angiopep-6
(SEQ ID NO:111), or Angiopep-7 (SEQ ID NO:112)). The amino acid
sequence or the compounds of the invention may be efficiently
transported into a particular cell type (e.g., any one, two, three,
four, or five of liver, ovary, lung, kidney, spleen, and muscle) or
may cross the mammalian BBB efficiently (e.g., Angiopep-1, -2, -3,
-4a, -4b, -5, and -6). In some embodiments, the cells are ovary
cells. In another embodiment, the conjugate is able to enter a
particular cell type (e.g., any one, two, three, four, or five of
liver, ovary, lung, kidney, spleen, and muscle) but does not cross
the BBB efficiently (e.g., a conjugate including Angiopep-7). In
some embodiments, the cells are ovary cells. The polypeptide may be
of any length, for example, at least 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 25, 35, 50, 75, 100, 200, or 500
amino acids. In certain embodiments, the polypeptide is 10 to 50
amino acids in length. The conjugate may be substantially pure. The
polypeptide may be produced by recombinant genetic technology or
chemical synthesis. The conjugate can be formulated with a
pharmaceutically acceptable carrier.
TABLE-US-00001 TABLE 1 Exemplary Polypeptides SEQ ID NO: 1 T F V Y
G G C R A K R N N F K S A E D 2 T F Q Y G G C M G N G N N F V T E K
E 3 P F F Y G G C G G N R N N F D T E E Y 4 S F Y Y G G C L G N K N
N Y L R E E E 5 T F F Y G G C R A K R N N F K R A K Y 6 T F F Y G G
C R G K R N N F K R A K Y 7 T F F Y G G C R A K K N N Y K R A K Y 8
T F F Y G G C R G K K N N F K R A K Y 9 T F Q Y G G C R A K R N N F
K R A K Y 10 T F Q Y G G C R G K K N N F K R A K Y 11 T F F Y G G C
L G K R N N F K R A K Y 12 T F F Y G G S L G K R N N F K R A K Y 13
P F F Y G G C G G K K N N F K R A K Y 14 T F F Y G G C R G K G N N
Y K R A K Y 15 P F F Y G G C R G K R N N F L R A K Y 16 T F F Y G G
C R G K R N N F K R E K Y 17 P F F Y G G C R A K K N N F K R A K E
18 T F F Y G G C R G K R N N F K R A K D 19 T F F Y G G C R A K R N
N F D R A K Y 20 T F F Y G G C R G K K N N F K R A E Y 21 P F F Y G
G C G A N R N N F K R A K Y 22 T F F Y G G C G G K K N N F K T A K
Y 23 T F F Y G G C R G N R N N F L R A K Y 24 T F F Y G G C R G N R
N N F K T A K Y 25 T F F Y G G S R G N R N N F K T A K Y 26 T F F Y
G G C L G N G N N F K R A K Y 27 T F F Y G G C L G N R N N F L R A
K Y 28 T F F Y G G C L G N R N N F K T A K Y 29 T F F Y G G C R G N
G N N F K S A K Y 30 T F F Y G G C R G K K N N F D R E K Y 31 T F F
Y G G C R G K R N N F L R E K E 32 T F F Y G G C R G K G N N F D R
A K Y 33 T F F Y G G S R G K G N N F D R A K Y 34 T F F Y G G C R G
N G N N F V T A K Y 35 P F F Y G G C G G K G N N Y V T A K Y 36 T F
F Y G G C L G K G N N F L T A K Y 37 S F F Y G G C L G N K N N F L
T A K Y 38 T F F Y G G C G G N K N N F V R E K Y 39 T F F Y G G C M
G N K N N F V R E K Y 40 T F F Y G G S M G N K N N F V R E K Y 41 P
F F Y G G C L G N R N N Y V R E K Y 42 T F F Y G G C L G N R N N F
V R E K Y 43 T F F Y G G C L G N K N N Y V R E K Y 44 T F F Y G G C
G G N G N N F L T A K Y 45 T F F Y G G C R G N R N N F L T A E Y 46
T F F Y G G C R G N G N N F K S A E Y 47 P F F Y G G C L G N K N N
F K T A E Y 48 T F F Y G G C R G N R N N F K T E E Y 49 T F F Y G G
C R G K R N N F K T E E D 50 P F F Y G G C G G N G N N F V R E K Y
51 S F F Y G G C M G N G N N F V R E K Y 52 P F F Y G G C G G N G N
N F L R E K Y 53 T F F Y G G C L G N G N N F V R E K Y 54 S F F Y G
G C L G N G N N Y L R E K Y 55 T F F Y G G S L G N G N N F V R E K
Y 56 T F F Y G G C R G N G N N F V T A E Y 57 T F F Y G G C L G K G
N N F V S A E Y 58 T F F Y G G C L G N R N N F D R A E Y 59 T F F Y
G G C L G N R N N F L R E E Y 60 T F F Y G G C L G N K N N Y L R E
E Y 61 P F F Y G G C G G N R N N Y L R E E Y 62 P F F Y G G S G G N
R N N Y L R E E Y 63 M R P D F C L E P P Y T G P C V A R I 64 A R I
I R Y F Y N A K A G L C Q T F V Y G 65 Y G G C R A K R N N Y K S A
E D C M R T C G 66 P D F C L E P P Y T G P C V A R I I R Y F Y 67 T
F F Y G G C R G K R N N F K T E E Y 68 K F F Y G G C R G K R N N F
K T E E Y 69 T F Y Y G G C R G K R N N Y K T E E Y 70 T F F Y G G S
R G K R N N F K T E E Y 71 C T F F Y G C C R G K R N N F K T E E Y
72 T F F Y G G C R G K R N N F K T E E Y C 73 C T F F Y G S C R G K
R N N F K T E E Y 74 T F F Y G G S R G K R N N F K T E E Y C 75 P F
F Y G G C R G K R N N F K T E E Y 76 T F F Y G G C R G K R N N F K
T K E Y 77 T F F Y G G K R G K R N N F K T E E Y 78 T F F Y G G C R
G K R N N F K T K R Y 79 T F F Y G G K R G K R N N F K T A E Y 80 T
F F Y G G K R G K R N N F K T A G Y 81 T F F Y G G K R G K R N N F
K R E K Y 82 T F F Y G G K R G K R N N F K R A K Y 83 T F F Y G G C
L G N R N N F K T E E Y 84 T F F Y G C G R G K R N N F K T E E Y 85
T F F Y G G R C G K R N N F K T E E Y 86 T F F Y G G C L G N G N N
F D T E E E 87 T F Q Y G G C R G K R N N F K T E E Y 88 Y N K E F G
T F N T K G C E R G Y R F 89 R F K Y G G C L G N M N N F E T L E E
90 R F K Y G G C L G N K N N F L R L K Y 91 R F K Y G G C L G N K N
N Y L R L K Y 92 K T K R K R K K Q R V K I A Y E E I F K N Y 93 K T
K R K R K K Q R V K I A Y 94 R G G R L S Y S R R F S T S T G R 95 R
R L S Y S R R R F 96 R Q I K I W F Q N R R M K W K K 97 T F F Y G G
S R G K R N N F K T E E Y 98 M R P D F C L E P P Y T G P C V A R I
I R Y F Y N A K A G L C Q T F V Y G O C R A K R N N F K S A E D C M
R T C G G A 99 T F F Y G G C R G K R N N F K T K E Y 100 R F K Y G
G C L G N K N N Y L R L K Y 101 T F F Y G G C R A K R N N F K R A K
Y 102 N A K A G L C Q T F V Y G G C L A K R N N F E S A E D C M R T
C G G A 103 Y G G C R A K R N N F K S A E D C M R T C G G A 104 G L
C Q T F V Y G G C R A K R N N F K S A E 105 L C Q T F V Y G G C E A
K R N N F K S A 107 T F F Y G G S R G K R N N F K T E E Y 108 R F F
Y G G S R G K R N N F K T E E Y 109 R F F Y G G S R G K R N N F K T
E E Y 110 R F F Y G G S R G K R N N F R T E E Y 111 T F F Y G G S R
G K R N N F R T E E Y 112 T F F Y G G S R G R R N N F R T E E Y 113
C T F F Y G G S R G K R N N F K T E E Y 114 T F F Y G G S R G K R N
N F K T E E Y C 115 C T F F Y G G S R G R R N N F R T E E Y 116 T F
F Y G G S R G R R N N F R T E E Y C Polypeptides Nos. 5, 67, 76,
and 91, include the sequences of SEQ ID NOS: 5, 67, 76, and 91,
respectively, and are amidated at the C-terminus. Polypeptides Nos.
107, 109, and 110 include the sequences of SEQ ID NOS: 97, 109, and
110, respectively, and are acetylated at the N-terminus.
[0052] In any of the above aspects, the polypeptide may include an
amino acid sequence having the formula:
X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19
where each of X1-X19 (e.g., X1-X6, X8, X9, X11-X14, and X16-X19)
is, independently, any amino acid (e.g., a naturally occurring
amino acid such as Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,
Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) or
absent and at least one (e.g., 2 or 3) of X1, X10, and X15 is
arginine. In some embodiments, X7 is Ser or Cys; or X10 and X15
each are independently Arg or Lys. In some embodiments, the
residues from X1 through X19, inclusive, are substantially
identical to any of the amino acid sequences of any one of SEQ ID
NOS:1-105 and 107-116 (e.g., Angiopep-1, Angiopep-2, Angiopep-3,
Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7).
In some embodiments, at least one (e.g., 2, 3, 4, or 5) of the
amino acids X1-X19 is Arg. In some embodiments, the polypeptide has
one or more additional cysteine residues at the N-terminal of the
polypeptide, the C-terminal of the polypeptide, or both.
[0053] In certain embodiments of any of the above aspects, the
polypeptide is modified (e.g., as described herein). The
polypeptide may be amidated, acetylated, or both. Such
modifications to polypeptides may be at the amino or carboxy
terminus of the polypeptide. The conjugates of the invention may
also include peptidomimetics (e.g., those described herein) of any
of the polypeptides described herein. The polypeptide may be in a
multimeric form, for example, dimeric form (e.g., formed by
disulfide bonding through cysteine residues).
[0054] In certain embodiments, the polypeptide has an amino acid
sequence described herein with at least one amino acid substitution
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 substitutions). The
polypeptide may contain, for example, 1 to 12, 1 to 10, 1 to 5, or
1 to 3 amino acid substitutions, for example, 1 to 10 (e.g., to 9,
8, 7, 6, 5, 4, 3, 2) amino acid substitutions. The amino acid
substitution(s) may be conservative or non-conservative. For
example, the polypeptide may gave an arginine at one, two, or three
of the positions corresponding to positions 1, 10, and 15 of the
amino acid sequence of any of SEQ ID NO:1, Angiopep-1, Angiopep-2,
Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6, and
Angiopep-7.
[0055] In any of the above aspects, the conjugate may specifically
exclude a polypeptide including or consisting of any of SEQ ID
NOS:1-105 and 107-116 (e.g., Angiopep-1, Angiopep-2, Angiopep-3,
Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7).
In some embodiments, the polypeptides and conjugates of the
invention exclude the polypeptides of SEQ ID NOs:102, 103, 104, and
105.
[0056] In some embodiments, the amino acid sequence has at least
35%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity
to an amino acid sequence selected from the group consisting of SEQ
ID NOS:1-105 and 107-116, or a functional derivative thereof. In
certain embodiments, the amino acid sequence has at least 35%, 40%,
50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino
acid sequence selected from the group consisting of Angiopep-2 (SEQ
ID NO:97), Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7 (SEQ
ID NOS:109-112). In still other embodiments, the amino acid
sequence has at least 35%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, or 95% identity to an amino acid sequence of Angiopep-2 (SEQ
ID NO:97).
[0057] In some embodiments, the amino acid sequence comprises the
amino acid sequence selected from the group consisting of SEQ ID
NOS:1-105 and 107-116, or a functional derivative thereof. In
certain embodiments, the amino acid sequence is that of Angiopep-2
(SEQ ID NO:97), Angiopep-4b, Angiopep-5, Angiopep-6, or Angiopep-7
(SEQ ID NOS:109-112).
[0058] In still other embodiments, the amino acid sequence consists
of the amino acid sequence selected from the group consisting of
SEQ ID NOS:1-105 and 107-116, or a functional derivative thereof.
In certain embodiments, the amino acid sequence is that of
Angiopep-2 (SEQ ID NO:97), Angiopep-4b, Angiopep-5, Angiopep-6, or
Angiopep-7 (SEQ ID NOS:109-112).
[0059] In some embodiments, the compounds of the invention can
alter the accumulation of a biologically active agent (e.g.,
podophyllotoxin derivatives such as the compounds of Formula (I) or
doxorubicin derivatives such as the compounds of Formula (II)) in a
target cell type or tissue relative to the corresponding
unconjugated biologically active agent. In still other embodiments,
the compound of the invention promotes accumulation of the
biologically active agent in a target cell type or tissue. In
certain embodiments, the concentration of the biologically active
agent increases by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%,
650%, 700%, 750%, 800%, 850%, 900%, 950%, 1,000%, 2,000%, 3,000%,
4,000%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, 10,000%, 12500%,
15,000%, 17,500%, or 20,000% relative to that observed with the
unconjugated biologically active agent. In some embodiments, the
target cell type or tissue is the brain, ovary, liver, lung,
kidney, spleen, or muscle. In some embodiments, the target cell
type is the brain or the ovary. In certain embodiments, the
biologically active agent is selected from etoposide, etoposide
phosphate, etoposide.sub.DMG, teniposide, doxorubicin, or
epirubicin. In other embodiments, the compound of the invention
includes the amino acid sequence of Angiopep-2 (SEQ ID NO:97),
Angiopep-4b, Angiopep-5, Angiopep-6, or Angiopep-7 (SEQ ID
NOS:109-112), or a functional derivative thereof.
[0060] In a third aspect, the invention features a pharmaceutical
composition that includes any compound of the invention as
described herein (e.g., a compound that includes an amino acid
sequence substantially identical to an amino acid sequence selected
from the group consisting of SEQ ID NOS:1-105 and 107-116, or a
functional derivative or pharmaceutically acceptable salt thereof,
where the amino acid sequence includes a covalent bond from an
amino acid of the amino acid sequence to a compound of Formulas (I)
or (II) (e.g., Compound (1) or (2)) and a pharmaceutically
acceptable carrier. In a fourth aspect, the invention features a
method of treating or treating prophylactically a cancer, where the
method includes administering to a patient a therapeutically
effective amount of any compound of the invention as described
herein (e.g., a compound that includes an amino acid sequence
substantially identical to an amino acid sequence selected from the
group consisting of SEQ ID NOS:1-105 and 107-116, or a functional
derivative thereof, where the amino acid sequence includes a
covalent bond from an amino acid of the amino acid sequence to a
compound of Formulas (I) or (II)). In some embodiments, the
compound is Compound (1) or (2). In some embodiments, the
podophyllotoxin derivative is selected from
##STR00022##
where
[0061] Y is H; each R.sub.2 is, independently, H or P(O)(OH).sub.2,
or --C(O)R.sub.8; each R.sub.6 is, independently, CH.sub.3 or
2-thiophene; each Y is --C(O)(CH.sub.2).sub.nC(O)--; each R.sub.8
is, independently, optionally substituted C.sub.1-6 alkyl; and each
n is, independently, 2, 3, or 4. In some embodiments, n is 3. In
some embodiments, each R.sub.2 is --C(O)R.sub.8. In some
embodiments, R.sub.8 is a C.sub.1-6 alkyl that includes an least
one optionally substituted amino group (e.g., NH.sub.2 or
N(CH.sub.3).sub.2). In certain embodiments --C(O)R.sub.8 is a
C-linked amino acid. In some embodiments, the podophyllotoxin
derivative is etoposide, etoposide phosphate, etoposide
4'-dimethylglycine (etoposide.sub.DMG), or teniposide. In still
other embodiments, the compound is doxorubicin or any of the
doxorubicin derivatives (e.g., a compound of Formula (II))
described herein.
[0062] In some embodiments, the method also includes the
administration of a second agent. In still other embodiments, the
agent is a therapeutic agent. In certain embodiments, the second
therapeutic agent is also covalently bonded to the compound of the
invention. In still other embodiments, the second therapeutic agent
is not covalently bonded to the compounds of the invention. In some
embodiments, the therapeutic agent is drug, a medicine, an agent
emitting radiation, a cellular toxin, a biologically active
fragment thereof, or a mixture thereof to treat a disease. In other
embodiments, the administering is concurrent with another
therapeutic regime. In some embodiments, the therapeutic regime is
radiation therapy, chemotherapy, stem cell transplantation, bone
marrow transplant, surgery, or hyperthermia treatment. In some
embodiments, the second therapeutic agent is a polypeptide that
includes or that consists of the sequence of Angiopep-2 (SEQ ID
NO:97), preferably where the Angiopep-2 is conjugated to an
anticancer agent (e.g., paclitaxel), e.g., ANG1005, which has the
following structure:
##STR00023##
Still other exemplary second therapeutic agents are described in
U.S. Pat. No. 7,557,182, herein incorporated by reference.
[0063] In some embodiments, the cancer is cancer of the brain. In
other embodiments, the cancer of the brain is glioblastoma, a
glioma, an acoustic neuroma, an adenoma, an astrocytoma, a choroid
plexus papilloma, CNS lymphoma, ependymoma, a gangliocytoma, a
ganglioglioma, a medulloblastoma (mdl), an anaplastic (malignant)
meningioma, or neurofibromatosis. In still other embodiments, the
cancer is acute lymphocytic leukemia, acute myeloblastic leukemia,
adrenocortical cancer, intravenous and intravesical bladder cancer,
bone sarcoma, breast cancer, carcinoid syndrome (small bowel),
endometrial cancer, Ewing's sarcoma, gynecological sarcoma, head
and neck cancer (squamous cell), hepatic cancer, Hodgkin's disease,
islet cell cancer, leukemia, lung cancer, malignant lymphoma,
multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma, pancreatic
cancer, prostate cancer, osteogenic sarcoma, ovarian cancer,
retinoblastoma, rhabdomyosarcoma, stomach cancer, testicular
cancer, thyroid cancer, transitional cell bladder carcinoma, soft
tissue sarcoma, or Wilms' tumor.
[0064] In any of therapeutic methods described herein, the compound
of the invention (e.g., Compound (1) or (2)), or a pharmaceutically
acceptable salt thereof, can be administered to a patient as a
subtherapeutic dose or a supertherapeutic dose relative to the
unconjugated therapeutic agent (e.g., etoposide, etoposide
phosphate, etoposide 4-dimethylglycine, or doxorubicin).
[0065] In another aspect, the invention features a method of making
any of the compounds of the invention described herein, where the
method includes the step of covalently binding a podophyllotoxin
derivative to any amino acid sequence described herein, or
functional derivative thereof, using a difunctional hydrolyzable
linking group. In some embodiments, the amino acid sequence is
selected from SEQ ID NOS:1-105 and 107-116, or a functional
derivative thereof. In other embodiments, the amino acid sequence
includes the amino acid sequence of Angiopep-2 (SEQ ID NO:97),
Angiopep-4b, Angiopep-5, Angiopep-6, or Angiopep-7 (SEQ ID
NOS:109-112).
[0066] In some embodiments, the method of making any of the
compounds of the invention includes the steps of [0067] (a)
combining said compound of Formula (I) with said difunctional
hydrolyzable linking group to form a covalent adduct; and [0068]
(b) combining the adduct of (a) with said amino acid sequence; and
[0069] where the adduct of (a) may be optionally purified prior to
use in (b).
[0070] In some embodiments, 1.0-10.0 equivalents of the
difunctional hydrolyzable linking group is used relative to the
compound of Formula (I). For example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or
3.0 equivalents can be used. In other embodiments, 3.5, 4.0, 4.5,
5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0
equivalents the difunctional hydrolyzable linking group is used. In
certain embodiments, the method includes the use of a peptide
coupling agent. In some embodiments, peptide coupling agent is
N,N,N',N'-Tetramethyl-O-(benzotriazol-1-yl)uronium
tetrafluoroborate (TBTU).
[0071] In some embodiments, the hydrolyzable difunctional linking
group is selected from dicarboxylic acids, dicarbonates, carboxylic
anhydrides, diisocyanates, or diphosphonic acids. In certain
embodiments, the hydrolyzable difunctional linking group is
selected from succinic acid, glutaric acid, glutaric anhydride, or
butaric acid.
[0072] In some embodiments, the podophyllotoxin derivative is
selected from:
##STR00024##
where
[0073] Y is H; each R.sub.2 is, independently, H or P(O)(OH).sub.2,
or --C(O)R.sub.8; each R.sub.6 is, independently, CH.sub.3 or
2-thiophene; each Y is --C(O)(CH.sub.2).sub.nC(O)--; each R.sub.8
is, independently, optionally substituted C.sub.1-6 alkyl; and each
n is, independently, 2, 3, or 4. In some embodiments, n is 3. In
some embodiments, each R.sub.2 is --C(O)R.sub.8. In some
embodiments, R.sub.8 is a C.sub.1-6 alkyl that includes an least
one optionally substituted amino group (e.g., NH.sub.2 or
N(CH.sub.3).sub.2). In certain embodiments --C(O)R.sub.8 is a
C-linked amino acid. In some embodiments, the podophyllotoxin
derivative is etoposide, etoposide phosphate, etoposide.sub.DMG, or
teniposide.
[0074] In any of the methods or compositions described herein, the
pharmaceutically acceptable salt of the compound can be the mono-,
di-, tri-, or tetra acid addition salt (e.g., the trihydrochloride
salt). In any of the embodiments described herein, any of the
compounds of Formula (I) or (II) (e.g., etoposide,
etoposide.sub.DMG, or doxorubicin) that is covalently bonded to the
polypeptide is the site of protonation. For example, in Compound
(1), 1, 2, or 3 of the etoposide.sub.DMG moieties is protonated, or
in Compound (2), 1, 2, or 3 of the doxorubicin moieties, is
protonated to form the acid addition salt (e.g., the mono-, di-, or
trihydrochloride salt).
[0075] The term "C.sub.1-6 alkyl" or "alkyl" as used herein refers
to an optionally substituted C.sub.1-6 saturated hydrocarbon group.
An alkyl group may be linear or branched. Examples of alkyl
radicals include, but are not limited to, methyl, ethyl, n-propyl,
isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl,
tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl,
n-octyl, n-decyl, n-undecyl, dodecyl, and the like, which may bear
one or more substituents. For example, substituted alkyl groups may
have 1, 2, 3, 4, 5, or 6 substituents.
[0076] The term "aryl" as used herein refers to an optionally
substituted mono- or polycyclic, aromatic all-carbon moiety having
5-14 carbon atoms. In certain embodiments of the present invention,
"aryl" refers to a substituted or unsubstituted monocyclic or
bicyclic group. Exemplary aryl groups include, but are not limited
to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the
like, which may bear one or more substituents. Aryls also include
heteroaryls.
[0077] The term "C-linked amino acid" as used herein refers to an
amino acid that is covalently attached to another compound (e.g.,
any of the podophyllotoxin derivatives described herein) by the
C-terminus of the amino acid.
[0078] The term "C.sub.3-10 cycloalkyl" or "cycloalkyl" as used
herein refers to an optionally substituted saturated 3- to
10-membered monocyclic or bicyclic hydrocarbon ring system.
Exemplary cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, and cycloheptyl. A substituted cycloalkyl can have, for
example, 1, 2, 3, 4, 5, 6, or 7 substituents.
[0079] The term "heteroaryl" as used herein refers to a substituted
or unsubstituted mono- or polycyclic, aromatic moiety having 5-14
ring atoms of which one, two, three, or four ring atoms may be
selected from S, O, and N and the remaining ring atoms are carbon.
Exemplary heteroaryl groups include, but are not limited to,
pyrrolyl, pyrazolyl, imadazolyl, pyridinyl, pyrimidinyl, pyrazinyl,
pyridazinyl, triazinyl, tetrazinyl, pryyrolizinyl, indolyl,
quinolinyl, isoquinolynyl, benzimidazolyl, indazolyl, quinolizinyl,
cinnolinyl, quinazolinyl, phthalazinyl, napthyridinyl,
quinoxalinyl, thiophenyl, thiepinyl, furanyl, benzofuranyl,
thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl,
oxadiazolyl, and the like, which may bear one or more
substituents.
[0080] The term "heterocyclic," or "heterocyclyl," as used herein,
refers to an optionally substituted non-aromatic, partially
unsaturated or fully saturated, 3- to 10-membered ring system,
which includes single rings of 3 to 8 atoms in size, and bi- and
tri-cyclic ring systems which may include aromatic five- or
six-membered aryl or heteroaryl groups fused to a non-aromatic
ring. These heterocyclic rings include those having from one to
three heteroatoms independently selected from oxygen, sulfur, and
nitrogen, in which the nitrogen and sulfur heteroatoms may
optionally be oxidized and the nitrogen heteroatom may optionally
be quaternized or substituted. In certain embodiments, the term
heterocyclic refers to a non-aromatic 5-, 6-, or 7-membered
monocyclic ring wherein at least one ring atom is a heteroatom
selected from O, S, and N (wherein the nitrogen and sulfur
heteroatoms may be optionally oxidized), and the remaining ring
atoms are carbon, the radical being joined to the rest of the
molecule via any of the ring atoms. Exemplary heterocyclics
include, but are not limited to, azacyclopropanyl, azacyclobutanyl,
1,3-diazatidinyl, pyrrolidinyl, piperidinyl, piperazinyl, thiranyl,
thietanyl, tetrahydrothiophenyl, dithiolanyl,
tetrahydrothiopyranyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, dioxanyl, oxathiolanyl, morpholinyl,
thiomorpholinyl, thioxanyl, quinuclidinyl, and the like, which may
bear one or more substituents.
[0081] The term "pharmaceutically acceptable salt," as use herein,
represents those acid addition salts which are, within the scope of
sound medical judgment, suitable for use in contact with the
tissues of humans and animals without undue toxicity, irritation,
allergic response and the like and are commensurate with a
reasonable benefit/risk ratio. Pharmaceutically acceptable salts
are well known in the art. For example, pharmaceutically acceptable
salts are described in: Berge et al., J. Pharmaceutical Sciences
66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection,
and Use, (Eds. P. H. Stahl and C. G. Wermuth), Wiley-VCH, 2008. The
salts can be prepared in situ during the final isolation and
purification of the compounds described herein or separately by
reacting a compound having one or basic groups (e.g., 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10) with the desired equivalents of a suitable
organic or inorganic acid. Representative acid addition salts
include acetate, adipate, alginate, ascorbate, aspartate,
benzenesulfonate, benzoate, bisulfate, borate, butyrate,
camphorate, camphorsulfonate, citrate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, fumarate,
glucoheptonate, glycerophosphate, hemisulfate, heptonate,
hexanoate, hydrobromide, hydrochloride, hydroiodide,
2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl
sulfate, malate, maleate, malonate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate,
palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,
phosphate, picrate, pivalate, propionate, stearate, succinate,
sulfate, tartrate, thiocyanate, toluenesulfonate, trifluoroacetate,
trifluoromethylsulfonate, undecanoate, and valerate salts, and the
like. Representative alkali or alkaline earth metal salts include
sodium, lithium, potassium, calcium, magnesium and the like, as
well as nontoxic ammonium, quaternary ammonium, and amine cations,
including, but not limited to ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine,
triethylamine, ethylamine and the like. Desirably, the
"pharmaceutically acceptable acid addition salt" is the mono-,
bis-, tri-, or tetra acid addition salt of any of the compounds
described herein (e.g., a mono-, bis-, tris-, or tetrahydrochloride
salt of any of the compounds described herein).
[0082] The term "phosphate" as used herein refers to a pentavalent
phosphorous group having the formula --OP(.dbd.O)(OR')(OR''), where
each R' and R'' is selected, independently, from hydrogen,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-10
cycloalkyl, heterocyclyl, aryl, or heteroaryl.
[0083] Where a group is described as "optionally substituted," the
optional substituents may be selected, independently, from groups
that include, but are not limited to: C.sub.1-6 alkyl; halogen;
azido (--N.sub.3), nitro (--NO.sub.2), cyano (--CN), acyloxy, acyl
(--C(O)R), (--OC(O)R), alkoxy (--OR), amido (--NRC(O)R' or
--C(O)NRR'), amino (--NRR'), aryl, carboxylic acid (--CO.sub.2H),
carboxylic ester (--CO.sub.2R), carbamoyl (--OC(O)NRR' or
--NRC(O)OR'), cycloalkyl, heterocyclyl, hydroxy (--OH), isocyano
(--NC), phosphate (--P(O)(OR)(OR')), sulfonate (--SO.sub.2OR), or
sulfonyl (--SO.sub.2R), where each R or R' is selected,
independently, from H, C.sub.1-6 alkyl, cycloalkyl, heterocyclyl,
aryl, or heteroaryl. A substituted group may have, for example, 1,
2, 3, 4, 5, 6, 7, 8, or 9 substituents. In some embodiments, a
substituent group may itself be further substituted by replacing a
hydrogen with a substituent group such as those described
herein.
[0084] By "vector" is meant a compound or molecule such as a
polypeptide that is able to be transported into a particular cell
type (e.g., liver, ovary, lungs, kidney, spleen, or muscle) or
across the BBB. The vector may be attached to (covalently or not)
or conjugated to an agent and thereby may be able to transport the
agent into a particular cell type or across the BBB. In certain
embodiments, the vector may bind to receptors present on cancer
cells or brain endothelial cells and thereby be transported into
the cancer cell or across the BBB by transcytosis. The vector may
be a molecule for which high levels of transendothelial transport
may be obtained, without affecting the cell or BBB integrity. The
vector may be a polypeptide or a peptidomimetic and may be
naturally occurring or produced by chemical synthesis or
recombinant genetic technology.
[0085] By "conjugate" is meant a vector linked to an agent. The
conjugation may be chemical in nature, such as via a linker, or
genetic in nature for example by recombinant genetic technology,
such as in a fusion protein with for example a reporter molecule
(e.g., green fluorescent protein, .beta.-galactosidase, Histag,
etc.).
[0086] By a vector which is "efficiently transported across the
BBB" is meant a vector that is able to cross the BBB at least as
efficiently as AngioPep-6 (i.e., greater than 38.5% that of
AngioPep-1 (250 nM) in the in situ brain perfusion assay described
herein). Accordingly, a vector or conjugate which is "not
efficiently transported across the BBB" is transported to the brain
at lower levels (e.g., transported less efficiently than
AngioPep-6).
[0087] By a vector or conjugate which is "efficiently transported
to a particular cell type" is meant a vector or conjugate that is
able to accumulate (e.g., either due to increased transport into
the cell, decreased efflux from the cell, or a combination thereof)
in that cell type at least 10% (e.g., 25%, 50%, 100%, 200%, 500%,
1,000%, 5,000%, or 10,000%) greater extent than either a control
substance, or, in the case of a conjugate, as compared to the
unconjugated agent.
[0088] By "substantially pure" or "isolated" is meant a compound
(e.g., a polypeptide or conjugate) that has been separated from
other chemical components. Typically, the compound is substantially
pure when it is at least 30%, by weight, free from other
components. In certain embodiments, the preparation is at least
50%, 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by weight, free
from other components. A purified polypeptide may be obtained, for
example, by expression of a recombinant polynucleotide encoding
such a polypeptide or by chemically synthesizing the polypeptide.
Purity can be measured by any appropriate method, for example,
column chromatography, polyacrylamide gel electrophoresis, or by
HPLC analysis.
[0089] By "analogue" is meant a polypeptide originating from an
original sequence or from a portion of an original sequence and
which may include one or more modification; for example, one or
more modification in the amino acid sequence (e.g., an amino acid
addition, deletion, insertion, or substitution), one or more
modification in the backbone or side-chain of one or more amino
acid, or an addition of a group or another molecule to one or more
amino acids (side-chains or backbone). An analogue may have one or
more amino acid insertions, at either or at both of the ends of the
polypeptide or inside the amino acid sequence of the polypeptide.
An analogue may have sequence similarity and/or sequence identity
(e.g., may be substantially identical) with that of an original
sequence or a portion of an original sequence. Analogues may
include a modification of its structure, e.g., as described herein.
The degree of similarity between two sequences is base upon the
percentage of identities (identical amino acids) and of
conservative substitution. An analogue may have at least 35%, 50%,
60%, 70%, 80%, 90%, or 95% (e.g., 96%, 97%, 98%, 99%, and 100%)
sequence similarity to an original sequence with a combination of
one or more modifications in a backbone or side-chain of an amino
acid, or an addition of a group or another molecule. Exemplary
amino acids which are intended to be similar (a conservative amino
acid) to others are known in the art and include, for example,
those listed in Table 3.
[0090] By "substantially identical" is meant a polypeptide or
nucleic acid exhibiting at least 35%, 40%, 50%, 55%, 60%, 65%, 70%,
75%, 85%, 90%, 95%, or even 99% identity to a reference amino acid
or nucleic acid sequence. For polypeptides, the length of
comparison sequences will generally be at least 4 (e.g., at least
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50,
or 100) amino acids. For nucleic acids, the length of comparison
sequences will generally be at least 60 nucleotides, preferably at
least 90 nucleotides, and more preferably at least 120 nucleotides,
or full length. It is to be understood herein that gaps may be
found between the amino acids of an analogs which are identical or
similar to amino acids of the original polypeptide. The gaps may
include no amino acids, one or more amino acids which are not
identical or similar to the original polypeptide. Biologically
active analogs of the vectors (polypeptides) of the invention are
encompassed herewith. Percent identity may be determined, for
example, with n algorithm GAP, BESTFIT, or FASTA in the Wisconsin
Genetics Software Package Release 7.0, using default gap
weights.
[0091] By "functional derivative" is meant a "chemical derivative,"
"fragment," or "variant" biologically active sequence or portion of
a vector or agent or conjugate and a salt thereof of the invention.
A vector functional derivative may be able to be attached to or
conjugated to an agent and enter a particular cell type, thereby
transporting the agent into that cell.
[0092] By "chemical derivative" is meant a vector, an agent, or a
conjugate of the invention, which contains additional chemical
moieties not a part of the vector, agent or vector-agent conjugate,
including covalent modifications. A chemical derivative may be
prepared by direct chemical synthesis using methods known in the
art. Such modifications may be introduced into a protein or peptide
vector, agent, or vector-agent conjugate by reacting targeted amino
acid residues with an organic derivatizing agent capable of
reacting with selected side chains or terminal residues. A vector
chemical derivative may be able to cross the BBB or to enter or
accumulate in a particular cell type (e.g., those described herein
such as the ovary). In a preferred embodiment, very high levels of
transendothelial transport across the BBB are obtained without
effecting BBB integrity.
[0093] By "fragment" is meant a polypeptide originating from a
portion of an original or parent sequence or from an analogue of
said parent sequence. Fragments encompass polypeptides having
truncations of one or more amino acids, wherein the truncation may
originate from the amino terminus (N-terminus), carboxy terminus
(C-terminus), or from the interior of the protein. A fragment may
include the same sequence as the corresponding portion of the
original sequence. Functional fragments of the vector (polypeptide)
described herein are encompassed by the invention. Fragments may be
at least 5 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 25, 28, 30, 35, 40, 45, 50, 60, 75,
100, or 150) amino acids. Fragments of the invention may include,
for example, a polypeptide of 7, 8, 9 or 10 amino acids to 18 amino
acids. Fragments may contain any of the modifications described
herein (e.g., acetylation, amidation, amino acid substitutions)
[0094] A "non-naturally occurring amino acid" is an amino acid
which is not naturally produced or found in a mammal.
[0095] By "agent" is meant, any compound, for example, an antibody,
or a therapeutic agent, a marker, a tracer, or an imaging
compound.
[0096] By "therapeutic agent" is meant an agent having a biological
activity. In some cases, the therapeutic agent is used to treat the
symptoms of a disease, a physical or mental condition, an injury,
or an infection and includes anti-cancer agents, antibiotics,
anti-angiogenic agents, and molecules active at the level of the
central nervous system.
[0097] By "small molecule drug" is meant a drug having a molecular
weight of 1000 g/mol or less (e.g., less than 800, 600, 500, 400,
or 200 g/mol).
[0098] By "subject" is meant a human or non-human animal (e.g., a
mammal).
[0099] By "treating" a disease, disorder, or condition in a subject
is meant reducing at least one symptom of the disease, disorder, or
condition by administrating a therapeutic agent to the subject.
[0100] By "treating prophylactically" a disease, disorder, or
condition in a subject is meant reducing the frequency of
occurrence of (e.g., preventing) a disease, disorder or condition
by administering a therapeutic agent to the subject.
[0101] By "cancer" is meant any cellular proliferation whose unique
trait is the loss of normal controls which can result in
unregulated growth, lack of differentiation, or ability to invade
tissues and metastasize. Cancer can develop in any tissue or in any
organ. Cancer is intended to include, without limitation, cancer of
the brain, ovary, liver, lungs, kidney, or spleen. Additional
cancers are described herein.
[0102] By "providing" is meant, in the context of a vector or
conjugate of the invention, to bring the vector or conjugate into
contact with a target cell or tissue either in vivo or in vitro. A
vector or conjugate may be provided by administering the vector or
conjugate to a subject.
[0103] By "administering" and "administration" is meant a mode of
delivery including, without limitation, intra-arterially,
intra-nasally, intraperitoneally, intravenously, intramuscularly,
subcutaneously, transdermally or per os. A daily dosage can be
divided into one, two or more doses in a suitable form to be
administered at one, two or more times throughout a time
period.
[0104] By "therapeutically effective" or "effective amount" is
meant an amount of a therapeutic agent sufficient to improve,
decrease, prevent, delay, suppress, or arrest any symptom of the
disease or condition being treated. A therapeutically effective
amount of an agent need not cure a disease or condition but will
provide a treatment for a disease or condition such that the onset
of the disease or condition is delayed, hindered, or prevented, or
the disease or condition symptoms are ameliorated, or the term of
the disease or condition is changed or, for example, is less severe
or recovery is accelerated in an individual. A "subtherapeutic
dose" is a dose less than the minimum effective amount of a
therapeutic agent that has been approved for clinical use by a
patient. A "supertherapeutic dose" is a dose greater than the
maximum effective amount of a therapeutic agent that has been
approved for clinical use by a patient. The amount of a
subtherapeutic dose or a supertherapeutic dose may vary according
to the patient demographics (e.g., adult, pediatric, or geriatric)
or when used in conjunction with the administration of additional
therapeutic agents (e.g., when administered concurrently with other
therapeutic agents or treatment regimes such as, for example, in
cancer chemotherapy).
[0105] By "condition" is meant any situation causing pain,
discomfort, sickness, disease or disability (mental or physical) to
or in an individual, including neurological disease, injury,
infection, or chronic or acute pain. Neurological diseases include
brain tumors, brain metastases, schizophrenia, epilepsy,
Alzheimer's disease, Parkinson's disease, Huntington's disease, and
stroke.
[0106] By "pharmaceutical composition" is meant a therapeutically
effective amount of an agent together with a pharmaceutically
acceptable diluents, preservative, solubilizer, emulsifier, or
adjuvant, for example, any of those described herein.
[0107] By "therapeutic dose" is meant the dosage of a agent such as
a drug (without the vector) acceptable for use clinically with
respect to its toxicity or efficacy. By conjugation of an agent to
a vector of the invention, it may be possible to administer the
agent at a dosage either lower or higher dosage than the
therapeutic dose.
[0108] If a "range" or "group of substances" is mentioned with
respect to a particular characteristic (e.g., temperature,
concentration, time and the like), the invention relates to and
explicitly incorporates herein each and every specific member and
combination of sub-ranges or sub-groups therein. Thus, for example,
with respect to a length of from 9 to 18 amino acids, is to be
understood as specifically incorporating herein each and every
individual length, e.g., a length of 18, 17, 15, 10, 9, and any
number there between. Therefore, unless specifically mentioned,
every range mentioned herein is to be understood as being
inclusive. For example, in the expression from 5 to 19 amino acids
long is to be as including 5 and 19. This similarly applies with
respect to other parameters such as sequences, length,
concentrations, elements, and the like.
[0109] The sequences, regions, portions defined herein each include
each and every individual sequence, region, and portion described
thereby as well as each and every possible sub-sequence,
sub-region, and sub-portion whether such sub-sequences,
sub-regions, and sub-portions are defined as positively including
particular possibilities, as excluding particular possibilities or
a combination thereof. For example, an exclusionary definition for
a region may read as follows: "provided that said polypeptide is no
shorter than 4, 5, 6, 7, 8 or 9 amino acids. A further example of a
negative limitation is the following; a sequence including SEQ ID
NO:X with the exclusion of a polypeptide of SEQ ID NO:Y; etc. An
additional example of a negative limitation is the following;
provided that said polypeptide is not (does not include or consist
of) SEQ ID NO:Z.
[0110] Other features and advantages of the invention will be
apparent from the following Detailed Description, the drawings, and
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0111] FIG. 1 shows inhibition of subcutaneous U87 (s.c. U87)
xenograft tumor growth using the doxorubicin-An2(3:1)
("Doxorubicin-An2(3:1)") conjugate.
[0112] FIG. 2A shows the brain uptake of the 3:1
Etoposide:Angiopep-2 conjugate ("Etop-An2(3:1)") measured by in
situ brain perfusion.
[0113] FIG. 2B shows the brain uptake of the 3:1 etoposide
4'-dimethylglycine:Angiopep-2 conjugate ("Etop.sub.DMG-An2(3:1)")
measured by in situ brain perfusion.
[0114] FIG. 2C shows the brain uptake of the Doxorubicin-An2(3:1)
measured by in situ brain perfusion.
[0115] FIG. 3 shows the in situ perfusion of Etop-An2(3:1).
[0116] FIG. 4A shows the parenchymal uptake of unconjugated
etoposide compared to Etop-An2(3:1).
[0117] FIG. 4B shows the brain repartition of Etop-An2(3:1)
following brain capillary depletion.
[0118] FIG. 5 shows the in situ brain perfusion of Etop-An2(3:1)
compared to unconjugated etoposide in CD-1 versus P-gp knock-out
mice.
[0119] FIG. 6 shows the inhibition of brain uptake of Etop-An2(3:1)
by Angiopep-2.
[0120] FIG. 7 shows the in situ brain perfusion of Etop.sub.DMG-An2
compared to unconjugated Etop.sub.DMG in CD-1 versus P-gp knock-out
mice.
[0121] FIG. 8 shows data on the data on plasma kinetics of
Etop-An2(3:1).
[0122] FIG. 9 shows the brain distribution of Etop.sub.DMG-An2
following IV bolus administration in mice.
[0123] FIG. 10 shows the brain distribution of Etop-An2(3:1).
[0124] FIG. 11 shows the brain distribution of Etop-An2(3:1)
compared to unconjugated etoposide thirty minutes after IV bolus
administration.
[0125] FIG. 12 shows the tissue distribution of Etop-An2(3:1)
compared to unconjugated etoposide thirty minutes after IV bolus
administration.
[0126] FIG. 13A and FIG. 13B each show the in vivo effect of
(DoxSu).sub.3-An2 in mice that have been intracranially injected
with U87 glioblastoma cells. FIG. 13A shows the results obtained in
the first trial (Trial 1). FIG. 13B shows the results obtained in
the second trial (Trial 2) showing that the administration of
Compound (2) results in a statistically significant extension of
mean survival time.
DETAILED DESCRIPTION OF THE INVENTION
[0127] The invention features compounds, or any pharmaceutically
acceptable salt thereof, that include an amino acid sequence
substantially identical to an amino acid sequence selected from the
amino acid sequences described herein (e.g., SEQ ID NOS:1-105 and
107-116), or a functional derivative thereof, where said amino acid
sequence includes a covalent bond from an amino acid of the amino
acid sequence to an anti-cancer agent (e.g., podophyllotoxin
derivatives, doxorubicin, or doxorubicin derivatives). Exemplary
podophyllotoxin derivatives include, for example, a compound having
a structure according to Formula (I):
##STR00025##
or a stereoisomer or pharmaceutically acceptable salt thereof,
where
[0128] each R.sub.1, R.sub.2, and R.sub.3 is selected,
independently, from H, optionally substituted C.sub.1-6 alkyl,
C(O)R.sub.8 (e.g., C(O)CH.sub.2N(CH.sub.3).sub.2),
P(O)(OR.sub.9)(OR.sub.10), S(O).sub.2(OR.sub.9), or a hydrolyzable
linker Y that includes a covalent bond to an amino acid of the
polypeptide;
[0129] X is O or NR.sub.7;
[0130] each R.sub.4, R.sub.5, and R.sub.7 is selected,
independently, from H, optionally substituted C.sub.1-6 alkyl,
C(O)R.sub.8, or a hydrolyzable linker Y that includes a covalent
bond to an amino acid of the polypeptide;
[0131] R.sub.6 is H, optionally substituted C.sub.1-6 alkyl,
optionally substituted aryl, optionally substituted heteroaryl,
[0132] R.sub.8 is selected from optionally substituted C.sub.1-6
alkyl (e.g., CH.sub.2N(CH.sub.3).sub.2) or optionally substituted
aryl;
[0133] each R.sub.9 and R.sub.10 is selected, independently, from
H, optionally substituted C.sub.1-6 alkyl, or optionally
substituted aryl; and
[0134] n is 2, 3, or 4; and
where one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and
R.sub.7 is Y. In some embodiments, no more than one of R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.7 is Y. In some
embodiments, Y is --C(O)(CH.sub.2).sub.nC(O)--. In some
embodiments, each R.sub.2 is H or C(O)CH.sub.2N(CH.sub.3).sub.2. In
certain embodiments, the polypeptide may have at least 35%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, 99%, or even 100% identity to a
polypeptide described herein. The polypeptide may have one or more
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15)
substitutions relative to one of the sequences described herein. In
certain embodiments, the amino acid sequence is covalently bonded
to additional podophyllotoxin derivatives (e.g., a compound of
Formula (I)) through a second, third, fourth, fifth, or even sixth
amino acid of said amino acid sequence and at any position of the
amino acid sequence.
[0135] Exemplary compounds of the invention include, but are not
limited to, those having a polypeptide sequence according to (SEQ
ID NO:97). In some embodiments, the compounds have the following
structure:
##STR00026##
wherein each (-(Formula(I)) represents a covalent bond between the
indicated amino acid and a compound of Formula (I). In certain
embodiments, the compounds of Formula (I) have the following
structure:
##STR00027##
where n is 1, 2, or 3, R.sub.6 is CH.sub.3 or 2-thienyl, and
R.sub.2 is H, --OP(O)(OH).sub.2, or
--C(O)CH.sub.2N(CH.sub.3).sub.2, or any pharmaceutically acceptable
salts thereof.
[0136] In some embodiments, n is 3, R.sub.6 is CH.sub.3, and
R.sub.2 is H. In other embodiments, n is 3, R.sub.6 is CH.sub.3,
and R.sub.2 is --C(O)CH.sub.2N(CH.sub.3).sub.2.
[0137] Other embodiments are described in greater detail below.
Podophyllotoxin Derivatives
[0138] Podophyllotoxin derivatives include compounds such as those
described by Formula (I), e.g., etoposide, teniposide, and
derivatives thereof, or a pharmaceutically acceptable salt thereof.
Podophyllotoxin derivatives are exemplary therapeutic agents and
can be covalently bonded to an amino acid in any of the
polypeptides described herein (e.g., Angiopep-2). These compounds
can have, for example, antineoplastic activity, inhibit the
activity of topoisomerase II, or have antiviral activity.
[0139] Etoposide and Etoposide Derivatives
[0140] Etoposide (also known as Toposar, Vepesid, or VP16) is a
podophyllotoxin derivative having the following structure
##STR00028##
The chemical structure of etoposide can be varied to afford
etoposide derivatives. An exemplary derivative of etoposide is
etoposide phosphate (ETOPOPHOS.RTM.), where the phenolic --OH is
replaced with --OP(O)(OH).sub.2, or any pharmaceutically acceptable
salt thereof (e.g., --OP(O)(ONa).sub.2). Etoposide phosphate has
improved water solubility compared to etoposide.
[0141] Other etoposide derivatives include those where the phenolic
--OH is replaced with an acyloxy group (e.g., --OC(O)R.sub.8, as
described herein) such as the following compound:
##STR00029##
("etoposide 4'-dimethylglycine" or "etoposide.sub.DMG"). These
acylated etoposide derivatives can also show improved water
solubility relative to etoposide when covalently attached to any of
the polypeptides described herein.
[0142] Etoposide, etoposide phosphate, etoposide.sub.DMG, or
derivatives thereof, can be covalently attached to an amino acid in
a polypeptide by attaching a hydrolyzable covalent linker Y to, for
example, the 2'' hydroxyl or the 3'' hydroxyl of the molecule.
Exemplary linkers may be derived, for example, from dicarboxylic
acids such as succinic, glutaric, and butaric acids, or any
anhydrides thereof. Additionally, a covalent linker can be attached
to etoposide, or derivatives thereof, at the phenol --OH group.
[0143] Etoposide derivatives can be described generally by the
following formula:
##STR00030##
(I-A), or any stereoisomer thereof, wherein
[0144] each R.sub.1, R.sub.2, and R.sub.3 is selected,
independently, from H, optionally substituted C.sub.1-6 alkyl,
C(O)R.sub.8, P(O)(OR.sub.9)(OR.sub.10), or
S(O).sub.2(OR.sub.9);
[0145] X is O or NR.sub.7;
[0146] each R.sub.4, R.sub.5, and R.sub.7 is selected,
independently, from H, optionally substituted C.sub.1-6 alkyl, or
C(O)R.sub.8;
[0147] R.sub.6 is H, optionally substituted C.sub.1-6 alkyl,
optionally substituted aryl, optionally substituted heteroaryl,
[0148] R.sub.8 is selected from optionally substituted C.sub.1-6
alkyl or optionally substituted aryl; and
[0149] each R.sub.9 and R.sub.10 is selected, independently, from
H, optionally substituted C.sub.1-6 alkyl, or optionally
substituted aryl.
[0150] When the compounds of the invention includes an etoposide
derivative according to Formula (I), one of R.sub.1-R.sub.6
includes a hydrolyzable linker Y as described herein. In some
embodiments, Y is --C(O)(CH.sub.2).sub.nC(O)-- and n is 2, 3, or 4.
In exemplary, non-limiting embodiments where R.sub.2 is
C(O)R.sub.8, R.sub.8 can be C.sub.1-6 alkyl including an amino
substituent and having optional additional substituents. In some
embodiments C(O)R.sub.8 is a C-linked .alpha.-amino acid. The
C-linked .alpha.-amino acid can be a natural or an unnatural amino
acid.
[0151] Other exemplary podophyllotoxin derivatives of Formula (I)
that can be covalently attached to any of the polypeptides
described herein include teniposide and NK611(Scheme 3).
##STR00031##
[0152] Additional Podophyllotoxin Derivatives
[0153] Still other podophyllotoxin derivatives suitable for use in
the invention are described in U.S. Pat. Nos. 4,567,253; 4,609,644;
4,900,814; 4,958,010; 5,489,698; 5,536,847; 5,571,914; 6,051,721;
6,107,284; 6,475,486; 6,610,299; 6,878,746; 6,894,075; 7,087,641;
7,176,236; 7,241,595; 7,342,114; and 7,378,419; and in U.S. Patent
Publication Nos. 20030064482, 20030162722, 20040044058,
20060148728, and 20070249651, each of which is hereby incorporated
by reference.
[0154] For example, the etoposide derivatives described in U.S.
Pat. No. 7,176,236 can be covalently bonded to an amino acid in any
of the polypeptides described herein (e.g., Angiopep-2).
Accordingly, in one embodiment, the compounds of the invention
include a structure according to Formula (I)
##STR00032##
wherein
[0155] R.sub.2 and Y are as described for Formula (I);
[0156] X.sub.2 is --O--, --S--, --NH--, --CO--, --CH.dbd.N--, or
--CH.sub.2NH--;
[0157] X.sub.3 is OR.sub.2 or N(R.sub.2).sub.2;
[0158] Z.sub.1 is a covalent bond, --NHCO--, --CONH--, --OCO--, or
--COO--;
[0159] Z.sub.2 is a covalent --(CH.sub.2).sub.oR.sub.15, or
--(CH.sub.2).sub.o is incorporated into Z.sub.2 as a 5-8 membered
ring;
[0160] R.sub.14 is a covalent bond or optionally substituted alkyl,
alkenyl, or phenyl; and
[0161] R.sub.15 is substituted alkyl, substituted alkenyl, or
substituted aryl, wherein the substituted group includes at least
one amino group.
[0162] In some embodiments, X.sub.3 is --OH,
--OC(O)CH.sub.2NH.sub.2, --OC(O)CH.sub.2NHCH.sub.3, or
--OC(O)CH.sub.2N(CH.sub.3).sub.2. In other embodiments, X is
--NH--. In some embodiments, --R.sub.14--Z.sub.1--Z.sub.2-- is
-(p-C.sub.6H.sub.4--R.sub.16)--, where R.sub.16 is --NO.sub.2, --F,
--CONHCH.sub.2CH.sub.2C.sub.6H.sub.5, or
--CONHCH.sub.2CH.sub.2(p-C.sub.6H.sub.4OH).
[0163] In any compounds of Formulas (I) or (I-A), the group
OR.sub.2 may be --OC(O)R.sub.8.
[0164] In some embodiments, the compound of Formula (I), or a
pharmaceutically acceptable salt thereof, that is used can allow
for improved physicochemical (e.g., solubility properties). For
example, when increased solubility is desired, the compound of
Formula (I) is preferably Etoposide.sub.DMG.
Doxorubicin Derivatives
[0165] In some embodiments, the anti-cancer agent is doxorubicin
(hydroxydaunorubicin or Adriamycin.RTM.) or a doxorubicin
derivative such as epirubicin (Ellence.RTM. or Pharmorubicin.RTM.),
or a pharmaceutically acceptable salt thereof. The structures of
these exemplary compounds are shown in Scheme 4. Doxorubicin and
doxorubicin derivatives can be covalently attached to an amino acid
in any of the polypeptides described herein through a hydrolyzable
covalent linker Y, as defined herein, covalently bonded to, for
example, the 14-hydroxyl group.
##STR00033##
[0166] Doxorubicin derivatives can be described generally by the
following Formula (II):
##STR00034##
wherein
[0167] each X.sub.1, X.sub.2, X.sub.3, X.sub.4, and X.sub.5 is
selected, independently, from a covalent bond, O, or NR.sub.25;
[0168] each R.sub.17, R.sub.18, R.sub.19, R.sub.20, R.sub.20,
R.sub.21, R.sub.22, R.sub.23, R.sub.24, and R.sub.25, is selected,
independently, from H, optionally substituted C.sub.1-6 alkyl,
optionally substituted C.sub.2-6 alkenyl, optionally substituted
C.sub.2-6 alkynyl, optionally substituted cycloalkyl, optionally
substituted heterocyclyl, or is a hydrolyzable linker Y as defined
herein.
When a compound of Formula (II) is attached to any of the
polypeptides described herein, one of R.sub.17, R.sub.18, R.sub.19,
R.sub.20, R.sub.20, R.sub.21, R.sub.22, R.sub.23, R.sub.24, and
R.sub.25 is Y. In certain embodiments, R.sub.21 is Y. Compounds of
Formula (II) include compounds having a structure according to
Formula (II-A)
##STR00035##
wherein Y is a hydrolyzable linker as described herein;
X.sub.2R.sub.18 is H or NH.sub.2; X.sub.3R.sub.19 is H or OH; and
X.sub.4R.sub.20 is H or optionally substituted C.sub.1-3 alkyl. In
some embodiments, the hydrolyzable linker Y is
--C(O)(CH.sub.2).sub.nC(O)-- and n is 2, 3, or 4. In certain
embodiments, the compound of Formula (II) is:
##STR00036##
[0169] Other doxorubicin derivatives can be found in U.S. Pat. Nos.
4,098,884, 4,301,277, 4,314,054, 4,464,529, 4,585,859, 4,672,057,
4,684,629, 4,826,964, 5,200,513, 5,304,687, 5,594,158, 5,625,043,
and 5,874,412, each of which is hereby incorporated by
reference.
[0170] In some embodiments, the compound of Formula (II), or a
pharmaceutically acceptable salt thereof, that is used can allow
for improved physicochemical (e.g., solubility properties). For
example, when increased solubility is desired, the compound of
Formula (II) is preferably the hydrochloride salt of
doxorubicin.
[0171] In addition to Angiopep-2, podophyllotoxin derivatives such
as etoposide, etoposide phosphate, etoposide.sub.DMG, teniposide,
NK611, and other compounds of Formulas (I) and (I-A), or
doxorubicin, epirubicin, and other doxorubicin derivatives (e.g.,
compounds of Formula (II)) can also be conjugated to any of the
polypeptides described herein (e.g., Angiopep-4b, Angiopep-5,
Angiopep-6, or Angiopep-7). Hydrolysable linkers, such as linkers
that include ester groups, can be used to covalently bind the
anticancer agent (e.g., podophyllotoxin derivatives or doxorubicin
derivatives) to a polypeptide (e.g., Example 1 described herein).
Etoposide, etoposide phosphate, etoposide.sub.DMG, other
podophyllotoxin derivatives thereof, doxorubicin, epirubicin, and
other doxorubicin derivatives have multiple strategic positions
(e.g., the 2'' and 3'' hydroxyls of etoposide, etoposide phosphate,
and etoposide.sub.DMG, and the 14 hydroxyl of doxorubicin and
epirubicin). For example, a difunctional group (e.g., a reagent
derived from succinic acid, glutaric acid, glutaric anhydride, or
butaric acid, or any anhydrides thereof) can be attached to
etoposide at the 2'' hydroxyl or to doxorubicin at the 14 hydroxyl.
These exemplary intermediates can then be activated with a
peptide-coupling reagent such as BTTU and treated with a
polypeptide. Other peptide coupling agents include carbodiimides
(e.g., dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide
(DIC), and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride) (EDC-HCl)), triazoles (e.g., 1-hydroxy-benzotriazole
(HOBt) and 1-hydroxy-7-aza-benzotriazole (HOAt)), related
benzotriazole peptide coupling agents such as
O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate
(HBTU),
2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HCTU),
2-(1H-9-Azobenzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HATU),
Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorophosphate (BOP reagent), and
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
(PyBOP), and 3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one
(DEPBT). The conjugate can then be purified. Each intermediate or
product of this synthetic procedure is purified and validated using
different approaches such as HPLC, thin liquid chromatography, NMR
(.sup.13C or .sup.1H exchange), melting point, mass spectrometry.
The final conjugate is analyzed by mass spectrometry and
SDS-polyacrylamide gel electrophoresis. This allows the
determination of the number of molecules (e.g., of etoposide,
etoposide phosphate, etoposide.sub.DMG, doxorubicin, or epirubicin)
conjugated to each vector.
Hydrolyzable Linkers
[0172] When a compound of Formula (I) is covalently attached by a
hydrolyzable linker Y to an amino acid in a polypeptide, the linker
can be located at R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, or
R.sub.7. Similarly, when a compound of Formula (II) is covalently
attached by a hydrolyzable linker Y to an amino acid in a
polypeptide, the linker can be located at any of R.sub.17,
R.sub.18, R.sub.19, R.sub.20, R.sub.20, R.sub.21, R.sub.22,
R.sub.23, R.sub.24, and R.sub.25. Exemplary, non-limiting
hydrolyzable linkers may be prepared from dicarboxylic acids,
dicarbonates, carboxylic anhydrides, diisocyanates, or diphosphonic
acids. A compound that includes a compound of Formula (I) or (II)
that is covalently attached to an amino acid in any of the amino
acid sequences described herein may also be described by the
following formula
D-G-X-G'-A (III),
[0173] where each G and G' is a group selected, independently, from
--C(O)--, --C(O)O--, --OC(O)--, --S(O).sub.2O--, --OS(O).sub.2--,
--S(O).sub.2NH--, --NHS(O).sub.2--, and --OP(O)(OR.sub.11)O--;
[0174] G is covalently bonded to D, where D is a podophyllotoxin
derivative (e.g., a compound of Formula (I)) or doxorubicin or a
doxorubicin derivative (e.g., a compound of Formula (II);
[0175] G' is covalently bonded to A, where A is an amino acid in an
amino acid sequence described herein (e.g., the amino acid
sequences described in Table 1, or functional derivatives thereof);
and
[0176] X is -(optionally substituted aryl)-,
--(CR.sub.12R.sub.13).sub.n--,
--O{(CR.sub.12R.sub.13).sub.2O}.sub.n--,
--{(CR.sub.12R.sub.13).sub.2O(CR.sub.12R.sub.13).sub.2}.sub.n--, or
--(CR.sub.12R.sub.13).sub.oY(CR.sub.12R.sub.13).sub.p--, where each
n, o, and p is, independently, an integer between 1-10;
[0177] R.sub.11 is H or lower C.sub.1-6 alkyl;
[0178] R.sub.12 and R.sub.13 are each selected, independently, from
H, OH, or lower C.sub.1-6 alkyl; and
[0179] Y is O, NH, N(lower C.sub.1-6 alkyl), or -optionally
substituted aryl.
[0180] Each n, o, and p may be, independently, 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10.
[0181] In some embodiments, the G-X-G' moiety in Formula (III) is
selected from --C(O)CH.sub.2C(O)--, --C(O)(CH.sub.2).sub.2C(O)--,
--C(O)(CH.sub.2).sub.3C(O)--, --C(O)(CH.sub.2).sub.4C(O)--,
--C(O)(CH.sub.2).sub.5C(O)--, --C(O)(CH.sub.2).sub.6C(O)--,
--C(O)(OCH.sub.2CH.sub.2)OC(O)--,
--C(O)(OCH.sub.2CH.sub.2).sub.2OC(O)--,
--C(O)(OCH.sub.2CH.sub.2).sub.3OC(O)--, and
--C(O)(OCH.sub.2CH.sub.2).sub.4OC(O)--.
Polypeptides
[0182] Exemplary amino acid sequences useful in the compounds of
the invention include, but are not limited to, the amino acid
sequences described in Table 1.
[0183] In addition to the amino acid sequences described in Table
1, the invention also features fragments of these amino acid
sequences (e.g., a functional fragment). In certain embodiments,
the fragments are capable of entering or accumulating in a
particular cell type (e.g., ovary, liver, lung, kidney, spleen, or
muscle) or capable of crossing the BBB. Truncations of the
polypeptide may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more
amino acids from either the N-terminus of the polypeptide, the
C-terminus of the polypeptide, or a combination thereof. Other
fragments include sequences where internal portions of the
polypeptide are deleted.
[0184] Additional polypeptides of the invention may be identified
by using one of the assays or methods described in U.S. Patent
Application Publication No. 2006/0189515, which is hereby
incorporated by reference, or by any method known in the art. For
example, a candidate vector may be produced by conventional
polypeptide synthesis and conjugated with, for example, a compound
of Formulas (I) or (II), and administered to a laboratory animal. A
biologically active vector may be identified, for example, based on
its efficacy to increase survival of an animal injected with tumor
cells and treated with the conjugate as compared to a control which
has not been treated with a conjugate (e.g., treated with the
unconjugated agent).
[0185] In another example, a biologically active polypeptide of the
invention may be identified based on its location in the parenchyma
in an in situ cerebral perfusion assay. In vitro BBB assays, such
as the model developed by CELLIAL.TM. Technologies, may be used to
identify such vectors.
[0186] Assays to determine accumulation in other tissues may be
performed as well and exemplary assays are described herein.
Labeled polypeptides of the invention can be administered to an
animal, and accumulation in different organs can be measured. For
example, a polypeptide conjugated to a detectable label (e.g., a
near-IR fluorescence spectroscopy label such as Cy5.5) allows live
in vivo visualization. Such a polypeptide can be administered to an
animal, and the presence of the polypeptide in an organ can be
detected, thus allowing determination of the rate and amount of
accumulation of the polypeptide in the desired organ. In other
embodiments, the polypeptide of the invention can be labeled with a
radioactive isotope (e.g., .sup.125I). The polypeptide is then
administered to an animal. After a period of time, the animal is
sacrificed, and the animal's organs are extracted. The amount of
radioisotope in each organ can then measured using any means known
in the art. By comparing the amount of a labeled candidate
polypeptide in a particular organ without amount of labeled
control, the ability of the candidate polypeptide the rate or
amount of accumulation of a candidate polypeptide in a particular
tissue can be ascertained. Appropriate negative controls include
any polypeptide known not be transported into a particular cell
type.
[0187] For example, the amine groups of Angiopep-1 (SEQ ID NO:67)
and Angiopep-2 (SEQ ID NO:97) can be used as sites for conjugation
of agents. To study the role of amine groups in conjugation and
their impact in the overall transport capacity of these vectors,
other vectors have been developed based on the Angiopep-1 and
Angiopep-2 sequence. These vectors variable reactive amine groups
and variable overall charge. These polypeptides are shown in Table
2.
TABLE-US-00002 TABLE 2 Vectors with variable amine group targets
Reactive Polypeptide amines SEQ ID Name Polypeptide Sequences
(positions) Charge No. Angiopep-3* Ac.sup.1-TFFYGGSRGKRNNFKTEEY 2
(10,15) +1 107 Angiopep-4b RFFYGGSRGKRNNFKTEEY 3 (1, 10, 15) +3 108
Angiopep-4a Ac.sup.1-RFFYGGSRGKRNNFKTEEY 2 (10, 15) +2 109
Angiopep-5 Ac.sup.1-RFFYGGSRGKRNNFRTEEY 1 (10) +2 110 Angiopep-6
TFFYGGSRGKRNNFRTEEY 2 (1, 10) +2 111 Angiopep-7 TFFYGGSRGRRNNFRTEEY
1 (1) +2 112 *Angiopep-3 is an acetylated form of Angiopep-2.
.sup.1Ac represents acetylation.
Modified Polypeptides
[0188] The invention can also include polypeptides having a
modification of an amino acid sequence described herein (e.g.,
polypeptide having a sequence described in any one of SEQ ID
NOS:1-105 and 107-116 such as AngioPep-3, -4a, -4b, -5, -6, or -7)
and in which the polypeptide includes an amino acid that is
covalently bonded to a compound of Formulas (I) or (II). In certain
embodiments, the modification does not destroy significantly a
desired biological activity. In some embodiments, the modification
may cause a reduction in biological activity (e.g., by at least 5%,
10%, 20%, 25%, 35%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%). In other
embodiments, the modification has no effect on the biological
activity or may increase (e.g., by at least 5%, 10%, 25%, 50%,
100%, 200%, 500%, or 1000%) the biological activity of the original
polypeptide. The modified polypeptide may have or may optimize one
or more of the characteristics of a polypeptide of the invention
which, in some instance might be needed or desirable. Such
characteristics include in vivo stability, bioavailability,
toxicity, immunological activity, or immunological identity.
[0189] Polypeptides of the invention may include amino acids or
sequences modified either by natural processes, such as
posttranslational processing, or by chemical modification
techniques known in the art. Modifications may occur anywhere in a
polypeptide including the polypeptide backbone, the amino acid
side-chains and the amino- or carboxy-terminus. The same type of
modification may be present in the same or varying degrees at
several sites in a given polypeptide, and a polypeptide may contain
more than one type of modification. Polypeptides may be branched as
a result of ubiquitination, and they may be cyclic, with or without
branching. Cyclic, branched, and branched cyclic polypeptides may
result from posttranslational natural processes or may be made
synthetically. Other modifications include pegylation, acetylation,
acylation, addition of acetomidomethyl (Acm) group,
ADP-ribosylation, alkylation, amidation, biotinylation,
carbamoylation, carboxyethylation, esterification, covalent
attachment to fiavin, covalent attachment to a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of drug, covalent attachment of a marker (e.g.,
fluorescent or radioactive), covalent attachment of a lipid or
lipid derivative, covalent attachment of phosphatidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent crosslinks, formation of
cystine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation and ubiquitination.
[0190] A modified polypeptide of the invention may further include
an amino acid insertion, deletion, or substitution, either
conservative or non-conservative (e.g., D-amino acids, desamino
acids) in the polypeptide sequence (e.g., where such changes do not
substantially alter the biological activity of the
polypeptide).
[0191] For example, in some embodiments, the amino acid sequence
(e.g., SEQ ID NOS 1-105 or 107-116) is modified by inserting one or
more additional cysteine residues at the N-terminal of the peptide,
the C-terminal of the peptide, or both. The addition of one or more
cysteine residues to the amino or carboxy terminus of any of the
amino acid sequences described herein can facilitate conjugation of
these polypeptides to nucleic acids (e.g., siRNA molecules) or
lipid vectors by, e.g., disulfide bonding. For example, Angiopep-1
(SEQ ID NO:67), Angiopep-2 (SEQ ID NO:97), or Angiopep-7 (SEQ ID
NO:112) can be modified to include a single cysteine residue at the
amino-terminus (SEQ ID NOS: 71, 113, and 115, respectively) or a
single cysteine residue a the carboxy-terminus (SEQ ID NOS: 72,
114, and 116, respectively).
[0192] Substitutions may be conservative (i.e., wherein a residue
is replaced by another of the same general type or group) or
non-conservative (i.e., wherein a residue is replaced by an amino
acid of another type). In addition, a non-naturally occurring amino
acid may be substituted for a naturally occurring amino acid (i.e.,
non-naturally occurring conservative amino acid substitution or a
non-naturally occurring non-conservative amino acid
substitution).
[0193] Polypeptides made synthetically may include substitutions of
amino acids not naturally encoded by DNA (e.g., non-naturally
occurring or unnatural amino acid). Examples of non-naturally
occurring amino acids include D-amino acids, an amino acid having
an acetylaminomethyl group attached to a sulfur atom of a cysteine,
a pegylated amino acid, the omega amino acids of the formula
NH.sub.2(CH.sub.2).sub.nCOOH wherein n is 2-6, neutral nonpolar
amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine,
N-methyl isoleucine, and norleucine. Phenylglycine may substitute
for Trp, Tyr, or Phe; citrulline and methionine sulfoxide are
neutral nonpolar, cysteic acid is acidic, and ornithine is basic.
Proline may be substituted with hydroxyproline and retain the
conformation conferring properties.
[0194] Analogues may be generated by substitutional mutagenesis and
retain the biological activity of the original polypeptide.
Examples of substitutions identified as "conservative
substitutions" are shown in Table 3. If such substitutions result
in a change not desired, then other type of substitutions,
denominated "exemplary substitutions" in Table 3, or as further
described herein in reference to amino acid classes, are introduced
and the products screened.
[0195] Substantial modifications in function or immunological
identity are accomplished by selecting substitutions that differ
significantly in their effect on maintaining (a) the structure of
the polypeptide backbone in the area of the substitution, for
example, as a sheet or helical conformation. (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk
of the side chain. Naturally occurring residues are divided into
groups based on common side chain properties:
[0196] (1) hydrophobic: norleucine, methionine (Met), Alanine
(Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile), Histidine
(His), Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe),
[0197] (2) neutral hydrophilic: Cysteine (Cys), Serine (Ser),
Threonine (Thr)
[0198] (3) acidic/negatively charged: Aspartic acid (Asp), Glutamic
acid (Glu)
[0199] (4) basic: Asparagine (Asn), Glutamine (Gln), Histidine
(His), Lysine (Lys), Arginine (Arg)
[0200] (5) residues that influence chain orientation: Glycine
(Gly), Proline (Pro);
[0201] (6) aromatic: Tryptophan (Trp), Tyrosine (Tyr),
Phenylalanine (Phe), Histidine (His),
[0202] (7) polar: Ser, Thr, Asn, Gln
[0203] (8) basic positively charged: Arg, Lys, His, and;
[0204] (9) charged: Asp, Glu, Arg, Lys, His
[0205] Other conservative amino acid substitutions are listed in
Table 3.
TABLE-US-00003 TABLE 3 Original residue Exemplary substitution
Conservative substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys,
Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C)
Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H)
Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu
norleucine Leu (L) Norleucine, Ile, Val, Met, Ile Ala, Phe Lys (K)
Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile,
Ala Leu Pro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr
Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala,
Leu norleucine
Additional Polypeptide Analogues
[0206] The compounds of the invention may include polypeptide
analogs of aprotinin known in the art where the analogs include an
amino acid that is covalently bonded to a podophyllotoxin
derivative (e.g., a compound of Formula (I)) or to doxorubicin or a
doxorubicin derivative (e.g., a compound of Formula (II)). For
example, U.S. Pat. No. 5,807,980 describes Bovine Pancreatic
Trypsin Inhibitor (aprotinin)-derived inhibitors as well as a
method for their preparation and therapeutic use, including the
polypeptide of SEQ ID NO:102. These polypeptides have been used for
the treatment of a condition characterized by an abnormal
appearance or amount of tissue factor and/or factor VIIIa such as
abnormal thrombosis. U.S. Pat. No. 5,780,265 describes serine
protease inhibitors capable of inhibiting plasma kallikrein,
including SEQ ID NO:103. U.S. Pat. No. 5,118,668 describes Bovine
Pancreatic Trypsin Inhibitor variants, including SEQ ID NO:105. The
aprotinin amino acid sequence (SEQ ID NO:98), the Angiopep-1 amino
acid sequence (SEQ ID NO:67), and SEQ ID NO:104, as well as some
sequences of biologically active analogs may be found in
International Application Publication No. WO 2004/060403.
[0207] An exemplary nucleotide sequence encoding an aprotinin
analogue is illustrated in SEQ ID NO:106 (atgagaccag atttctgcct
cgagccgccg tacactgggc cctgcaaagc tcgtatcatc cgttacttct acaatgcaaa
ggcaggcctg tgtcagacct tcgtatacgg cggctgcaga gctaagcgta acaacttcaa
atccgcggaa gactgcatgc gtacttgcgg tggtgcttag; Genbank accession No.
X04666). This sequence encodes a lysine at position 16 instead of a
valine, as found in SEQ ID NO:98. A mutation in the nucleotide
sequence of SEQ ID NO:106 may be introduced by methods known in the
art to change the produce the polypeptide of SEQ ID NO:98 having a
valine in position 16. Additional mutations or fragments may be
obtained using any technique known in the art.
[0208] Other examples of aprotinin analogs may be found by
performing a protein BLAST (Genebank: www.ncbi.nlm.nih.gov/BLAST/)
using the synthetic aprotinin sequence (or portion thereof)
disclosed in International Application No. PCT/CA2004/000011.
Exemplary aprotinin analogs are found under accession Nos. CAA37967
(GI:58005) and 1405218C (GI:3604747).
Preparation of Polypeptide Derivatives and Peptidomimetics
[0209] In addition to polypeptides consisting only of naturally
occurring amino acids, peptidomimetics or polypeptide analogs are
also encompassed by the present invention. Polypeptide analogs are
commonly used in the pharmaceutical industry as non-polypeptide
drugs with properties analogous to those of the template
polypeptide. The non-polypeptide compounds are termed "polypeptide
mimetics" or peptidomimetics (Fauchere et al., Infect. Immun.
54:283-287, 1986; Evans et al., J. Med. Chem. 30:1229-1239, 1987).
Polypeptide mimetics that are structurally related to
therapeutically useful polypeptides may be used to produce an
equivalent or enhanced therapeutic or prophylactic effect.
Generally, peptidomimetics are structurally similar to the paradigm
polypeptide (i.e., a polypeptide that has a biological or
pharmacological activity) such as naturally-occurring
receptor-binding polypeptides, but have one or more peptide
linkages optionally replaced by linkages such as --CH.sub.2NH--,
--CH.sub.2S--, --CH.sub.2--CH.sub.2--, --CH.dbd.CH--(cis and
trans), --CH.sub.2SO--, --CH(OH)CH.sub.2--, --COCH.sub.2-- etc., by
methods well known in the art (Spatola, Peptide Backbone
Modifications, Vega Data, 1(3):267, 1983); Spatola et al. (Life
Sci. 38:1243-1249, 1986); Hudson et al. (Int. J. Pept. Res.
14:177-185, 1979); and Weinstein. B., 1983, Chemistry and
Biochemistry, of Amino Acids, Peptides and Proteins, Weinstein eds,
Marcel Dekker, New York). Such polypeptide mimetics may have
significant advantages over naturally-occurring polypeptides
including more economical production, greater chemical stability,
enhanced pharmacological properties (e.g., half-life, absorption,
potency, efficiency), reduced antigenicity and others.
[0210] While the polypeptides of the invention may be effective in
entering particular cell types (e.g., those described herein),
their effectiveness may be reduced by the presence of proteases.
Serum proteases have specific substrate requirements. The substrate
must have both L-amino acids and peptide bonds for cleavage.
Furthermore, exopeptidases, which represent the most prominent
component of the protease activity in serum, usually act on the
first peptide bond of the polypeptide and require a free N-terminus
(Powell et al., Pharm. Res. 10:1268-1273, 1993). In light of this,
it is often advantageous to use modified versions of polypeptides.
The modified polypeptides retain the structural characteristics of
the original L-amino acid polypeptides that confer biological
activity with regard to IGF-1, but are advantageously not readily
susceptible to cleavage by protease and/or exopeptidases.
[0211] Systematic substitution of one or more amino acids of a
consensus sequence with D-amino acid of the same type (e.g.,
D-lysine in place of L-lysine) may be used to generate more stable
polypeptides. Thus, a polypeptide derivative or peptidomimetic of
the present invention may be all L, all D or mixed D, L
polypeptide. The presence of an N-terminal or C-terminal D-amino
acid increases the in vivo stability of a polypeptide because
peptidases cannot utilize a D-amino acid as a substrate (Powell et
al., Pharm. Res. 10:1268-1273, 1993). Reverse-D polypeptides are
polypeptides containing D-amino acids, arranged in a reverse
sequence relative to a polypeptide containing L-amino acids. Thus,
the C-terminal residue of an L-amino acid polypeptide becomes
N-terminal for the D-amino acid polypeptide, and so forth. Reverse
D-polypeptides retain the same tertiary conformation and therefore
the same activity, as the L-amino acid polypeptides, but are more
stable to enzymatic degradation in vitro and in vivo, and thus have
greater therapeutic efficacy than the original polypeptide (Brady
and Dodson, Nature 368:692-693, 1994; Jameson et al., Nature
368:744-746, 1994). In addition to reverse-D-polypeptides,
constrained polypeptides comprising a consensus sequence or a
substantially identical consensus sequence variation may be
generated by methods well known in the art (Rizo and Gierasch, Ann.
Rev. Biochem. 61:387-418, 1992). For example, constrained
polypeptides may be generated by adding cysteine residues capable
of forming disulfide bridges and, thereby, resulting in a cyclic
polypeptide. Cyclic polypeptides have no free N- or C-termini.
Accordingly, they are not susceptible to proteolysis by
exopeptidases, although they are, of course, susceptible to
endopeptidases, which do not cleave at peptide termini. The amino
acid sequences of the polypeptides with N-terminal or C-terminal
D-amino acids and of the cyclic polypeptides are usually identical
to the sequences of the polypeptides to which they correspond,
except for the presence of N-terminal or C-terminal D-amino acid
residue, or their circular structure, respectively.
[0212] A cyclic derivative containing an intramolecular disulfide
bond may be prepared by conventional solid phase synthesis while
incorporating suitable S-protected cysteine or homocysteine
residues at the positions selected for cyclization such as the
amino and carboxy termini (Sah et al., J. Pharm. Pharmacol. 48:197,
1996). Following completion of the chain assembly, cyclization can
be performed either (1) by selective removal of the S-protecting
group with a consequent on-support oxidation of the corresponding
two free SH-functions, to form a S--S bonds, followed by
conventional removal of the product from the support and
appropriate purification procedure or (2) by removal of the
polypeptide from the support along with complete side chain
de-protection, followed by oxidation of the free SH-functions in
highly dilute aqueous solution.
[0213] The cyclic derivative containing an intramolecular amide
bond may be prepared by conventional solid phase synthesis while
incorporating suitable amino and carboxyl side chain protected
amino acid derivatives, at the position selected for cyclization.
The cyclic derivatives containing intramolecular --S-alkyl bonds
can be prepared by conventional solid phase chemistry while
incorporating an amino acid residue with a suitable amino-protected
side chain, and a suitable S-protected cysteine or homocysteine
residue at the position selected for cyclization.
[0214] Another effective approach to confer resistance to
peptidases acting on the N-terminal or C-terminal residues of a
polypeptide is to add chemical groups at the polypeptide termini,
such that the modified polypeptide is no longer a substrate for the
peptidase. One such chemical modification is glycosylation of the
polypeptides at either or both termini. Certain chemical
modifications, in particular N-terminal glycosylation, have been
shown to increase the stability of polypeptides in human serum
(Powell et al., Pharm. Res. 10:1268-1273, 1993). Other chemical
modifications which enhance serum stability include, but are not
limited to, the addition of an N-terminal alkyl group, consisting
of a lower alkyl of from one to twenty carbons, such as an acetyl
group, and/or the addition of a C-terminal amide or substituted
amide group. In particular, the present invention includes modified
polypeptides consisting of polypeptides bearing an N-terminal
acetyl group and/or a C-terminal amide group.
[0215] Also included by the present invention are other types of
polypeptide derivatives containing additional chemical moieties not
normally part of the polypeptide, provided that the derivative
retains the desired functional activity of the polypeptide.
Examples of such derivatives include (1) N-acyl derivatives of the
amino terminal or of another free amino group, wherein the acyl
group may be an alkanoyl group (e.g., acetyl, hexanoyl, octanoyl)
an aroyl group (e.g., benzoyl) or a blocking group such as F-moc
(fluorenylmethyl-O|CO|); (2) esters of the carboxy terminal or of
another free carboxy or hydroxyl group; (3) amide of the
carboxy-terminal or of another free carboxyl group produced by
reaction with ammonia or with a suitable amine; (4) phosphorylated
derivatives; (5) derivatives conjugated to an antibody or other
biological ligand and other types of derivatives.
[0216] Longer polypeptide sequences which result from the addition
of additional amino acid residues to the polypeptides of the
invention are also encompassed in the present invention. Such
longer polypeptide sequences would be expected to have the same
biological activity (e.g., entering particular cell types) as the
polypeptides described above. While polypeptides having a
substantial number of additional amino acids are not excluded, it
is recognized that some large polypeptides may assume a
configuration that masks the effective sequence, thereby preventing
binding to a target (e.g., a member of the LRP receptor family such
as LRP or LRP2). These derivatives could act as competitive
antagonists. Thus, while the present invention encompasses
polypeptides or derivatives of the polypeptides described herein
having an extension, desirably the extension does not destroy the
cell targeting activity of the polypeptide or derivative.
[0217] Other derivatives included in the present invention are dual
polypeptides consisting of two of the same, or two different
polypeptides of the present invention covalently linked to one
another either directly or through a spacer, such as by a short
stretch of alanine residues or by a putative site for proteolysis
(e.g., by cathepsin, see e.g., U.S. Pat. No. 5,126,249 and European
Patent No. 495 049). Multimers of the polypeptides of the present
invention consist of polymer of molecules formed from the same or
different polypeptides or derivatives thereof.
[0218] The present invention also features polypeptide derivatives
that are chimeric or fusion proteins containing a polypeptide
described herein, or fragment thereof, linked at its amino- or
carboxy-terminal end, or both, to an amino acid sequence of a
different protein. Such a chimeric or fusion protein may be
produced by recombinant expression of a nucleic acid encoding the
protein. For example, a chimeric or fusion protein may contain at
least 6 amino acids of a polypeptide of the present invention and
desirably has a functional activity equivalent or greater than a
polypeptide of the invention.
[0219] Polypeptide derivatives of the present invention can be made
by altering the amino acid sequences by substitution, addition, or
deletion or an amino acid residue to provide a functionally
equivalent molecule, or functionally enhanced or diminished
molecule, as desired. The derivative of the present invention
include, but are not limited to, those containing, as primary amino
acid sequence, all or part of the amino acid sequence of the
polypeptides described herein (e.g., any one of SEQ ID NOS:1-105
and 107-112) including altered sequences containing substitutions
of functionally equivalent amino acid residues. For example, one or
more amino acid residues within the sequence can be substituted by
another amino acid of a similar polarity that acts as a functional
equivalent, resulting in a silent alteration. Substitution for an
amino acid within the sequence may be selected from other members
of the class to which the amino acid belongs. For example, the
positively charged (basic) amino acids include, arginine, lysine
and histidine. The nonpolar (hydrophobic) amino acids include,
leucine, isoleucine, alanine, phenylalanine, valine, proline,
tryptophan and methionine. The uncharged polar amino acids include
serine, threonine, cysteine, tyrosine, asparagine and glutamine.
The negatively charged (acid) amino acids include glutamic acid and
aspartic acid. The amino acid glycine may be included in either the
nonpolar amino acid family or the uncharged (neutral) polar amino
acid family Substitutions made within a family of amino acids are
generally understood to be conservative substitutions.
[0220] Assays to Identify Peptidomimetics
[0221] As described above, non-peptidyl compounds generated to
replicate the backbone geometry and pharmacophore display
(peptidomimetics) of the polypeptides identified by the methods of
the present invention often possess attributes of greater metabolic
stability, higher potency, longer duration of action and better
bioavailability.
[0222] The peptidomimetics compounds of the present invention can
be obtained using any of the numerous approaches in combinatorial
library methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to polypeptide libraries, while the
other four approaches are applicable to polypeptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, Anticancer
Drug Des. 12:145, 1997). Examples of methods for the synthesis of
molecular libraries can be found in the art, for example, in:
DeWitt et al. (Proc. Natl. Acad. Sci. USA 90:6909, 1993); Erb et
al. (Proc. Natl. Acad. Sci. USA 91:11422, 1994); Zuckermann et al.,
J. Med. Chem. 37:2678, 1994); Cho et al. (Science 261:1303, 1993);
Carell et al. (Angew. Chem, Int. Ed. Engl. 33:2059, 1994 and ibid
2061); and in Gallop et al. (Med. Chem. 37:1233, 1994). Libraries
of compounds may be presented in solution (e.g., Houghten,
Biotechniques 13:412-421, 1992) or on beads (Lam, Nature 354:82-84,
1991), chips (Fodor, Nature 364:555-556, 1993), bacteria or spores
(U.S. Pat. No. 5,223,409), plasmids (Cull et al., Proc. Natl. Acad.
Sci. USA 89:1865-1869, 1992) or on phage (Scott and Smith, Science
249:386-390, 1990), or luciferase, and the enzymatic label detected
by determination of conversion of an appropriate substrate to
product.
[0223] Once a polypeptide of the present invention is identified,
it may be isolated and purified by any number of standard methods
including, but not limited to, differential solubility (e.g.,
precipitation), centrifugation, chromatography (e.g., affinity, ion
exchange, size exclusion, and the like) or by any other standard
techniques used for the purification of polypeptides,
peptidomimetics or proteins. The functional properties of an
identified polypeptide of interest may be evaluated using any
functional assay known in the art. Desirably, assays for evaluating
downstream receptor function in intracellular signaling are used
(e.g., cell proliferation).
[0224] For example, the peptidomimetics compounds of the present
invention may be obtained using the following three-phase process:
(1) scanning the polypeptides of the present invention to identify
regions of secondary structure necessary for targeting the
particular cell types described herein; (2) using conformationally
constrained dipeptide surrogates to refine the backbone geometry
and provide organic platforms corresponding to these surrogates;
and (3) using the best organic platforms to display organic
pharmacophores in libraries of candidates designed to mimic the
desired activity of the native polypeptide. In more detail the
three phases are as follows. In phase 1, the lead candidate
polypeptides are scanned and their structure abridged to identify
the requirements for their activity. A series of polypeptide
analogs of the original are synthesized. In phase 2, the best
polypeptide analogs are investigated using the conformationally
constrained dipeptide surrogates. Indolizidin-2-one,
indolizidin-9-one and quinolizidinone amino acids (I.sup.2aa,
I.sup.9aa and Qaa respectively) are used as platforms for studying
backbone geometry of the best polypeptide candidates. These and
related platforms (reviewed in Halab et al., Biopolymers
55:101-122, 2000; and Hanessian et al. Tetrahedron 53:12789-12854,
1997) may be introduced at specific regions of the polypeptide to
orient the pharmacophores in different directions. Biological
evaluation of these analogs identifies improved lead polypeptides
that mimic the geometric requirements for activity. In phase 3, the
platforms from the most active lead polypeptides are used to
display organic surrogates of the pharmacophores responsible for
activity of the native polypeptide. The pharmacophores and
scaffolds are combined in a parallel synthesis format. Derivation
of polypeptides and the above phases can be accomplished by other
means using methods known in the art.
[0225] Structure function relationships determined from the
polypeptides, polypeptide derivatives, peptidomimetics or other
small molecules of the present invention may be used to refine and
prepare analogous molecular structures having similar or better
properties. Accordingly, the compounds of the present invention
also include molecules that share the structure, polarity, charge
characteristics and side chain properties of the polypeptides
described herein.
[0226] In summary, based on the disclosure herein, those skilled in
the art can develop polypeptides and peptidomimetics screening
assays which are useful for identifying compounds for targeting an
agent to particular cell types (e.g., those described herein). The
assays of this invention may be developed for low-throughput,
high-throughput, or ultra-high throughput screening formats. Assays
of the present invention include assays that are amenable to
automation.
Polypeptide Conjugates Covalently Bonded to Additional Agents
[0227] The compounds described herein, or functional derivatives
thereof, in addition to including an amino acid sequence that is
covalently bonded through an amino acid to a podophyllotoxin
derivative (e.g., a compound having a structure according to
Formula (I)) or to doxorubicin or a doxorubicin derivative (e.g., a
compound of Formula (II)), may also include a covalent bond to
another agent (e.g., another therapeutic agent, a diagnostic agent,
or to a label). In certain embodiments, the amino acid sequence is
also linked to or labeled with a detectable label, such as a
radioimaging agent, for diagnosis of a disease or condition.
Examples of these agents include a radioimaging
agent-antibody-vector conjugate, where the antibody binds to a
disease or condition-specific antigen (e.g., for diagnosis or
therapy). Other binding molecules are also contemplated by the
invention. In other cases, the compound of the invention, or a
functional derivative thereof, is linked to another therapeutic
agent, to treat a disease or condition, or may be linked to or
labeled with mixtures thereof. The disease or condition may be
treated by administering a vector-agent conjugate to an individual
under conditions which allow transport of the agent across the BBB
or into a particular cell type. Each polypeptide may include at
least 1, 2, 3, 4, 5, 6, or 7 additional agents. In other
embodiments, each agent has at least 1, 2, 3, 4, 5, 6 7, 10, 15,
20, or more polypeptides attached thereto. The conjugates of the
invention may be able to promote accumulation (e.g., due to
increased uptake or reduced removal) of the agent in a particular
cell type or tissue such as the brain, ovary, liver, lung, kidney,
spleen or muscle of a subject.
[0228] An agent (e.g., a podophyllotoxin derivative such as a
compound of Formula (I) or doxorubicin or a doxorubicin derivative
(e.g., a compound of Formula (II)), another therapeutic agent, a
diagnostic agent, or a label) that has a covalent bond to an amino
acid in any of the amino acid sequences described herein (e.g.,
those listed in Table 1, or functional derivatives thereof) may be
releasable from the vector after transport into a particular cell
type or across the BBB. The agent can be released, for example, by
enzymatic cleavage or other breakage of a chemical bond between the
vector and the agent. The released agent may then function in its
intended capacity in the absence of the vector.
[0229] Other methods and cross-linkers can be used to conjoin the
polypeptides and RNAi agents of the invention. For example, a 5' or
3' thiol-containing siRNA sense strand can be linked by a disulfide
bond to a cysteine residue placed at either the amino or carboxy
terminus of the polypeptide. Muratovska et al. (FEBS Letters
558:63-68, 2004) and Turner et al. (Blood Cells, Molecules, and
Diseases 38:1-7, 2007) provide exemplary chemical bonding methods
for conjugating polypeptides to RNA molecules and are hereby
incorporated by reference.
Therapeutic Agents
[0230] A therapeutic agent may be any biologically active agent.
For example, a therapeutic may be a drug, a medicine, an agent
emitting radiation, a cellular toxin (for example, a
chemotherapeutic agent), a biologically active fragment thereof, or
a mixture thereof to treat a disease (e.g., to killing cancer
cells) or it may be an agent to treat a disease or condition in an
individual. Podophyllotoxin derivatives (e.g., the compounds of
Formula (I)) and doxorubicin and doxorubicin derivatives (e.g.,
compounds of Formula (II)) are exemplary useful classes of
therapeutic agents. A therapeutic agent may be a synthetic product
or a product of fungal, bacterial or other microorganism (e.g.,
mycoplasma or virus), animal, such as reptile, or plant origin. A
therapeutic agent and/or biologically active fragment thereof may
be an enzymatically active agent and/or fragment thereof, or may
act by inhibiting or blocking an important and/or essential
cellular pathway or by competing with an important and/or essential
naturally occurring cellular component. Other therapeutic agents
include antibodies and antibody fragments.
[0231] Any anticancer agent known in the art may be part of a
conjugate of the invention. Podophyllotoxin derivatives (e.g., the
compounds of Formula (I)) and doxorubicin and doxorubicin
derivatives (e.g., compounds of Formula (II)) can be anticancer
agents. Additional anticancer agents may also be conjugated to the
compounds of the invention as described herein. Cancers of the
brain may be treated with a conjugate containing a vector that is
efficiently transported across the BBB (e.g., AngioPep-2,
AngioPep-3, AngioPep-4a, AngioPep-4b, AngioPep-5, or AngioPep-6).
Ovary, liver, lung, kidney, or spleen cancers may be treated with
an anticancer agent conjugated to a vector that is transported
efficiently into the appropriate cell type (e.g., AngioPep-7).
Conjugate Activities
[0232] Compounds, or a pharmaceutically acceptable salt thereof,
that include an amino acid sequence, where the amino acid sequence
is covalently bonded through an amino acid to a podophyllotoxin
derivative (e.g., a compound having a structure according to
Formula (I)) or to doxorubicin or doxorubicin derivatives (e.g.,
compounds of Formula (II)) can achieve desirable properties, such
as altered pharmacokinetics or altered tissue distribution (e.g.,
increased delivery to particular tissues or cell types such as
ovary, liver, brain, lung, spleen, or kidney) relative to the
unconjugated biologically active agent. Accordingly, the compounds
of the invention can be used as vectors. Polypeptides such as
AngioPep-3, AngioPep-4a, AngioPep-4b, AngioPep-5, and AngioPep-6
efficiently transport agents across the BBB. Like AngioPep-2, these
polypeptides may also be capable of targeting agents to other cell
types or tissues (e.g., ovary, liver, lung, kidney, spleen, or
muscle). The AngioPep-7 polypeptide, which is not efficiently
transported across the BBB, is transported to particular tissues
(e.g., ovary, liver, lung, kidney, spleen, or muscle). This
activity may be useful where transport across the BBB is not
desired. For example, the use of a compound of the invention can
increase the concentration of the therapeutic agent in the target
tissue by anywhere from 10%-20,000% relative to that observed with
the unconjugated biologically active agent (for example, etoposide,
etoposide phosphate, etoposide.sub.DMG, teniposide, doxorubicin, or
epirubicin).
[0233] Because the compounds of the invention can transport agents
to specific tissues, conjugated agents may result in lower toxicity
(e.g., fewer side effects), higher efficacy (e.g., because the
agent is concentrated into a target tissue due to increased uptake
or decreased efflux from the tissue or because the agent has
greater stability when conjugated), or a combination thereof. Such
activities are described below and in International Publication No.
WO 2007/009229, hereby incorporated by reference.
[0234] In some cases, conjugation of an agent to a vector allows
the agent to escape the action of P-glycoprotein (P-gp), an efflux
pump capable of expelling certain agents from a cell. By decreasing
the ability of P-gp to expel an agent from a cell, the potency of
that agent in a cell can be increased. These conjugates can thus
actively inhibit cancer cell proliferation. Moreover, results
obtained for in vivo tumor growth indicate that the vectors of the
invention may target the receptor LRP. Also, conjugation may modify
the pharmacokinetics or biodistribution of the unconjugated
agent.
[0235] Taken together, conjugates can be used against primary
tumors including ovary, breast, lung, and skin cancers as well as
metastasis originating from primary tumors.
Methods of Treatment
[0236] The invention also features methods of treatment using the
compounds of the invention, or pharmaceutical compositions thereof,
described herein. The compounds of the invention (e.g., compounds
that include an amino acid sequence that is covalently bonded
through an amino acid to a podophyllotoxin derivative such as a
compound of Formula (I) or to doxorubicin or doxorubicin
derivatives (e.g., compounds of Formula (II)) that are efficiently
transported across the BBB (e.g., AngioPep-2, AngioPep-3,
AngioPep-4a, AngioPep-4b, AngioPep-5, and AngioPep-6) may be used
to treat any brain or central nervous system disease. Exemplary
neurological diseases include, but are not limited to, brain
cancers such as a brain tumor, a spinal cord tumor (e.g.,
chordoma), and a brain metastasis
[0237] Brain tumors may be primary metastatic brain tumors. Brain
tumors that originate in the brain are primary brain tumors. Brain
tumors caused by the spread of cancer elsewhere in the body (e.g.,
lung, breast, melanoma, colon, kidney, and other cancers) are
metastatic brain tumors. Exemplary categories of tumors, as
described by their location in the brain, include brain stem
tumors, cerebellopontine angle tumors (e.g., acoustic nerve
tumors), cerebral hemisphere tumors, frontal lobe tumors, parietal
lobe tumors, pineal region tumors, occipital lobe tumors, temporal
lobe tumors, subcortical tumors, meningeal brain tumors, midline
tumors (e.g., craniopharyngioma, optic nerve glioma, and tumors of
the thalamus and sellar areas), posterior fossa tumors (e.g.,
tumors of the fourth ventricle, andcerebellar tumors).
[0238] Exemplary brain tumors include acoustic neuroma
(neurilemmoma, schwannoma, neurinoma), adenoma, astrocytoma (e.g.,
juvenile pilocytic astrocytomas, subependymal giant cell
astrocytomas, gemistocytic astrocytoma, anaplastic astrocytoma,
malignant astrocytoma, glioblastoma multiforme, and gliosarcoma),
brain stem glioma which may be anastrocytoma, anaplastic
astrocytoma, glioblastoma multiforme, or a mixed tumor, choroid
plexus papilloma, cns lymphoma, ependymoma (e.g., anaplastic
ependymoma), gangliocytoma, ganglioglioma, glioma, glioblastoma
multiforme, medulloblastoma (mdl), anaplastic (malignant)
meningioma, mixed glioma, neurofibromatosis (von Recklinghausen's
Disease), oligodendroglioma, and optic nerve glioma (e.g.,
pilocytic astrocytoma).
[0239] Conjugates can also be efficiently transported to the liver,
ovary, lung, kidney, spleen or muscle and therefore may also be
used, in conjunction with an appropriate therapeutic agent, to
treat a disease associated with these tissues (e.g., a cancer).
Because AngioPep-7 is not efficiently transported to the brain, but
is transported efficiently to tissues and cells such as liver,
lung, kidney, spleen and muscle, compounds of the invention that
include AngioPep-7 may be especially well suited as a vector
treatment of diseases associated with these tissues when targeting
the agent to the brain is not desired. Exemplary diseases of the
liver include hepatocellular carcinoma (hepatoma) and liver cancer.
Exemplary lung diseases include lung cancers such as small cell
carcinoma (e.g., oat cell cancer), mixed small cell/large cell
carcinoma, combined small cell carcinoma, and metastatic tumors.
Metastatic tumors can originate from cancer of any tissue,
including breast cancer (e.g., metastatic breast carcinoma), colon
cancer, prostate cancer (e.g., metastatic prostate carcinoma),
sarcoma, bladder cancer, neuroblastoma, and Wilms' tumor
(nephroblastoma). Spleen diseases include cancers such as lymphoma,
non-Hodgkin's lymphoma, and certain T-cell lymphomas.
[0240] Additional exemplary cancers that may be treated using a
conjugate or composition of the invention include breast cancer,
cancers of the head and neck including various lymphomas such as
mantle cell lymphoma, adenoma, squamous cell carcinoma, laryngeal
carcinoma, cancers of the retina, cancers of the esophagus,
multiple myeloma, ovarian cancer (e.g., ovarian germ-cell tumors
and ovarian carcinoma), uterine cancer, melanoma, colorectal
cancer, bladder cancer, prostate cancer, lung cancer (including
small cell lung carcinoma and non-small cell lung carcinoma),
pancreatic cancer, cervical cancer, head and neck cancer, skin
cancers, nasopharyngeal carcinoma, liposarcoma, epithelial
carcinoma, renal cell carcinoma, gallbladder adenocarcinoma,
parotid adenocarcinoma, endometrial sarcoma, multidrug resistant
cancers; and proliferative diseases and conditions, such as
neovascularization associated with tumor angiogenesis, macular
degeneration (e.g., wet/dry AMD), comeal neovascularization,
diabetic retinopathy, neovascular glaucoma, myopic degeneration and
other proliferative diseases and conditions such as restenosis and
polycystic kidney disease.
[0241] As described herein, brain cancers that can be treated with
the compounds or the compositions of the invention that are
transported efficiently across the BBB include astrocytoma,
pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor,
oligodendrogliomas, ependymoma, glioblastoma multiforme, mixed
gliomas, oligoastrocytomas, medulloblastoma, retinoblastoma,
neuroblastoma, germinoma, and teratoma. Other exemplary cancers
that may be treated with the compounds or compositions of the
invention include mycosis fungoides (also known as Alibert-Bazin
syndrome or granuloma fungoides), Hodgkin's disease (Hodgkin's
lymphoma), acute myelogenous leukemia, acute lymphocytic leukemia,
chronic myelogenous leukemia, Kaposi's sarcoma related to acquired
immune deficiency syndrome (AIDS), AIDS-related non-Hodgkin's
lymphoma, gestational trophoblastic tumors, Ewing's sarcoma,
rhabdomyosarcoma, refractory advanced breast cancer, testicular
cancer (e.g., malignant tumor of testis, refractory testicular
neoplasm, and testicular germ cell tumor carcinoma), refractory
advanced malignant neoplasms, diffuse large B-cell lymphoma,
osteosarcoma, Burkitt's lymphoma, adult acute lymphocytic leukemia,
Burkitt's leukemia, mediastinal neoplasms, lymphoblastic lymphoma,
large cell anaplastic lymphoma, plasma cell neoplasm.
[0242] A compound or composition of the invention may be
administered by any means known in the art; e.g., orally,
intraarterially, intranasally, intraperitoneally, intravenously,
intramuscularly, subcutaneously, transdermally or per os to the
subject. The agent may be, for example, an anti-angiogenic
compound.
[0243] Combination Therapies
[0244] The compounds of the invention may be administered
concurrently with other therapeutic agents or other therapeutic
regimes. In some embodiments, the additional therapeutic agent or
agents may also have a covalent bond to the polypeptides, or
derivatives thereof, described herein (e.g., the polypeptides of
Table 1 and derivatives thereof). In other embodiments, the
additional therapeutic agent or agents are not covalently bound to
the polypeptides described herein. Exemplary therapeutic regimes
and therapeutic agents that can be used in combination therapy with
the compounds of the invention include, but are not limited to:
radiation therapy, chemotherapy, high dose chemotherapy, stem cell
transplant (e.g., autologous stem cell transplant), bone marrow
transplant, surgery, surgery to remove tumors, hyperthermia
treatment, cisplatin, irinotecan, irinotecan hydrochloride,
carboplatin, chlorambucil (Leukeran.RTM.), tositumomab
(Bexxar.RTM.), rituximab (Rituxan.RTM. and MabThera.RTM.),
bleomycin, vincristine, vinblastine, cyclophosphamide,
procarbazine, mitoxantrone, prednisone, prednisolone, gemcitabine
(Gemzar.RTM.), paclitaxel (Taxon, ifosfamide, methotrexate,
doxorubicin, (Adriamycin.RTM.), dexamethasone, cyclosporin, Rad-001
(Certican), cytarabine (Ara-C), daunorubicin, fludarabine,
idarubicin, vorinostat (SAHA), niacinamide, AZD2171, mitotane,
Gemtuzumab ozogamicin (Mylotarg.RTM.), mitoxantrone, clofarabine,
asparaginase, mercaptopurine, granulocyte colony-stimulating factor
(G-CSF or GCSF), vindesine, thioguanine, VM26, VP16, dacarbazine,
dactinomycin, temozolomide, thiotepa, epirubicin hydrochloride,
carmustine, filgrastim, docetaxel, gefitinib, or pharmaceutically
acceptable salts thereof, or any combination thereof.
[0245] The second therapeutic agents used in the methods described
herein can also be a polypeptide that includes or that consists of
the sequence of Angiopep-2 (SEQ ID NO:97), preferably where the
Angiopep-2 is conjugated to an anticancer agent (e.g., paclitaxel).
An exemplary therapeutic agent that can be used in combination with
any of the compounds described herein is ANG1005, which has the
following structure:
##STR00037##
Still other exemplary second therapeutic agents are described in
U.S. Pat. No. 7,557,182, herein incorporated by reference.
Pharmaceutical Compositions
[0246] Pharmaceutical compositions of the invention include a
compound of the invention as described herein, in association with
a pharmaceutically acceptable carrier. Such compositions are
liquids or lyophilized or otherwise dried formulations and include
diluents of various buffer content (e.g., Tris-HCl, acetate,
phosphate), pH and ionic strength, additives such as albumin or
gelatin to prevent absorption to surfaces, detergents (e.g., Tween
20, Tween 80, Pluronic F68, bile acid salts). Solubilizing agents
(e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g.,
ascorbic acid, sodium metabisulfite), preservatives (e.g.,
thimerosal, benzyl alcohol, parabens), bulking substances or
tonicity modifiers (e.g., lactose, mannitol), covalent attachment
of polymers such as polyethylene glycol to the protein,
complexation with metal ions, or incorporation of the material into
or onto particulate preparations of polymeric compounds such as
polylactic acid, polyglycolic acid, hydrogels, etc, or onto
liposomes, microemulsions, micelles, unilamellar or multilamellar
vesicles, erythrocyte ghosts, or spheroplasts. Such compositions
will influence the physical state, solubility, stability, rate of
in vivo release, and rate of in vivo clearance. Controlled or
sustained release compositions include formulation in lipophilic
depots (e.g., fatty acids, waxes, oils). Also comprehended by the
invention are particulate compositions coated with polymers (e.g.,
poloxamers or poloxamines). Other embodiments of the compositions
of the invention incorporate particulate forms protective coatings,
protease inhibitors or permeation enhancers for various routes of
administration, including parenteral, pulmonary, nasal, oral,
vaginal, rectal routes. In one embodiment the pharmaceutical
composition is administered parenterally, paracancerally,
transmucosally, transdermally, intramuscularly, intravenously,
intradermally, subcutaneously, intraperitonealy,
intraventricularly, intracranially, and intratumorally.
[0247] Pharmaceutically acceptable carriers further include
0.01-0.1 M or 0.05 M phosphate buffer or 0.8% saline. Additionally,
such pharmaceutically acceptable carriers may be aqueous or
non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol,
vegetable oils such as olive oil, and injectable organic esters
such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers such as those
based on Ringer's dextrose, and the like. Preservatives and other
additives may also be present, such as, for example,
antimicrobials, antioxidants, collating agents, inert gases and the
like.
[0248] Other formulations include poly-oxyethylene esters of a
fatty acid (e.g., 12-hydroxystearic acid) such as Solutol.RTM.
HS15. Thus, in some embodiments, a pharmaceutical composition may
comprise a) a conjugate described herein, b) Solutol.RTM. HS 15 and
c) an aqueous solution or buffer (e.g., Ringer/Hepes solution at a
pH of 5 to 7). The concentration of Solutol.RTM. HS 15 in the
formulation may be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, or 60% (e.g., 30%) or within any range between any two of
these numbers. The concentration of conjugate may be determined
based upon the dose required for efficiently treating a subject, or
the amount the ester required for solubility of the conjugate being
administered. The use of Solutol in a formulation for
administration of a Taxol conjugate is described, for example, in
International Publication No. WO 2007/009229, hereby incorporated
by reference.
Parenteral Compositions
[0249] The pharmaceutical composition may be administered
parenterally by injection, infusion, or implantation (subcutaneous,
intravenous, intramuscular, intraperitoneal, or the like) in dosage
forms, formulations, or via suitable delivery devices or implants
containing conventional, non-toxic pharmaceutically acceptable
carriers and adjuvants. The formulation and preparation of such
compositions are well known to those skilled in the art of
pharmaceutical formulation.
[0250] Compositions for parenteral use may be provided in unit
dosage forms (e.g., in single-dose ampoules), or in vials
containing several doses and in which a suitable preservative may
be added (see below). The composition may be in form of a solution,
a suspension, an emulsion, an infusion device, or a delivery device
for implantation, or it may be presented as a dry powder to be
reconstituted with water or another suitable vehicle before use.
Apart from the active agent(s), the composition may include
suitable parenterally acceptable carriers and/or excipients. The
active agent(s) may be incorporated into microspheres,
microcapsules, nanoparticles, liposomes, or the like for controlled
release. Furthermore, the composition may include suspending,
solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting
agents, and/or dispersing agents.
[0251] As indicated above, the pharmaceutical compositions
according to the invention may be in a form suitable for sterile
injection. To prepare such a composition, the suitable active
agent(s) are dissolved or suspended in a parenterally acceptable
liquid vehicle. Among acceptable vehicles and solvents that may be
employed are water, water adjusted to a suitable pH by addition of
an appropriate amount of hydrochloric acid, sodium hydroxide or a
suitable buffer, 1,3-butanediol, Ringer's solution, dextrose
solution, and isotonic sodium chloride solution. The aqueous
formulation may also contain one or more preservatives (e.g.,
methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of
the compounds is only sparingly or slightly soluble in water, a
dissolution enhancing or solubilizing agent can be added, or the
solvent may include 10-60% w/w of propylene glycol or the like.
[0252] The administration of a parenteral composition or
formulation that includes a compound of the invention may be
administered to a patient over a time period that is, for example,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, 100, 105, 110, 115, or 120 minutes or, for example, over 0.5,
1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 hours.
[0253] Controlled Release Parenteral Compositions
[0254] Controlled release parenteral compositions may be in form of
aqueous suspensions, microspheres, microcapsules, magnetic
microspheres, oil solutions, oil suspensions, or emulsions. The
composition may also be incorporated in biocompatible carriers,
liposomes, nanoparticles, implants, or infusion devices.
[0255] Materials for use in the preparation of microspheres and/or
microcapsules are, e.g., biodegradable/bioerodible polymers such as
polygalactin, poly-(isobutyl cyanoacrylate),
poly(2-hydroxyethyl-L-glutamine), poly(lactic acid), polyglycolic
acid, and mixtures thereof. Biocompatible carriers that may be used
when formulating a controlled release parenteral formulation are
carbohydrates (e.g., dextrans), proteins (e.g., albumin),
lipoproteins, or antibodies. Materials for use in implants can be
non-biodegradable (e.g., polydimethyl siloxane) or biodegradable
(e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid)
or poly(ortho esters)) or combinations thereof.
Solid Dosage Forms for Oral Use
[0256] Formulations for oral use include tablets containing the
active ingredient(s) in a mixture with non-toxic pharmaceutically
acceptable excipients, and such formulations are known to the
skilled artisan (e.g., U.S. Pat. Nos. 5,817,307, 5,824,300,
5,830,456, 5,846,526, 5,882,640, 5,910,304, 6,036,949, 6,036,949,
6,372,218, hereby incorporated by reference). These excipients may
be, for example, inert diluents or fillers (e.g., sucrose,
sorbitol, sugar, mannitol, microcrystalline cellulose, starches
including potato starch, calcium carbonate, sodium chloride,
lactose, calcium phosphate, calcium sulfate, or sodium phosphate);
granulating and disintegrating agents (e.g., cellulose derivatives
including microcrystalline cellulose, starches including potato
starch, croscarmellose sodium, alginates, or alginic acid); binding
agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid,
sodium alginate, gelatin, starch, pregelatinized starch,
microcrystalline cellulose, magnesium aluminum silicate,
carboxymethylcellulose sodium, methylcellulose, hydroxypropyl
methylcellulose, ethylcellulose, polyvinylpyrrolidone, or
polyethylene glycol); and lubricating agents, glidants, and
anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic
acid, silicas, hydrogenated vegetable oils, or talc). Other
pharmaceutically acceptable excipients can be colorants, flavoring
agents, plasticizers, humectants, buffering agents, and the
like.
[0257] The tablets may be uncoated or they may be coated by known
techniques, optionally to delay disintegration and absorption in
the gastrointestinal tract and thereby providing a sustained action
over a longer period. The coating may be adapted to release the
agent in a predetermined pattern (e.g., in order to achieve a
controlled release formulation) or it may be adapted not to release
the agent(s) until after passage of the stomach (enteric coating).
The coating may be a sugar coating, a film coating (e.g., based on
hydroxypropyl methylcellulose, methylcellulose, methyl
hydroxyethylcellulose, hydroxypropylcellulose,
carboxymethylcellulose, acrylate copolymers, polyethylene glycols,
and/or polyvinylpyrrolidone), or an enteric coating (e.g., based on
methacrylic acid copolymer, cellulose acetate phthalate,
hydroxypropyl methylcellulose phthalate, hydroxypropyl
methylcellulose acetate succinate, polyvinyl acetate phthalate,
shellac, and/or ethylcellulose). Furthermore, a time delay material
such as, e.g., glyceryl monostearate or glyceryl distearate, may be
employed.
[0258] The solid tablet compositions may include a coating adapted
to protect the composition from unwanted chemical changes, (e.g.,
chemical degradation prior to the release of the active
substances). The coating may be applied on the solid dosage form in
a similar manner as that described in Encyclopedia of
Pharmaceutical Technology, supra.
[0259] The compositions of the invention may be mixed together in
the tablet, or may be partitioned. In one example, a first agent is
contained on the inside of the tablet, and a second agent is on the
outside, such that a substantial portion of the second agent is
released prior to the release of the first agent.
[0260] Formulations for oral use may also be presented as chewable
tablets, or as hard gelatin capsules wherein the active ingredient
is mixed with an inert solid diluent (e.g., potato starch, lactose,
microcrystalline cellulose, calcium carbonate, calcium phosphate,
or kaolin), or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example,
peanut oil, liquid paraffin, or olive oil. Powders and granulates
may be prepared using the ingredients mentioned above under tablets
and capsules in a conventional manner using, e.g., a mixer, a fluid
bed apparatus, or spray drying equipment.
Dosage Regimens
[0261] The dosage of any compound, conjugate, or composition
described herein or identified using the methods described herein
depends on several factors, including: the administration method,
the disease (e.g., cancer) to be treated, the severity of the
disease, whether the cancer is to be treated or prevented, and the
age, weight, and health of the subject to be treated.
[0262] With respect to the treatment methods of the invention, it
is not intended that the administration of a vector, conjugate, or
composition to a subject be limited to a particular mode of
administration, dosage, or frequency of dosing; the invention
contemplates all modes of administration. The conjugate, or
composition may be administered to the subject in a single dose or
in multiple doses. For example, a compound described herein or
identified using screening methods of the invention may conjugate
be administered, for example, once a week for, e.g., 2, 3, 4, 5, 6,
7, 8, 10, 15, 20, or more weeks. A compound of the invention may
also be administered, for example, daily for 1, 2, 3, 4, 5, 6, or 7
days or for 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks.
[0263] Time periods during which the compound of the invention are
administered may be followed or preceded by time periods during
which the compound is not administered. For example, following
administration of the compound as described herein, the compound of
the invention is not administered to the patient for 1, 2, 3, 4, 5,
6, or 7 days or for 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more
weeks. In some embodiments, the patient can receive other
therapeutic agents during said time period. These cycles of
chemotherapy that include a period of time during which the
compound of the invention is administered to a patient that is
followed by a period of time during which the compound of the
invention is not administered to said patient can be repeated as
medically necessary (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times).
[0264] It is to be understood that, for any particular subject,
specific dosage regimes should be adjusted over time according to
the individual need and the professional judgment of the person
administering or supervising the administration of the vector,
conjugate, or composition. For example, the dosage of a conjugate
can be increased if the lower dose does not provide sufficient
activity in the treatment of a disease or condition described
herein (e.g., cancer). Conversely, the dosage of the compound can
be decreased if the disease (e.g., cancer) is reduced or
eliminated.
[0265] While the attending physician ultimately will decide the
appropriate amount and dosage regimen, a therapeutically effective
amount of a compound, vector, conjugate, or composition described
herein, may be, for example, in the range of 0.0035 .mu.g to 20
.mu.g/kg body weight/day or 0.010 .mu.g to 140 .mu.g/kg body
weight/week. Desirably a therapeutically effective amount is in the
range of 0.025 .mu.g to 10 .mu.g/kg, for example, at least 0.025,
0.035, 0.05, 0.075, 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,
4.0, 5.0, 6.0, 7.0, 8.0, or 9.0 .mu.g/kg body weight administered
daily, every other day, or twice a week. In addition, a
therapeutically effective amount may be in the range of 0.05 .mu.g
to 20 .mu.g/kg, for example, at least 0.05, 0.7, 0.15, 0.2, 1.0,
2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.0, 14.0, 16.0, or 18.0
.mu.g/kg body weight administered weekly, every other week, or once
a month. Furthermore, a therapeutically effective amount of a
compound may be, for example, in the range of 0.100 mg/m.sup.2 to
2000 mg/m.sup.2 administered every other day, once weekly, or every
other week. In a desirable embodiment, the therapeutically
effective amount is in the range of 1 mg/m.sup.2 to 1000
mg/m.sup.2, for example, at least 100, 150, 400, or 800 mg/m.sup.2
of the compound administered daily, every other day, twice weekly,
weekly, or every other week.
[0266] For example, the compounds of the invention (e.g., Compound
(1)) can be used to administer podophyllotoxin derivatives (e.g., a
compound of Formula (I) such as etoposide, etoposide phosphate,
etoposide.sub.DMG, and teniposide) or to administer doxorubicin or
doxorubicin derivatives (e.g., Compound (2) or a compound of the
invention that includes a compound of Formula (II))) to a patient
using any of the methods of administration, dosage amounts, and
dosage schedules described herein. Because the compounds of the
invention can include covalent bonds to, for example, 1, 2, 3, 4,
or 5 molecules of a podophyllotoxin derivative such as those of
Formula (I) (e.g., etoposide, etoposide phosphate,
etoposide.sub.DMG, and teniposide) or of Formula (II) (e.g.,
doxorubicin or epirubicin), this stoichiometry can be used to
calculate and to adjust the dosage amount to be administered
("equivalent dose"). For example, the Etoposide:Angiopep-2 (3:1)
conjugate ("Etop-An2(3:1)") has a molecular weight of 4354 g/mol,
with the etoposide content accounting for 40% of the molecular
weight. Similarly, the Doxorubicin:Angiopep-2 (3:1) conjugate has a
molecular weight of 4178 g/mol, with the doxorubicin accounting for
40% of the molecular weight. Using this information, the amount of
a compound of the invention that is included in a composition for
administration (e.g., parenteral or oral) may be calculated in
order to administer 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,
115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,
180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400,
425, 450, 475, or 500 mgs/m.sup.2/day of a podophyllotoxin
derivative (e.g., etoposide, etoposide phosphate,
etoposide.sub.DMG, or etoposide) to a patient. This dosage can be
administered to a patient daily for 2, 3, 4, 5, 6, or 7 days. The
administration period can be followed by 1, 2, 3, 4, 5, 6, or 7
days or by 2, 3, 4, 5, 6, 7, or 8 weeks of rest without
administration of a compound of the invention, with the cycle of
administration periods/rest periods repeated for, e.g., 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10 additional times.
[0267] The compounds of the invention (e.g., Compounds (1) and (2),
or a pharmaceutically acceptable salt thereof) can show improved
physicochemical and pharmaceutical properties relative to the
respective unconjugated therapeutic agent (e.g., etoposide,
etoposide 4-dimethylglycine, or doxorubicin). For example,
increased targeting of exemplary cell types, tissues, or organs as
described herein (e.g., brain, ovary, liver, lung, kidney, spleen,
or muscle) allows for subtherapeutic doses of the compound (e.g.,
Compounds (1) or (2)) to be administered to a patient. Compounds of
the invention can also exhibit reduced toxicity relative to the
respective unconjugated therapeutic agent and thus allow for
supertherapeutic doses of the compound to be administered to a
patient. For example, unconjugated doxorubicin is typically
administered in dose schedules that range from 60-75 mg/m.sup.2
when administered alone as a single intravenous injection or from
40-50 mg/m.sup.2 when administered in combination with another
chemotherapeutic agent. Typical dose schedules for unconjugated
etoposide or etoposide phosphate can range from 1-5 mg/m.sup.2/day,
1-50 mg/m.sup.2/day, 35-50 mg/m.sup.2/day, or 50-100
mg/m.sup.2/day. Improved targeting of cells types, tissues, or
organs by the compounds of the invention (e.g., Compounds (1) or
(2)) can allow for reduced doses of the therapeutic agent relative
to those used for the corresponding unconjugated therapeutic agent
("subtherapeutic doses," i.e., an effective dose that is, for
example, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,
325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625,
650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950,
975, 1000, 2000, 3000, 4000, or 5000 times less than the minimum
effective dose of the corresponding unconjugated therapeutic
agent). Reduced toxicity that can be associated with the compounds
of the invention (e.g., Compounds (1) or (2)) can allow for
increased doses to be safely administered to a patient relative to
those used for the corresponding unconjugated therapeutic agent
("supertherapeutic doses," i.e., an effective dose that is, for
example, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,
325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625,
650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950,
975, 1000, 2000, 3000, 4000, or 5000 times more than the maximum
allowed dose of the unconjugated therapeutic agent). Similarly,
improved physicochemical properties can also affect the doses
administered to a patient. For example, improved solubility can
allow for the administration of subtherapeutic doses. These
improved physicochemical pharmaceutical characteristics can allow
for the safe administration of the compounds of the invention
(e.g., Compounds (1) or (2)) to, for example, pediatric or
geriatric patient populations.
[0268] The following examples are intended to illustrate, rather
than limit the invention.
EXAMPLES
Example 1
Synthesis of a 3:1 Etoposide:Angiopep-2 Conjugate
[0269] Synthesis of the compounds of the invention can be
accomplished by the combination of a podophyllotoxin derivative
(e.g., a compound of Formula (I) such as etoposide, etoposide
phosphate, etoposide.sub.DMG, or teniposide), a difunctional
hydrolyzable linking group (e.g., a dicarboxylic acid or a
diisocyanate), and any amino acid sequence described herein, or a
functional derivative thereof. Variation of the equivalents of the
derivatized podophyllotoxin intermediate (e.g., 2''-glutaryl
etoposide) relative to the polypeptide can allow the synthesis of
polypeptide conjugates with different stoichiometries (for example,
"Etop-An2(1:1)" or "Eto-An2(1:1)", where one molecule of etoposide
is bound to the Angiopep-2 polypeptide).
[0270] Scheme 5 shows the synthesis of a compound that includes an
Angiopep-2 polypeptide covalently bonded to three etoposide
molecules ("Etop-An2(3:1)" or ("Eto-An2(3:1)").
##STR00038##
[0271] (2''-Glutaryl)-Etoposide.
[0272] To a solution of Etoposide (10 g, 17 mmol) in CHCl.sub.3
(120 mL) were added anhydride glutaric (3.13 g, 27.4 mmol) and DMAP
(115 mg, 0.942 mmol). After 72 hours at room temperature, the
mixture was evaporated and purified by reverse phase chromatography
using a polystyrene/DVB column (15 to 35% acetonitrile (ACN) in
H.sub.2O, no trifluoroacetic acid (TFA)). HPLC of the crude product
showed a mixture of regioisomers 2''-Glutaryl Etoposide
(2''-glu-Etop) and 3''-Glutaryl Etoposide (3''-glu-Etop) in a 2:1
ratio. After evaporation and lyophilisation,
(2''-Glutaryl)-Etoposide was obtained as a white powder (4.1 g,
34%). The regioisomeric (3''-glutaryl)-etoposide was also isolated
as a white solid (2.4 g, 20%). The purities of 2''-glu-Etop and
3''-glu-Etop were determined by RP-HPLC. Using a MetaChem Taxsil-3
column and gradient elution (1 mL/min; 10% to 65% (0.05% TFA in
H.sub.2O):(0.05% TFA in ACN) over 15 minutes), 2''-glu-Etop has a
retention time of 9.00 minutes and 3''-glu-Etop has a retention
time of 9.42 minutes. The purity of 2''-glu-Etop (retention
time=9.00 minutes) was >98% (99.4%) and the purity of
3''-glu-Etop was >98% (99.6%).
[0273] ((2''-Glutaryl)-Etop).sub.3-(Angiopep2) (Etop-An2(3:1)).
[0274] To a solution of (2''-Glutaryl)-Etoposide (2.83 g, 4.025
mmol) in anhydrous DMF (400 mL) were added triethylamine
(Et.sub.3N; 0.84 mL, 6.037 mmol) and
N,N,N',N'-Tetramethyl-O-(benzotriazol-1-yeuronium tetrafluoroborate
(TBTU; 1.32 g, 4.628 mmol). The reaction mixture was stirred at
room temperature for 1 hour (pH=9.3). A solution of AN2
(Angiopep-2, 75% in content, 3.68 g, 1.207 mmol) in PBS 10.times.
buffer (pH 7.3; 200 mL) was prepared by adjusting pH with 10N NaOH
(to pH 7.3). After cooling with dry iced bath, the previously
activated acid was added (4.times.100 mL) to Angiopep-2, and the pH
of the mixture was adjusted to 7.2 by addition of 10N NaOH. After 1
hour at room temperature, the reaction was filtered to eliminate
phosphate salts and evaporated to obtain 35 mL of the crude
mixture. HPLC of the crude product showed a mixture of (3:1)- and
(2:1) conjugates in a 3:1 ratio. The residue was purified by
reverse phase chromatography using a polystyrene/DVB column (15 to
37.5% ACN in H.sub.2O). After purification, 0.1% acetic acid was
added to the combined fractions in order to increase the
solubility. Evaporation and lyophilisation of the mixture afforded
the Etop-An2(3:1) product as a white solid (596 mg, 12%). Using a
MetaChem Taxsil-3 column and gradient elution (1 mL/min; 10% to 65%
(0.05% TFA in H.sub.2O):(0.05% TFA in ACN) over 15 minutes), the
isolated product was found to have a retention time of 9.36 minutes
and to be >95% (97.3%) pure. m/z (ESI-TOF): 2178 (+2), 1452
(+3).
Example 2
Synthesis of a 3:1 Etoposide.sub.DMG:Angiopep-2 Conjugate
[0275] The procedure described in Example 1 can be used to prepare
compounds that include peptides conjugated to other podophyllotoxin
derivatives. For example, Etoposide.sub.DMG can be used to prepare
the conjugate shown in Scheme 7 ("Etop.sub.DMG-An2 (3:1)") using
the synthetic procedure shown in Scheme 6.
##STR00039##
##STR00040##
[0276] Etoposide 4'-Dimethylglycine:
[0277] A mixture of etoposide (235 mg, 0.4 mmol) and DMAP (73 mg,
0.6 mmol) in DMF (4 mL) was stirred at room temperature for 20
minutes, and then N,N-dimethylacetyl chloride (96 mg, 0.52 mmol)
was added in one pot with stirring. After 30 minutes, the reaction
was complete according to HPLC. Formic acid (1M in DMF, 0.5 mL) was
added, and the solvent was concentrated to 1 mL The resulting
solution was loaded onto an AKTA RPC column for purification
(gradient 10% to 30% MeCN in H.sub.2O with 0.1% Formic acid). After
lyophilization, etoposide 4'-dimethylglycine ("Etoposide.sub.DMG"
or "Etop.sub.DMG"; 180 mg, 67%) was obtained as a colorless powder.
.sup.1H NMR (CD.sub.3OD) .delta. 7.01 (1H, s), 6.56 (1H, s), 6.39
(2H, s), 5.98 (2H, d, J=2.9 Hz), 5.05 (1H, d, J=3.4 Hz), 4.77 (1H,
q, J=4.9 Hz), 4.68 (1H, d, J=5.4 Hz), 4.66 (1H, d, J=7.8 Hz), 4.46
(2H, s), 4.45 (1H, dd, J=10.3, 8.8 Hz), 4.31 (1H, t, J=8.0 Hz),
4.17 (1H, dd, J=10.3, 4.9 Hz), 3.68 (6H, s), 3.56 (1H, q, J=10 Hz),
3.54 (1H, t, J=9.3 Hz), 3.52 (1H, dd, J=14.2, 5.6 Hz), 3.32 (1H,
m), 3.26 (1H, dd, J=9.1, 4.1 Hz), 3.24 (1H, dd, J=9.2, 5.4 Hz),
3.02 (6H, s), 2.96 (1H, m), 1.33 (3H, d, J=4.9 Hz). .sup.13C NMR
(DMSO) .delta. 175.26, 168.68, 151.35, 148.49, 147.01, 139.39,
132.74, 129.6, 127.28, 110.65, 110.45, 108.02, 102.19, 102.02,
99.25, 80.78, 75.06, 73.39, 72.41, 68.43, 68.01, 66.44, 59.73,
56.63, 56.47, 45.03, 43.86, 37.89, 20.99; HRMS (MicroTOF) calcd.
for C.sub.33H.sub.39NO.sub.14 673.2371. found 274.2534 (M+1).
[0278] Etoposide 4'-Dimethylglycine 2''-Glutaric acid:
[0279] A mixture of etoposide 4'-dimethylglycine (655 mg, 0.97
mmol) and DMAP (18 mg, 0.15 mmol) in chloroform (11 mL) was cooled
to 0.degree. C. DMF (3 mL) and N,N-diisopropylethylamine (DIEA;
0.25 mL, 1.46 mmol) were added consecutively, followed by glutaric
anhydride (222 mg, 1.94 mmol). The reaction mixture was stirred at
room temperature, monitored by HPLC. After 2 days, the solvent was
concentrated to 3 mL The resulting solution was loaded to an AKTA
RPC column for purification (gradient elution, 10% to 30% MeCN in
H.sub.2O), and Etoposide 4'-Dimethylglycine 2''-Glutaric acid (305
mg, 40%) was obtained as a white powder after lyophilization.
.sup.1H NMR (CD.sub.3OD) .delta. 7.0 (1H, s), 6.53 (1H, s), 6.39
(2H, s), 5.99 (2H, d, J=4.6 Hz), 4.97 (1H, q, J=7.9 Hz), 4.78 (1H,
q, J=4.75 Hz), 4.74 (1H, d, J=7.9 Hz), 4.68 (1H, d, J=5.6 Hz), 4.45
(2H, s), 4.41 (1H, dd, J=9.6, 8.8 Hz), 4.29 (1H, t. J=8.2 Hz), 4.15
(1H, dd, J=10.0, 4.5 Hz), 3.78 (1H, t, J=9.4 Hz), 3.69 (6H, s),
3.61 (1H, t, J=10.2 Hz), 3.42 (1H, td, J=9.6, 5.2 Hz), 3.33 (1H,
dd, J=8.7, 8.2 Hz), 3.3 (1H, dd, J=13.4, 5.3 Hz), 3.02 (6H, s),
2.93 (1H, m), 2.26 (1H, m), 2.16 (2H, m), 2.02 (1H, m), 1.64 (2H,
m), 1.32 (3H, d, J=4.9 Hz). .sup.13C NMR (DMSO) .delta. 175.96,
175.33, 172.46, 163.74, 151.14, 148.96, 147.43, 139.53, 131.90,
129.83, 126.42, 110.20, 109.18, 107.40, 101.91, 100.65, 99.63,
80.39, 74.55, 73.95, 71.55, 71.29, 68.43, 67.82, 66.46, 56.43,
55.35, 43.90, 43.15, 40.82, 38.0, 32.86, 32.59, 19.93, 19.39. HRMS
(MicroTOF) calcd. for C.sub.38H.sub.45NO.sub.17 787.2687. found
788.2432 (M+1).
[0280] (Etoposide-4'-Dimethylglycine-2''-Glutaric).sub.3-Angiopep-2
Conjugate ("Etop-4'-DMGly-2"-Glu).sub.3-An2'' or
"Etop.sub.DMG-An2(3:1)"):
[0281] DIEA (0.17 mL, 0.98 mmol) was added dropwise to a mixture of
Etoposide 4'-Dimethylglycine 2''-Glutaric acid (330 mg, 0.42 mmol)
and TBTU (145 mg, 0.46 mmol) in DMF (24 mL) The mixture was stirred
at room temperature for 50 minutes. A solution of Angpep-2 (422 mg,
0.14 mmol) in DMSO (1.5 mL) and DMF (9 mL) was then added, followed
by DIEA (0.084 mL, 0.48 mmol). The mixture was stirred at room
temperature for 20 minutes. An aliquot (10 mL) was taken for UPLC
analysis, and it showed the reaction was complete. After stirring
for another 10 minutes, the reaction solution was concentrated to 3
mL and purified using AKTA RPC column (gradient elution, 10% to 25%
MeCN in H.sub.2O with 0.05% formic acid).
(Etop-4'-DMGly-2''-Glu).sub.3-An2 (172 mg, 26%) was yielded as a
colorless powder after lyophilization. MS (MicroTOF), m/z,
2305.9327 (2+), 1537.6443 (3+), 1153.7463 (4+), 922.7970 (5+).
Example 3
Synthesis of of a 3:1 Doxorubicin:Angiopep-2 Conjugate
("(DoxSu).sub.3-An2")
[0282] A 3:1 doxorubicin:angiopep-2 conjugate be prepared according
to the synthetic scheme shown in Scheme 8 and described herein.
##STR00041##
[0283] FmocDoxorubicin:
[0284] DIEA (1.5 mL, 8.63 mmol) was added dropwise to a solution of
doxorubicin (2.0 g, 3.45 mmol) and 9-fluorenylmethyl N-succinimidyl
carbonate (FmocOSu; 2.32 g, 6.9 mmol) in DMF (35 mL) with stirring.
The mixture was stirred at room temperature for 3 hours and
concentrated. The resulting residue was triturated with 0.1% TFA in
H.sub.2O (3.times.20 mL), washed with Et.sub.2O (8.times.20 mL) The
resulting red solid was collected and dried over vacuum to give
FmocDoxorubicin as a red powder (2.1 g, 80% yield). UPLC purity,
98%. MS (ESI, MicroTOF), 788. 2411 (M+Na).
[0285] FmocDoxSuOH:
[0286] DIEA (0.17 mL, 1.0 mmol) was added dropwise to a solution of
FmocDoxorubicin (0.28 g, 0.366 mmol) and succinic anhydride (0.11
g, 1.1 mmol) in DMF (20 mL) under stirring. The mixture was stirred
at room temperature and monitored by UPLC. After two days, the
solvent was removed and the resulting residue was purified using a
Biotage column (silica gel, 2% to 9% MeOH in DCM) to give
FmocDoxSuOH as a red powder (100 mg, 33% yield). UPLC purity: 95%.
MS (ESI, MicroTOF), 888. 2577 (M+Na).
[0287] (FmocDoxSu).sub.3-An2:
[0288] DIEA (0.25 mL, 1.44 mmol) was added dropwise to a solution
of FmocDoxSuOH (599 mg, 0.692 mmol) and TBTU (231 mg, 0.72 mmol) in
DMF (21 mL) under stirring. The mixture was stirred at room
temperature for 50 minutes and then a solution of Angpep-2 (671 mg,
0.229 mmol) in DMSO (2 mL) and DMF (12 mL) was added. The mixture
was stirred at room temperature for 20 minutes, at which time HPLC
showed the reaction was complete. After stirring for another 10
minutes, the solvent was removed and the residue was purified using
a Biotage C18 column (40% to 80% MeCN in water and 0.05% TFA) to
give (FmocDoxSu).sub.3An-2 as a red powder (500 mg, 45% yield).
UPLC purity, 95%. MS (ESI, MicroTOF), m/z 2423.4239 (2+), 1615.6190
(3+).
[0289] (DoxSu).sub.3-An2:
[0290] Piperidine (20% in DMF, 1.5 mL) was added to a solution of
(FmocDoxSu).sub.3An-2 (260 mg, 0.053 mmol) in DMSO (1 mL) and DMF
(12 mL) The solution became blue. After stirring for 10 minutes,
the solution was cooled to 0.degree. C. and treated with formic
acid (0.5 M in DMF, 6 mL) to get a clear red solution. The solvent
was removed using vacuum pump, and the resulting residue was
triturated with Et.sub.2O (3.times.10 ml) and AcOEt (3.times.10
ml). The resulting red solid was purified using AKTA RPC 30 column
(10% to 40% MeCN in water and 0.15% formic acid) to give
(DoxSu).sub.3An-2 as a red powder (82 mg, 37% yield). UPLC purity:
95%. MS (ESI, MicroTOF), m/z 2089.9674 (2+), 1393.2419 (3+),
1045.4395 (4+).
Example 3
Effect of Etoposide, Etoposide-Angiopep Conjugates, Doxorubicin,
and Doxorubicin-Angiopep Conjugates on Cell Proliferation
[0291] For the in vitro cell proliferation assay, between 2.5 and
5.times.10.sup.4 of U87 or SK-HEP-1 cells were seeded in a 24 well
tissue culture microplate in a final volume of 1 mL of medium with
10% serum and incubated for 24 hours at 37.degree. C. and 5%
CO.sub.2. The medium was then replaced with serum-free medium and
incubated overnight. The next morning the agent was freshly
dissolved in dimethyl sulfoxide (DMSO) and the medium was replaced
with complete medium containing the agent at different
concentrations in triplicate. The final concentration of DMSO was
0.1%. The control used was a microplate well with cells and without
agent. The cells were incubated for 48 to 72 hrs at 37.degree. C.
and 5% CO.sub.2. After the incubation, the medium was changed and
replaced with 1 mL of complete medium containing
[.sup.3H]-thymidine (1 pCi/assay). The plate was incubated at
37.degree. C. and 5% CO.sub.2 for 4 hrs. The medium as removed, and
the cells were washed with PBS at 37.degree. C. The cells were
fixed with a mix of ethanol:acetic acid (3:1), washed with water,
and precipitated 3 times with 10% of ice-cold TCA (trichloroacetic
acid). Finally 500 .mu.L of PCA (perchloric acid) were added to the
wells and the microplates were heated for 30 min at 65.degree. C.
and 30 min at 75.degree. C. The contents of each well were then
transferred to a scintillation vial with 10 mL of scintillation
cocktail and the activity was measured in CPM (count per minute) on
a liquid scintillation counter Tri-Carb from Packard. The results
of the cell proliferation assay using unconjugated etoposide,
Etop-An2(1:1), and Etop-An2(3:1) are shown in Table 4. Table 5
shows the results obtained for Etop.sub.DMG-An2(3:1), unconjugated
etoposide.sub.DMG, the doxorubicin/Angiopep-2(3:1) conjugate
("Doxorubicin-An2 (3:1)"), and unconjugated doxorubicin.
[0292] In addition to the in vitro studies, the inhibition of cell
proliferation has been studied in xenograft tumor models and these
results are shown in FIG. 1. U87 glioblastoma cells (2.5.times.106)
were subcutaneously implanted in the right flank of nude mice.
Treatments started on day 15 after implantation (corresponding to
day 0 on the graph shown in FIG. 1) when tumor volume reached about
150-200 mm.sup.3. The mice were treated once a week for three weeks
by i.v. bolus injection with doxorubicin (6 mg/kg) and
doxorubicin-An2 conjugate (20 and 40 mg/kg). The doxorubicin-An2
conjugate was diluted in acidified D5W (5% dextrose in water) at 5
mg/ml.
TABLE-US-00004 TABLE 4 IC.sub.50 (nM) Drug 24 hours 48 hours 72
hours U-87 Cells Etoposide 160 221 145 Eto-An2 (1:1) 1313 722 550
Eto-An2 (3:1) 453 151 164 (Etoposide equiv.) (1359) (453) (492)
SK-HEP-1 Etoposide 116 56 50 Eto-An2 (1:1) 679 245 153 Eto-An2
(3:1) 160 59 52 (Etoposide equiv.) (480) (168) (156)
TABLE-US-00005 TABLE 5 IC.sub.50 (nM) Lung Glioblastoma
Hepatocarcinoma Carcinoma Drug (U87) (SK-Hep-1) (NCI-H460)
Etop.sub.DMG 145 62 90 Etop.sub.DMG-An2(3:1) 330 48 148 Doxorubicin
18 10 11 Doxorubicin-An2 6.0 4.6 7.3 (3:1)
Example 3
In Situ Brain Perfusion Studies
[0293] The procedures described in U.S. Patent Publication
20060189515, herein incorporated by reference, were used for the in
situ brain perfusion studies. These procedures are further
described herein.
Example 3a
Etop-An2(3:1)
[0294] The brain uptake of the compounds of the invention (e.g.,
Etop-An2(3:1), Etop.sub.DMG-An2(3:1), and Doxorubicin-An2 (3:1))
relative to the corresponding unconjugated drugs were measured
using in situ brain perfusion techniques described herein and in
Dagenais et al., J. Cereb. Blood Flow Metab. 20(2):381-386 (2000).
The uptake of [.sup.125I]-polypeptides to the luminal side of mouse
brain capillaries was measured using the in situ brain perfusion
method adapted in our laboratory for the study of agent uptake in
the mouse brain.
[0295] Polypeptides were iodinated with standard procedures using
iodo-beads from Sigma. Briefly polypeptides were diluted in 0.1 M
phosphate buffer, pH 6.5 (PB). Two iodo-beads were used for each
protein. These beads were washed twice with 3 mL of PB on a Whatman
filter and re-suspended in 60 .mu.L of PB. .sup.125I (1 mCi) from
Amersham-Pharmacia biotech was added to the bead suspension for 5
min at room temperature. Each iodination was initiated by the
addition of the polypeptide (100 .mu.g). After an incubation of 10
min at room temperature, the free iodine was removed by HPLC.
[0296] Briefly, the right common carotid of ketamine/xylazine
(140/8 mg/kg i.p.) anesthetized mice was exposed and ligated at the
level of the bifurcation of the common carotid, rostral to the
occipital artery. The common carotid was then catheterized
rostrally with polyethylene tubing filled with heparin (25 U/mL)
and mounted on a 26-gauge needle. The syringe containing the
perfusion fluid ([.sup.125I]-polypeptides or [.sup.14C]-inulin in
Krebs/bicarbonate buffer at a pH7.4 gassed with 95% O.sub.2 and 5%
CO.sub.2) was placed in an infusion pump (Harvard pump PHD 2000;
Harvard Apparatus) and connected to the catheter. Prior to the
perfusion, the contralateral blood flow contribution was eliminated
by severing heart ventricles. The brain was perfused for the
indicated times at a flow rate of 1.15 mL/min. After 14.5 min of
perfusion, the brain was further perfused for 60 seconds with Krebs
buffer to wash the excess of [.sup.125I]-proteins. Mice were then
decapitated to terminate perfusion and the right hemisphere was
isolated on ice before being subjected to capillary depletion.
Aliquots of homogenates, supernatants, pellets and perfusates were
taken to measure their contents in [.sup.125I]-conjugates by TCA
precipitation and to evaluate the apparent volume of
distribution.
[0297] The results of these experiments are illustrated in FIGS.
2A-D and 3. In FIG. 2A, in situ brain perfusion shows that the Vd
is higher for Etop-An2(3:1) than for the unconjugated etoposide.
Similar trends are observed for Etop.sub.DMG-An2(3:1) (FIG. 2B)
relative to unconjugated Etoposide.sub.DMG and for
doxorubicin-An2(3:1) relative to unconjugated doxorubicin (FIG.
2C). As can be see from FIG. 2C, the ratio of the K.sub.in for
Doxorubicin-An2(3:1):unconjugated doxorubicin is 15. This procedure
also distinguishes between compounds remaining in the brain
vascular compartment from those having crossed the abluminal
endothelial membrane to enter the brain parenchyma. FIG. 3 shows
the in situ perfusion of Etop-An2(3:1). The brain repartition of
Etop-An2(3:1) following brain capillary depletion is illustrated in
FIGS. 4A and 4B. Moreover, in contrast to etoposide the brain
uptake of Etop-An2(3:1) is similar in wild-type and P-gp knock-out
mice, indicating that Etop-An2(3:1) is not a P-gp substrate (FIG.
5). The brain uptake of Etop-An2(3:1) can be inhibited by the
coadministration of an unconjugated polypeptide. FIG. 6 shows that
the co-perfusion of [.sup.125I]-Etop-An2(3:1) with a two-fold
excess of unconjugated Angiopep-2 reduces the parenchyma Vd by
27%.
Example 3b
Etop.sub.DMG-An2 (3:1) and Doxorubicin-An2(3:1)
[0298] The brain uptake of Etop.sub.DMG-An2 (3:1) relative to the
unconjugated Etop.sub.DMG was measured using the methods described
for Example 3a, and these results are shown in Table 6 and in FIG.
7. Table 6 also includes the corresponding data for
Doxorubicin-An2(3:1) and doxorubicin. In contrast to unconjugated
Etop.sub.DMG, the brain uptake of doxorubicin-An2(3:1) is similar
in wild-type and P-gp knock-out mice, indicating that
doxorubicin-An2(3:1) is not a P-gp substrate.
TABLE-US-00006 TABLE 6 Drug Brain K.sub.in (ml/s/g)
Etop.sub.DMG-An2 1.4 .times. 10.sup.-3 Etop.sub.DMG 9.0 .times.
10.sup.-5 Doxorubicin-An2 (3:1) 3.7 .times. 10.sup.-3 Doxorubicin
2.8 .times. 10.sup.-4
Example 4
Plasma Kinetics of the 3:1 Etoposide-Angiopep-2 Conjugate
[0299] FIG. 8 describes the plasma kinetics of Etop-An2(3:1)
following administration as a bolus. Radiolabeled
(.sup.125I)Etop-An2 (20 mg/kg) was injected in bolus by intravenous
(i.v.) or intraperitoneal (i.p.) routes in CD-1 mice weighing about
25-30 g. The injection solution was composed of 12.5%
dimethylsulfoxide (DMSO), 12.5% anhydrous ethanol 25% polyethylene
glycol 400 (PEG400) and 50% NaCl/Glycine buffer. At several time
intervals (0.5, 1, 2, and 6 hours) the blood was collected by
cardiac puncture and animals were sacrificed. After blood
centrifugation, the plasma radioactivity was measured in a gamma
counter (Wizard 1470 Automatic Gamma Counter). The radioactivity
was interpreted as % of injected dose per gram of plasma. Results
were plotted using GraphPad prism software and the area under the
curve (AUC) for each injection mode was calculated. The
bioavailability of intraperitoneal Etop-An2 conjugate was then
estimated by dividing the AUC after i.p. injection by the AUC after
i.v. injection. The estimated bioavailability following i.p.
administration is calculated as 46%. Pharmacokinetic parameters of
Etop-An2(3:1) following IV bolus administration in mice are shown
in Table 7. Literature data for unconjugated etoposide shows a
T.sub.1/2.alpha.=0.13 hour (Reddy et al., Journal of drug
targeting, 13(10): 543-553 (2005)).
TABLE-US-00007 TABLE 7 Dose T.sub.max C.sub.max T.sub.1/2.alpha.
AUC.sub.0-.infin. Elim. Rate Molecule (mg/kg) (min) (.mu.g/mL) (hr)
(hr.degree..mu.g/mL) Const. (hr.sup.-1) Etop-An2 20 5 46 0.43 82
1.6 (3:1)
Example 5
Tissue Distribution of 3:1 Etoposide.sub.DMG-Angiopep-2 Conjugate
and 3:1 Etoposide-Angiopep-2 Conjugate
[0300] The effect of conjugation of an agent to a vector on
distribution of the agent or the pharmacokinetics of a polypeptide
that is conjugated to an agent was evaluated by administering a
labeled polypeptide or conjugate to an animal and measuring
distribution of the polypeptide or conjugate to organs (e.g., using
.sup.3H or .sup.125I labeled conjugates) to mice. Similar
experiments can be performed with compounds that include any of
polypeptides described herein (e.g., the polypeptides described in
Table 1 such as AngioPep-3, AngioPep-4a, AngioPep-4b, AngioPep-5,
AngioPep-6, and AngioPep-7, or analogs thereof). Here, the
unconjugated anticancer agent and the conjugates were injected
intravenously to mice as a bolus. Tissues were collected at
different times (0.25, 0.5, 1, and 4 hrs) and homogenized. To
quantify the amount of .sup.3H-labeled conjugate, tissue
homogenates were digested with tissue solubilizer, and 10 mL of
liquid scintillator was added to samples. The amount of the
.sup.125I labeled conjugate, in the different tissues is measured
after TCA precipitation. Radioactivity associated with the tissues
is quantified. The area under the curve (AUC 0-4) is estimated
using the Prism software and is plotted for the different
tissues.
[0301] FIG. 9 shows the brain distribution of Etop.sub.DMG-An2
following IV bolus administration in mice. FIG. 10 shows the brain
distribution of Etop-An2(3:1) following IV or IP bolus
administration in mice. FIG. 11 compares the brain distribution of
Etop-An2(3:1) versus unconjugated etoposide thirty minutes after IV
bolus administration to mice. FIG. 12 shows the tissue distribution
of Etop-An2(3:1) compared to unconjugated etoposide.
Example 6
Anti-Tumor Effect of Doxorubicin-an2(3:1) Conjugate in a Mouse
Model of Human Brain Tumor
[0302] All animals used in these studies were handled and
maintained in accordance to the Guidelines of the Canadian Council
on Animal Care (CCAC). Animal protocols were approved by the
Institutional Animal Care and Use Committee of Universite du Quebec
a Montreal.
[0303] The intracerebral human brain tumor model was established by
stereotactic inoculation of 5.times.10.sup.5 U87 cells in nude mice
brain. Female athymic nude mice (Crl:Nu/Nu-nuBR; 20-25 g, 4-6 weeks
old; Charles River Canada, St-Constant, QC) were used for tumor
models and were maintained in a pathogen-free environment. One hour
before surgery, mice received a subcutaneous injection of
buprenorphine (0.1 mg kg.sup.-1). For tumor cell inoculation, mice
were anesthetized by i.p. injection of ketamine/xylazine (120/10 mg
kg.sup.-1) and placed in a stereotactic apparatus (Kopf; Tujunga,
Calif.). A burr hole was drilled 1.5 mm anterior and 2.5 mm lateral
to the bregma. The cell suspension in 5 .mu.L of serum free cell
culture medium was injected over a 5 minute period using a Hamilton
syringe at a depth of 3.5 mm.
[0304] Drug treatment started 3 days post-inoculation (Table 8).
The therapeutic compound (e.g., doxorubicin or doxorubicin-An2(3:1)
conjugate) was given intravenously by bolus tail-vein injection
(once per week). Drug solutions were prepared in dextrose 5% water
(D5W). Injection solutions were freshly prepared before each
administration. Clinical signs of disease progression and body
weights were monitored everyday. When mice reached terminal
endpoints (20% of decrease in body weight), they were sacrificed by
carbon dioxide asphyxiation.
TABLE-US-00008 TABLE 8 Survival (days) Dose Median Mean Compound
(mg/kg) Administration Trial 1 Trial 2 Trial 1 Control 0 i.v. 18 22
18.5 Doxorubicin 6 i.v. (1.times. per week) 21 23 20.6
(DoxSu).sub.3-An2 40 i.v. (1.times. per week) 22 28 21.1
(DoxSu).sub.3-An2 + 60 + 40 i.p. (2.times. per week) + 22 -- 22.4
Angiopep2 + paclitaxel conjugate i.v. (1.times. per week)
[0305] Each of FIGS. 13A and 13B show the efficacy of the
(DoxSu).sub.3-An2 conjugate when administered alone or in
combination with a paclitaxel-Angiopep2 conjugate. FIG. 13A shows
results obtained in one trial and FIG. 13B shows results obtained
in a second set of experiments. Statistical analysis of the data
obtained from Trial 2 (FIG. 13B) showed that the observed 27%
improvement was statistically significant (p<0.007).
Other Embodiments
[0306] The content of each publication, patent, and patent
application mentioned in the present application is incorporated by
reference. Although the invention has been described in details
herein and illustrated in the accompanying drawings, it is to be
understood that the invention is not limited to the embodiments
described herein and that various changes and modifications may be
effected without departing from the scope or spirit of the
invention.
[0307] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth, and as follows in the scope of the appended
claims.
Sequence CWU 1
1
116119PRTArtificial sequencepolypeptide 1Thr Phe Val Tyr Gly Gly
Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser 1 5 10 15 Ala Glu Asp
219PRTArtificial sequencepolypeptide 2Thr Phe Gln Tyr Gly Gly Cys
Met Gly Asn Gly Asn Asn Phe Val Thr 1 5 10 15 Glu Lys Glu
319PRTArtificial sequencepolypeptide 3Pro Phe Phe Tyr Gly Gly Cys
Gly Gly Asn Arg Asn Asn Phe Asp Thr 1 5 10 15 Glu Glu Tyr
419PRTArtificial sequencepolypeptide 4Ser Phe Tyr Tyr Gly Gly Cys
Leu Gly Asn Lys Asn Asn Tyr Leu Arg 1 5 10 15 Glu Glu Glu
519PRTArtificial sequencepolypeptide 5Thr Phe Phe Tyr Gly Gly Cys
Arg Ala Lys Arg Asn Asn Phe Lys Arg 1 5 10 15 Ala Lys Tyr
619PRTArtificial sequencepolypeptide 6Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Arg Asn Asn Phe Lys Arg 1 5 10 15 Ala Lys Tyr
719PRTArtificial sequencepolypeptide 7Thr Phe Phe Tyr Gly Gly Cys
Arg Ala Lys Lys Asn Asn Tyr Lys Arg 1 5 10 15 Ala Lys Tyr
819PRTArtificial sequencepolypeptide 8Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Lys Asn Asn Phe Lys Arg 1 5 10 15 Ala Lys Tyr
919PRTArtificial sequencepolypeptide 9Thr Phe Gln Tyr Gly Gly Cys
Arg Ala Lys Arg Asn Asn Phe Lys Arg 1 5 10 15 Ala Lys Tyr
1019PRTArtificial sequencepolypeptide 10Thr Phe Gln Tyr Gly Gly Cys
Arg Gly Lys Lys Asn Asn Phe Lys Arg 1 5 10 15 Ala Lys Tyr
1119PRTArtificial sequencepolypeptide 11Thr Phe Phe Tyr Gly Gly Cys
Leu Gly Lys Arg Asn Asn Phe Lys Arg 1 5 10 15 Ala Lys Tyr
1219PRTArtificial sequencepolypeptide 12Thr Phe Phe Tyr Gly Gly Ser
Leu Gly Lys Arg Asn Asn Phe Lys Arg 1 5 10 15 Ala Lys Tyr
1319PRTArtificial sequencepolypeptide 13Pro Phe Phe Tyr Gly Gly Cys
Gly Gly Lys Lys Asn Asn Phe Lys Arg 1 5 10 15 Ala Lys Tyr
1419PRTArtificial sequencepolypeptide 14Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Gly Asn Asn Tyr Lys Arg 1 5 10 15 Ala Lys Tyr
1519PRTArtificial sequencepolypeptide 15Pro Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Arg Asn Asn Phe Leu Arg 1 5 10 15 Ala Lys Tyr
1619PRTArtificial sequencepolypeptide 16Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Arg Asn Asn Phe Lys Arg 1 5 10 15 Glu Lys Tyr
1719PRTArtificial sequencepolypeptide 17Pro Phe Phe Tyr Gly Gly Cys
Arg Ala Lys Lys Asn Asn Phe Lys Arg 1 5 10 15 Ala Lys Glu
1819PRTArtificial sequencepolypeptide 18Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Arg Asn Asn Phe Lys Arg 1 5 10 15 Ala Lys Asp
1919PRTArtificial sequencepolypeptide 19Thr Phe Phe Tyr Gly Gly Cys
Arg Ala Lys Arg Asn Asn Phe Asp Arg 1 5 10 15 Ala Lys Tyr
2019PRTArtificial sequencepolypeptide 20Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Lys Asn Asn Phe Lys Arg 1 5 10 15 Ala Glu Tyr
2119PRTArtificial sequencepolypeptide 21Pro Phe Phe Tyr Gly Gly Cys
Gly Ala Asn Arg Asn Asn Phe Lys Arg 1 5 10 15 Ala Lys Tyr
2219PRTArtificial sequencepolypeptide 22Thr Phe Phe Tyr Gly Gly Cys
Gly Gly Lys Lys Asn Asn Phe Lys Thr 1 5 10 15 Ala Lys Tyr
2319PRTArtificial sequencepolypeptide 23Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Asn Arg Asn Asn Phe Leu Arg 1 5 10 15 Ala Lys Tyr
2419PRTArtificial sequencepolypeptide 24Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Asn Arg Asn Asn Phe Lys Thr 1 5 10 15 Ala Lys Tyr
2519PRTArtificial sequencepolypeptide 25Thr Phe Phe Tyr Gly Gly Ser
Arg Gly Asn Arg Asn Asn Phe Lys Thr 1 5 10 15 Ala Lys Tyr
2619PRTArtificial sequencepolypeptide 26Thr Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Gly Asn Asn Phe Lys Arg 1 5 10 15 Ala Lys Tyr
2719PRTArtificial sequencepolypeptide 27Thr Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Arg Asn Asn Phe Leu Arg 1 5 10 15 Ala Lys Tyr
2819PRTArtificial sequencepolypeptide 28Thr Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Arg Asn Asn Phe Lys Thr 1 5 10 15 Ala Lys Tyr
2919PRTArtificial sequencepolypeptide 29Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Asn Gly Asn Asn Phe Lys Ser 1 5 10 15 Ala Lys Tyr
3019PRTArtificial sequencepolypeptide 30Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Lys Asn Asn Phe Asp Arg 1 5 10 15 Glu Lys Tyr
3119PRTArtificial sequencepolypeptide 31Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Arg Asn Asn Phe Leu Arg 1 5 10 15 Glu Lys Glu
3219PRTArtificial sequencepolypeptide 32Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Gly Asn Asn Phe Asp Arg 1 5 10 15 Ala Lys Tyr
3319PRTArtificial sequencepolypeptide 33Thr Phe Phe Tyr Gly Gly Ser
Arg Gly Lys Gly Asn Asn Phe Asp Arg 1 5 10 15 Ala Lys Tyr
3419PRTArtificial sequencepolypeptide 34Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Asn Gly Asn Asn Phe Val Thr 1 5 10 15 Ala Lys Tyr
3519PRTArtificial sequencepolypeptide 35Pro Phe Phe Tyr Gly Gly Cys
Gly Gly Lys Gly Asn Asn Tyr Val Thr 1 5 10 15 Ala Lys Tyr
3619PRTArtificial sequencepolypeptide 36Thr Phe Phe Tyr Gly Gly Cys
Leu Gly Lys Gly Asn Asn Phe Leu Thr 1 5 10 15 Ala Lys Tyr
3719PRTArtificial sequencepolypeptide 37Ser Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Lys Asn Asn Phe Leu Thr 1 5 10 15 Ala Lys Tyr
3819PRTArtificial sequencepolypeptide 38Thr Phe Phe Tyr Gly Gly Cys
Gly Gly Asn Lys Asn Asn Phe Val Arg 1 5 10 15 Glu Lys Tyr
3919PRTArtificial sequencepolypeptide 39Thr Phe Phe Tyr Gly Gly Cys
Met Gly Asn Lys Asn Asn Phe Val Arg 1 5 10 15 Glu Lys Tyr
4019PRTArtificial sequencepolypeptide 40Thr Phe Phe Tyr Gly Gly Ser
Met Gly Asn Lys Asn Asn Phe Val Arg 1 5 10 15 Glu Lys Tyr
4119PRTArtificial sequencepolypeptide 41Pro Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Arg Asn Asn Tyr Val Arg 1 5 10 15 Glu Lys Tyr
4219PRTArtificial sequencepolypeptide 42Thr Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Arg Asn Asn Phe Val Arg 1 5 10 15 Glu Lys Tyr
4319PRTArtificial sequencepolypeptide 43Thr Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Lys Asn Asn Tyr Val Arg 1 5 10 15 Glu Lys Tyr
4419PRTArtificial sequencepolypeptide 44Thr Phe Phe Tyr Gly Gly Cys
Gly Gly Asn Gly Asn Asn Phe Leu Thr 1 5 10 15 Ala Lys Tyr
4519PRTArtificial sequencepolypeptide 45Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Asn Arg Asn Asn Phe Leu Thr 1 5 10 15 Ala Glu Tyr
4619PRTArtificial sequencepolypeptide 46Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Asn Gly Asn Asn Phe Lys Ser 1 5 10 15 Ala Glu Tyr
4719PRTArtificial sequencepolypeptide 47Pro Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Lys Asn Asn Phe Lys Thr 1 5 10 15 Ala Glu Tyr
4819PRTArtificial sequencepolypeptide 48Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Asn Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Tyr
4919PRTArtificial sequencepolypeptide 49Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Asp
5019PRTArtificial sequencepolypeptide 50Pro Phe Phe Tyr Gly Gly Cys
Gly Gly Asn Gly Asn Asn Phe Val Arg 1 5 10 15 Glu Lys Tyr
5119PRTArtificial sequencepolypeptide 51Ser Phe Phe Tyr Gly Gly Cys
Met Gly Asn Gly Asn Asn Phe Val Arg 1 5 10 15 Glu Lys Tyr
5219PRTArtificial sequencepolypeptide 52Pro Phe Phe Tyr Gly Gly Cys
Gly Gly Asn Gly Asn Asn Phe Leu Arg 1 5 10 15 Glu Lys Tyr
5319PRTArtificial sequencepolypeptide 53Thr Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Gly Asn Asn Phe Val Arg 1 5 10 15 Glu Lys Tyr
5419PRTArtificial sequencepolypeptide 54Ser Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Gly Asn Asn Tyr Leu Arg 1 5 10 15 Glu Lys Tyr
5519PRTArtificial sequencepolypeptide 55Thr Phe Phe Tyr Gly Gly Ser
Leu Gly Asn Gly Asn Asn Phe Val Arg 1 5 10 15 Glu Lys Tyr
5619PRTArtificial sequencepolypeptide 56Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Asn Gly Asn Asn Phe Val Thr 1 5 10 15 Ala Glu Tyr
5719PRTArtificial sequencepolypeptide 57Thr Phe Phe Tyr Gly Gly Cys
Leu Gly Lys Gly Asn Asn Phe Val Ser 1 5 10 15 Ala Glu Tyr
5819PRTArtificial sequencepolypeptide 58Thr Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Arg Asn Asn Phe Asp Arg 1 5 10 15 Ala Glu Tyr
5919PRTArtificial sequencepolypeptide 59Thr Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Arg Asn Asn Phe Leu Arg 1 5 10 15 Glu Glu Tyr
6019PRTArtificial sequencepolypeptide 60Thr Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Lys Asn Asn Tyr Leu Arg 1 5 10 15 Glu Glu Tyr
6119PRTArtificial sequencepolypeptide 61Pro Phe Phe Tyr Gly Gly Cys
Gly Gly Asn Arg Asn Asn Tyr Leu Arg 1 5 10 15 Glu Glu Tyr
6219PRTArtificial sequencepolypeptide 62Pro Phe Phe Tyr Gly Gly Ser
Gly Gly Asn Arg Asn Asn Tyr Leu Arg 1 5 10 15 Glu Glu Tyr
6319PRTArtificial sequencepolypeptide 63Met Arg Pro Asp Phe Cys Leu
Glu Pro Pro Tyr Thr Gly Pro Cys Val 1 5 10 15 Ala Arg Ile
6421PRTArtificial sequencepolypeptide 64Ala Arg Ile Ile Arg Tyr Phe
Tyr Asn Ala Lys Ala Gly Leu Cys Gln 1 5 10 15 Thr Phe Val Tyr Gly
20 6522PRTArtificial sequencepolypeptide 65Tyr Gly Gly Cys Arg Ala
Lys Arg Asn Asn Tyr Lys Ser Ala Glu Asp 1 5 10 15 Cys Met Arg Thr
Cys Gly 20 6622PRTArtificial sequencepolypeptide 66Pro Asp Phe Cys
Leu Glu Pro Pro Tyr Thr Gly Pro Cys Val Ala Arg 1 5 10 15 Ile Ile
Arg Tyr Phe Tyr 20 6719PRTArtificial sequencepolypeptide 67Thr Phe
Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15
Glu Glu Tyr 6819PRTArtificial sequencepolypeptide 68Lys Phe Phe Tyr
Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu
Tyr 6919PRTArtificial sequencepolypeptide 69Thr Phe Tyr Tyr Gly Gly
Cys Arg Gly Lys Arg Asn Asn Tyr Lys Thr 1 5 10 15 Glu Glu Tyr
7019PRTArtificial sequencepolypeptide 70Thr Phe Phe Tyr Gly Gly Ser
Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Tyr
7120PRTArtificial sequencepolypeptide 71Cys Thr Phe Phe Tyr Gly Cys
Cys Arg Gly Lys Arg Asn Asn Phe Lys 1 5 10 15 Thr Glu Glu Tyr 20
7220PRTArtificial sequencepolypeptide 72Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Tyr Cys 20
7320PRTArtificial sequencepolypeptide 73Cys Thr Phe Phe Tyr Gly Ser
Cys Arg Gly Lys Arg Asn Asn Phe Lys 1 5 10 15 Thr Glu Glu Tyr 20
7420PRTArtificial sequencepolypeptide 74Thr Phe Phe Tyr Gly Gly Ser
Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Tyr Cys 20
7519PRTArtificial sequencepolypeptide 75Pro Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Tyr
7619PRTArtificial sequencepolypeptide 76Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Lys Glu Tyr
7719PRTArtificial sequencepolypeptide 77Thr Phe Phe Tyr Gly Gly Lys
Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Tyr
7819PRTArtificial sequencepolypeptide 78Thr Phe Phe Tyr Gly Gly Cys
Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Lys Arg Tyr
7919PRTArtificial sequencepolypeptide 79Thr Phe Phe Tyr Gly Gly Lys
Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Ala Glu Tyr
8019PRTArtificial sequencepolypeptide 80Thr Phe Phe Tyr Gly Gly Lys
Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Ala Gly Tyr
8119PRTArtificial sequencepolypeptide 81Thr Phe Phe Tyr Gly Gly Lys
Arg Gly Lys Arg Asn Asn Phe Lys Arg 1 5 10 15 Glu Lys Tyr
8219PRTArtificial sequencepolypeptide 82Thr Phe Phe Tyr Gly Gly Lys
Arg Gly Lys Arg Asn Asn Phe Lys Arg 1 5 10 15 Ala Lys Tyr
8319PRTArtificial sequencepolypeptide 83Thr Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Tyr
8419PRTArtificial sequencepolypeptide 84Thr Phe Phe Tyr Gly Cys Gly
Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Tyr
8519PRTArtificial sequencepolypeptide 85Thr Phe Phe Tyr Gly Gly Arg
Cys Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Tyr
8619PRTArtificial sequencepolypeptide 86Thr Phe Phe Tyr Gly Gly Cys
Leu Gly Asn Gly Asn Asn Phe Asp Thr 1 5 10 15 Glu Glu Glu
8719PRTArtificial sequencepolypeptide 87Thr Phe Gln Tyr Gly Gly Cys
Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Tyr
8819PRTArtificial sequencepolypeptide 88Tyr Asn Lys Glu Phe Gly Thr
Phe Asn Thr Lys Gly Cys Glu Arg Gly 1 5 10 15 Tyr Arg Phe
8919PRTArtificial sequencepolypeptide 89Arg Phe Lys Tyr Gly Gly Cys
Leu Gly Asn Met Asn Asn Phe Glu Thr 1 5 10 15 Leu Glu Glu
9019PRTArtificial sequencepolypeptide 90Arg Phe Lys Tyr Gly Gly Cys
Leu Gly Asn Lys Asn Asn Phe Leu Arg 1 5 10 15 Leu Lys Tyr
9119PRTArtificial sequencepolypeptide 91Arg Phe Lys Tyr Gly Gly Cys
Leu Gly Asn Lys Asn Asn Tyr Leu Arg 1 5 10 15 Leu Lys Tyr
9222PRTArtificial sequencepolypeptide 92Lys Thr Lys Arg Lys Arg Lys
Lys Gln Arg Val Lys Ile Ala Tyr Glu 1 5 10 15 Glu Ile Phe Lys Asn
Tyr 20 9315PRTArtificial sequencepolypeptide 93Lys Thr Lys Arg Lys
Arg Lys Lys Gln Arg Val Lys Ile Ala Tyr 1 5 10 15 9417PRTArtificial
sequencepolypeptide 94Arg Gly Gly Arg Leu Ser Tyr Ser Arg Arg Phe
Ser Thr Ser Thr Gly 1 5 10 15 Arg 9510PRTArtificial
sequencepolypeptide 95Arg Arg Leu Ser Tyr Ser Arg Arg Arg Phe 1 5
10 9616PRTArtificial sequencepolypeptide 96Arg Gln Ile Lys Ile Trp
Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 9719PRTArtificial
sequencepolypeptide 97Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg
Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Tyr 9859PRTArtificial
sequencepolypeptide 98Met Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr
Thr Gly Pro Cys Val 1 5 10 15 Ala Arg Ile Ile Arg Tyr Phe Tyr Asn
Ala Lys Ala Gly Leu Cys Gln 20 25 30 Thr Phe Val Tyr Gly Gly Cys
Arg Ala Lys Arg Asn Asn Phe Lys Ser 35 40 45 Ala Glu Asp Cys Met
Arg Thr Cys Gly Gly Ala 50 55 9919PRTArtificial
sequencepolypeptide 99Thr Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg
Asn Asn Phe Lys Thr 1 5 10 15 Lys Glu Tyr 10019PRTArtificial
sequencepolypeptide 100Arg Phe Lys Tyr Gly Gly Cys Leu Gly Asn Lys
Asn Asn Tyr Leu Arg 1 5 10 15 Leu Lys Tyr 10119PRTArtificial
sequencepolypeptide 101Thr Phe Phe Tyr Gly Gly Cys Arg Ala Lys Arg
Asn Asn Phe Lys Arg 1 5 10 15 Ala Lys Tyr 10235PRTArtificial
sequencepolypeptide 102Asn Ala Lys Ala Gly Leu Cys Gln Thr Phe Val
Tyr Gly Gly Cys Leu 1 5 10 15 Ala Lys Arg Asn Asn Phe Glu Ser Ala
Glu Asp Cys Met Arg Thr Cys 20 25 30 Gly Gly Ala 35
10324PRTArtificial sequencepolypeptide 103Tyr Gly Gly Cys Arg Ala
Lys Arg Asn Asn Phe Lys Ser Ala Glu Asp 1 5 10 15 Cys Met Arg Thr
Cys Gly Gly Ala 20 10422PRTArtificial sequencepolypeptide 104Gly
Leu Cys Gln Thr Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn 1 5 10
15 Asn Phe Lys Ser Ala Glu 20 10520PRTArtificial
sequencepolypeptide 105Leu Cys Gln Thr Phe Val Tyr Gly Gly Cys Glu
Ala Lys Arg Asn Asn 1 5 10 15 Phe Lys Ser Ala 20
106180DNAArtificial sequencesynthetic construct 106atgagaccag
atttctgcct cgagccgccg tacactgggc cctgcaaagc tcgtatcatc 60cgttacttct
acaatgcaaa ggcaggcctg tgtcagacct tcgtatacgg cggctgcaga
120gctaagcgta acaacttcaa atccgcggaa gactgcatgc gtacttgcgg
tggtgcttag 18010719PRTArtificial sequencepolypeptide 107Thr Phe Phe
Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu
Glu Tyr 10819PRTArtificial sequencepolypeptide 108Arg Phe Phe Tyr
Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu
Tyr 10919PRTArtificial sequencepolypeptide 109Arg Phe Phe Tyr Gly
Gly Ser Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Tyr
11019PRTArtificial sequencepolypeptide 110Arg Phe Phe Tyr Gly Gly
Ser Arg Gly Lys Arg Asn Asn Phe Arg Thr 1 5 10 15 Glu Glu Tyr
11119PRTArtificial sequencepolypeptide 111Thr Phe Phe Tyr Gly Gly
Ser Arg Gly Lys Arg Asn Asn Phe Arg Thr 1 5 10 15 Glu Glu Tyr
11219PRTArtificial sequencepolypeptide 112Thr Phe Phe Tyr Gly Gly
Ser Arg Gly Arg Arg Asn Asn Phe Arg Thr 1 5 10 15 Glu Glu Tyr
11320PRTArtificial sequencepolypeptide 113Cys Thr Phe Phe Tyr Gly
Gly Ser Arg Gly Lys Arg Asn Asn Phe Lys 1 5 10 15 Thr Glu Glu Tyr
20 11420PRTArtificial sequencepolypeptide 114Thr Phe Phe Tyr Gly
Gly Ser Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Tyr
Cys 20 11520PRTArtificial sequencepolypeptide 115Cys Thr Phe Phe
Tyr Gly Gly Ser Arg Gly Arg Arg Asn Asn Phe Arg 1 5 10 15 Thr Glu
Glu Tyr 20 11620PRTArtificial sequencepolypeptide 116Thr Phe Phe
Tyr Gly Gly Ser Arg Gly Arg Arg Asn Asn Phe Arg Thr 1 5 10 15 Glu
Glu Tyr Cys 20
* * * * *
References