U.S. patent application number 12/069727 was filed with the patent office on 2008-08-14 for multi-arm polymer prodrugs.
This patent application is currently assigned to Nektar Therapeutics AL, Corporation. Invention is credited to Michael D. Bentley, Zhongxu Ren, Tacey X. Viegas, Xuan Zhao.
Application Number | 20080194612 12/069727 |
Document ID | / |
Family ID | 34381082 |
Filed Date | 2008-08-14 |
United States Patent
Application |
20080194612 |
Kind Code |
A1 |
Zhao; Xuan ; et al. |
August 14, 2008 |
Multi-arm polymer prodrugs
Abstract
Provided herein are water-soluble prodrugs. The prodrugs of the
invention comprise a water-soluble polymer having three or more
arms, at least three of which are covalently attached to an active
agent, e.g., a small molecule. The conjugates of the invention
provide an optimal balance of polymer size and structure for
achieving improved drug loading, since the conjugates of the
invention possess three or more active agents releasably attached
to a multi-armed water soluble polymer. The prodrugs of the
invention are therapeutically effective, and exhibit improved
properties in-vivo when compared to unmodified parent drug.
Inventors: |
Zhao; Xuan; (Beijing,
CN) ; Bentley; Michael D.; (Huntsville, AL) ;
Ren; Zhongxu; (Madison, AL) ; Viegas; Tacey X.;
(Madison, AL) |
Correspondence
Address: |
NEKTAR THERAPEUTICS
201 INDUSTRIAL ROAD
SAN CARLOS
CA
94070
US
|
Assignee: |
Nektar Therapeutics AL,
Corporation
Huntsville
AL
|
Family ID: |
34381082 |
Appl. No.: |
12/069727 |
Filed: |
February 11, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10943799 |
Sep 17, 2004 |
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12069727 |
|
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60584308 |
Jun 30, 2004 |
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60503673 |
Sep 17, 2003 |
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Current U.S.
Class: |
514/283 ;
514/772.3 |
Current CPC
Class: |
A61K 47/595 20170801;
A61K 47/61 20170801; A61K 47/60 20170801; C08L 71/02 20130101; A61K
47/58 20170801; A61K 47/59 20170801; A61P 43/00 20180101; A61P
35/00 20180101 |
Class at
Publication: |
514/283 ;
514/772.3 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 31/436 20060101 A61K031/436 |
Claims
1. A polymer conjugate comprising a linear water-soluble and
non-peptidic polymer scaffold comprising from 3 to about 50 pendent
active agent moieties, each active agent moiety being covalently
attached to the scaffold via a spacer comprising a hydrolyzable
linkage, wherein: (i) the spacer comprises an amino acid, (ii) said
scaffold has a molecular weight ranging from about 1,000 daltons to
about 100,000 daltons, and (iii) said active agent moiety is a
camptothecin compound.
2. The conjugate of claim 1, wherein said amino acid is
glycine.
3. The conjugate of claim 1, wherein said spacer has an atom length
of from between about 5 and 25 atoms.
4. The conjugate of claim 1, wherein said hydrolyzable linkage is a
carboxylate ester.
5. The conjugate of claim 4, wherein said camptothecin compound is
released from said conjugate following administration via
hydrolysis of the ester linkage.
6. The conjugate of claim 1, wherein said scaffold comprises
molecules of a polyol.
7. The conjugate of claim 6, wherein said polyol is a
cyclodextrin.
8. The conjugate of claim 1, wherein said scaffold is a linear
water-soluble and non-peptidic copolymer.
9. The conjugate of claim 8, wherein said copolymer is a copolymer
of a polyethylene glycol and a poly(saccharide).
10. The conjugate of claim 9, wherein said poly(saccharide) is a
cyclodextrin.
11. The conjugate of claim 1, wherein said scaffold has a molecular
weight ranging from about greater than about 60,000 daltons to
about 100,000 daltons.
12. The conjugate of claim 1, which when evaluated in a suitable
animal model for solid tumor-type cancers and administered in a
therapeutically effective amount, is effective to suppress tumor
growth to an extent that is at least twice that observed for
camptothecin when evaluated over a time course of 30 days.
13. A conjugate having the following structure:
POLY.sub.1(X-D).sub.q wherein: POLY.sub.1 is a linear water-soluble
and non-peptidic copolymer; D is a camptothecin compound; X is a
spacer comprising an amino acid and a hydrolyzable linkage, said
camptothecin compound is released, and (q) is from 3 to about
50.
14. The conjugate of claim 13, wherein said amino acid is
glycine.
15. The conjugate of claim 13, wherein said hydrolyzable linkage is
a carboxylate ester.
16. The conjugate of claim 15, wherein said camptothecin compound
is released from said conjugate following administration via
hydrolysis of the ester linkage.
17. The conjugate of claim 13, wherein said copolymer is a
copolymer of a polyethylene glycol and a poly(saccharide).
18. The conjugate of claim 17, wherein said poly(saccharide) is a
cyclodextrin.
19. The conjugate of claim 13, wherein said spacer X has a
structure Y-Z where Y is a spacer fragment covalently attached to
Z, a hydrolyzable linkage.
20. The conjugate of claim 19, wherein Z is a carboxylate
ester.
21. The conjugate of claim 20, wherein Y has the structure
--(CR.sub.xR.sub.y).sub.a--K--(CR.sub.xR.sub.y).sub.b--(CH.sub.2CH.sub.2O-
).sub.c--, wherein each R.sub.x and R.sub.y, in each occurrence, is
independently H or an organic radical selected from the group
consisting of alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl,
a ranges from 0 to 12, b ranges from 0 to 12, c ranges from 0 to
25, K is selected from --C(O)--, --C(O)NH--, --NH--C(O)--, --O--,
--S--, O--C(O)--, C(O)--O--, O--C(O)--O--, O--C(O)--NH--,
NH--C(O)--O--.
22. The conjugate of claim 13, which when evaluated in a suitable
animal model for solid tumor-type cancers and administered in a
therapeutically effective amount, is effective to suppress tumor
growth to an extent that is at least twice that observed for
camptothecin when evaluated over a time course of 30 days.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 10/943,799, filed Sep. 17, 2004, which claims the benefit of
priority to U.S. Provisional Application No. 60/503,673, filed Sep.
17, 2003, and to U.S. Provisional Application Ser. No. 60/584,308,
filed Jun. 30, 2004, the contents of which are incorporated herein
by reference in their entirety.
FIELD
[0002] This invention relates to multi-arm, water-soluble polymer
drug conjugates, and in particular, to polymer-based prodrugs, and
to methods for preparing, formulating and administering
compositions comprising such prodrugs.
BACKGROUND
[0003] Over the years, numerous methods have been proposed for
improving the delivery of biologically active agents. Challenges
associated with the formulation and delivery of pharmaceutical
agents can include poor aqueous solubility of the pharmaceutical
agent, toxicity, low bioavailability, instability, and rapid
in-vivo degradation, to name just a few. Although many approaches
have been devised for improving the delivery of pharmaceutical
agents, no single approach is without its drawbacks. For instance,
commonly employed drug delivery approaches aimed at solving or at
least ameliorating one or more of these problems include drug
encapsulation, such as in a liposome, polymer matrix, or
unimolecular micelle, covalent attachment to a water-soluble
polymer such as polyethylene glycol, use of gene targeting agents,
and the like.
[0004] In looking more closely at some of these approaches,
liposome encapsulation is often plagued by low efficiencies of drug
loading, resulting in an oftentimes inefficient and cost
ineffective process. Moreover, the release rate of the active agent
in a liposomal formulation depends upon dissolution or
disintegration of the liposome, or diffusion of the active agent
through the liposomal layers, thereby limiting the practical
availability of the active agent to the biological system. In
addition, liposomal formulations are generally restricted to lipid
soluble drugs. Polymer matrix-based formulations can suffer from
similar shortcomings, such as the inability to well-characterize
such drug delivery systems, particular those that are cross-linked,
and the variable release rates associated with active agents that
must diffuse out of a hydrolytically degradable polymer matrix. In
comparison, conjugation of an active agent to a polymer such as
polyethylene glycol offers a more well-defined alternative, since
the conjugate itself is often although not necessarily
well-characterized, particularly in the case of site-specific
attachment of the polymer to the active agent. However,
protein-based compositions containing mixtures of positional
isomers varying in both the site(s) and number of polymer chains
attached to a particular protein are not uncommon. This can lead to
problems with reproducibly preparing such compositions.
[0005] While modification of therapeutic proteins for the purpose
of improving their pharmaceutical utility is perhaps one of the
most common applications of PEGylation, PEGylation has also been
used, albeit to a limited degree, to improve the bioavailability
and ease of formulation of small molecule therapeutics having poor
aqueous solubilities. For instance, water-soluble polymers such as
PEG have been covalently attached to artilinic acid to improve its
aqueous solubility (Bentley, et al., U.S. Pat. No. 6,461,603).
Similarly, PEG has been covalently attached to triazine-based
compounds such as trimelamol to improve their solubility in water
and enhance their chemical stability (Bentley, et al., WO
02/043772). Covalent attachment of PEG to bisindolyl maleimides has
been employed to improve poor bioavailability of such compounds due
to low aqueous solubility (Bentley, et al., WO 03/037384). Prodrugs
of camptothecin having one or two molecules of camptothecin
covalently attached to a linear polyethylene glycol have similarly
been prepared (Greenwald, et al, U.S. Pat. No. 5,880,131).
[0006] Camptothecin (often abbreviated as "CPT") is a phytotoxic
alkaloid first isolated from the wood and bark of Camptotheca
acuminata (Nyssaceae), and has been shown to exhibit antitumor
activity. The compound has a pentacyclic ring system with an
asymmetric center in lactone ring E with a 20 S configuration. The
pentacyclic ring system includes a pyrrolo[3,4-b]quinoline (rings
A, B and C), a conjugated pyridone (ring D), and a six-membered
lactone (ring E) with a 20-hydroxyl group. Due to its insolubility
in water, camptothecin was initially evaluated clinically in the
form of a water-soluble carboxylate salt having the lactone ring
open to form the sodium salt. The sodium salt, although exhibiting
much improved water solubility in comparison to camptothecin
itself, produced severe toxicity and demonstrated very little in
vivo anticancer activity, thus demonstrating the undesirability of
this approach.
[0007] It was later discovered that camptothecin and many of its
derivatives inhibit topoisomerase, an enzyme that is required for
swiveling and relaxation of DNA during molecular events such as
replication and transcription. Camptothecin stabilizes and forms a
reversible enzyme-camptothecin-DNA ternary complex. The formation
of the cleavable complex specifically prevents the reunion step of
the breakage/union cycle of the topoisomerase reaction.
Topoisomerase I inhibitors are also known to be useful in the
treatment of HIV.
[0008] In an effort to address the poor aqueous solubility
associated with camptothecin and many of its derivatives, a number
of synthetic efforts have been directed to derivatizing the A-ring
and/or B-ring or esterifying the 20-hydroxyl to improve
water-solubility while maintaining cytotoxic activity. For example,
topotecan (9-dimethylaminomethyl-10-hydroxy CPT) and irinotecan
(7-ethyl-10[4-(1-piperidino)-1-piperidino]carbonyloxy CPT),
otherwise known as CPT-11, are two water-soluble CPT derivatives
that have shown clinically useful activity. Conjugation of certain
camptothecin derivatives, such as 10-hydroxycamptothecin and
11-hydroxycamptothecin, to a linear poly(ethylene glycol) molecule
via an ester linkage has been described as a means to form water
soluble prodrugs (Greenwald, et al., U.S. Pat. No. 6,011,042).
[0009] The clinical effectiveness of many small molecule
therapeutics, and oncolytics in particular, is limited by several
factors. For instance, irinotecan and other camptothecin
derivatives undergo an undesirable hydrolysis of the E-ring lactone
under alkaline conditions. Additionally, administration of
irinotecan causes a number of troubling side effects, including
leukopenia and diarrhea. Due to its severe diarrheal side-effect,
the dose of irinotecan that can be administered in its
conventional, unmodified form is extremely limited, thus hampering
the efficacy of this drug and others of this type.
[0010] These associated side effects, when severe, can be
sufficient to arrest further development of such drugs as promising
therapeutics. Additional challenges facing small molecules include
high clearance rates, and in the instance of anticancer agents,
minimal tumor permeation and residence time. Approaches involving
the use of polymer attachment must balance the size of the polymer
against the molecular weight of the active agent in order to allow
therapeutically effective doses to be delivered. Finally, the
synthesis of a modified or drug-delivery enhanced active agent must
result in reasonable yields, to make any such approach economically
attractive. Thus, there exists a need for new methods for
effectively delivering drugs, and in particular small molecule
drugs, and even more particularly oncolytics, which can reduce
their adverse and often toxic side-effects, whilst simultaneously
improving their efficacy and ease of formulation. Specifically,
there is a need for improved methods for delivering drugs that
possess an optimal balance of bioavailability due to reduced
clearance times, bioactivity, and efficacy, coupled with reduced
side-effects. The present invention meets those needs.
SUMMARY
[0011] In one aspect, the present invention provides water-soluble
prodrugs. The prodrugs of the invention comprise a water-soluble
polymer having three or more arms, at least three of which are
covalently attached to an active agent, e.g., a small molecule. The
conjugates of the invention provide an optimal balance of polymer
size and structure for achieving improved drug loading, since the
conjugates of the invention possess three or more active agents
attached, preferably releasably, to a water soluble polymer. In one
embodiment, each of the arms of the water soluble polymer possesses
an active agent covalently attached thereto, preferably by a
hydrolyzable linkage.
[0012] In one embodiment, the prodrug conjugate comprises a
multi-arm polymer, i.e., having three or more arms, where the
conjugate comprises the following generalized structure:
R(-Q-POLY.sub.1-X-D).sub.q I
[0013] In structure I, R is an organic radical possessing from
about 3 to about 150 carbon atoms, preferably from about 3 to about
50 carbon atoms, and even more preferably from about 3 to about 10
carbon atoms, optionally containing one or more heteroatoms (e.g.,
O, S, or N). In one embodiment, R possesses a number of carbon
atoms selected from the group consisting of 3, 4, 5, 6, 7, 8, 9,
and 10. R may be linear or cyclic, and typically, emanating
therefrom are at least 3 independent polymer arms each having at
least one active agent moiety covalently attached thereto. Looking
at the above structure, "q" corresponds to the number of polymer
arms emanating from "R".
[0014] In structure I, Q is a linker, preferably one that is
hydrolytically stable. Typically, Q contains at least one
heteroatom such as O, or S, or NH, where the atom proximal to R in
Q, when taken together with R, typically represents a residue of
the core organic radical R. Illustrative examples are provided
below. Generally, Q contains from 1 to about 10 atoms, or from 1 to
about 5 atoms. More particularly, Q typically contains one of the
following number of atoms: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a
particular embodiment, Q is O, S, or --NH--C(O)--.
[0015] In structure I, POLY.sub.1 represents a water-soluble and
non-peptidic polymer. Representative polymers include poly(alkylene
glycol), poly(olefinic alcohol), poly(vinylpyrrolidone),
poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),
poly(saccharide), poly(.alpha.-hydroxy acid), poly(acrylic acid),
poly(vinyl alcohol), polyphosphazene, polyoxazoline,
poly(N-acryloylmorpholine), or copolymers or terpolymers thereof.
In a particular embodiment of structure I, POLY.sub.1 is a
polyethylene glycol, preferably a linear polyethylene glycol (i.e.,
in each arm of the overall multi-arm structure). In yet another
embodiment, POLY.sub.1 corresponds to the structure,
--(CH.sub.2CH.sub.2O).sub.n--, where n ranges from about 10 to
about 400, preferably from about 50 to about 350.
[0016] In structure I, X is a spacer that comprises a hydrolyzable
linkage, where the hydrolyzable linkage is attached directly to the
active agent, D. Typically, at least one atom of the hydrolyzable
linkage is contained in the active agent, D, in its unmodified
form, such that upon hydrolysis of the hydrolyzable linkage
comprised within X, the active agent, D, is released. Generally
speaking, the spacer, X, has an atom length of from about 4 atoms
to about 50 atoms, or more preferably from about 5 atoms to about
25 atoms, or even more preferably from about 5 atoms to about 20
atoms. Representative spacers have a length of from about 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20
atoms.
[0017] In yet another particular embodiment, X possesses the
structure: Y-Z, where Y is a spacer fragment covalently attached to
Z, a hydrolytically degradable linkage. In certain embodiments, Z
itself may not constitute a hydrolytically degradable linkage,
however, when taken together with Y, or at least a portion of Y,
forms a linkage that is hydrolytically degradable.
[0018] In yet a more particular embodiment of the spacer, X, Y has
the structure:
--(CR.sub.xR.sub.y).sub.a--K--(CR.sub.xR.sub.y).sub.b--(CH.sub.2CH.sub.2O-
).sub.c--, wherein each R.sub.x and R.sub.y, in each occurrence, is
independently H or an organic radical selected from the group
consisting of alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl,
a ranges from 0 to 12 (i.e., can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12), b ranges from 0 to 12 (i.e., can be 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, or 12), K is selected from --C(O)--,
--C(O)NH--, --NH--C(O)--, --O--, --S--, O--C(O)--, C(O)--O--,
O--C(O)--O--, O--C(O)--NH--, NH--C(O)--O--, c ranges from 0 to 25,
and Z is selected from C(O)--O--, O--C(O)--O--, --O--C(O)--NH--,
and NH--C(O)--O--. The particular structure of K and of Z will
depend upon the values of each of a, b, and c, such that none of
the following linkages result in the overall structure of spacer X,
--O--O--, NH--O--, NH--NH--.
[0019] Preferably, Y comprises
(CH.sub.2).sub.a--C(O)NH--(CH.sub.2).sub.0,1--(CH.sub.2CH.sub.2O).sub.0-1-
0.
[0020] In yet another embodiment of the spacer, X, Y has the
structure:
--(CR.sub.xR.sub.y).sub.a--K--(CR.sub.xR.sub.y).sub.b--(CH.sub.2CH.sub.2N-
H).sub.c--, where the variables have the values previously
described. In certain instances, the presence of the short ethylene
oxide or ethyl amino fragments in spacer, X, can be useful in
achieving good yields during preparation of the prodrug conjugate,
since the presence of the linker can help to circumvent problems
associated with steric hindrance, due to the multi-armed reactive
polymer, the structure of the active agent, or a combination of
both. Preferably, c is selected from the group consisting of 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, and 10.
[0021] Preferably, R.sub.x and R.sub.y in each occurrence are
independently H or lower alkyl. In one embodiment, R.sub.x and
R.sub.y are in each occurrence H. In yet another embodiment, a
ranges from 0 to 5. In yet another embodiment, b ranges from 0 to
5. In yet another embodiment, c ranges from 0 to 10. In yet another
embodiment, K is --C(O)--NH. Any of the embodiments described
herein is meant to apply not only to generalized structure I, but
also to extend to particular combinations of embodiments.
[0022] In yet another embodiment, R.sub.x and R.sub.y in each
occurrence are H, a is 1, K is --C(O)--NH, and b is 0 or 1.
[0023] Representative examples of X include
--CH.sub.2--C(O)--NH--CH.sub.2--C(O)O-- (here, Y corresponds to
--CH.sub.2--C(O)--NH--CH.sub.2-- and Z corresponds to --C(O)--O--),
and --CH.sub.2--C(O)--NH--(CH.sub.2CH.sub.2O).sub.2--C(O)--O--
(here, Y corresponds to
--CH.sub.2--C(O)--NH--(CH.sub.2CH.sub.2O).sub.2-- and Z corresponds
to --C(O)--O--).
[0024] Returning now to structure I, D is an active agent moiety,
and q (the number of independent polymer arms) ranges from about 3
to about 50. Preferably, q ranges from about 3 to about 25. More
preferably, q is from 3 to about 10, and possesses a value of 3, 4,
5, 6, 7, 8, 9, or 10.
[0025] In accordance with one embodiment of the invention, the
conjugate comprises a polymer having from about 3 to about 25
active agent molecules covalently attached thereto. More
particularly, the conjugate comprises a water soluble polymer
having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25 active agent molecules covalently
attached thereto. In a further embodiment, the conjugate of the
invention has from about 3 to about 8 active agent molecules
covalently attached to the water-soluble polymer. Typically,
although not necessarily, the number of polymer arms will
correspond to the number of active agents covalently attached to
the water soluble polymer.
[0026] The active agent moiety, D, is an active agent comprising a
functional group suitable for covalent attachment to the
multi-armed polymer described herein to form a hydrolyzable
linkage, such that upon hydrolysis, the active agent is released in
its unmodified form.
[0027] Preferred active agent moieties include anticancer
agents.
[0028] In one embodiment, the active agent is a small molecule. In
a particular embodiment, the active agent moiety is a small
molecule possessing a molecular weight of less than about 1000. In
yet additional embodiments, the small molecule drug possesses a
molecular weight of less than about 800, or even less than about
750. In yet another embodiment, the small molecule drug possesses a
molecular weight of less than about 500 or, in some instances, even
less than about 300.
[0029] In yet another embodiment, the small molecule is an
oncolytic drug having at least one hydroxyl group.
[0030] In yet a further embodiment, D represents a camptothecin
compound having the structure:
##STR00001##
wherein R.sub.1-R.sub.5 are each independently selected from the
group consisting of hydrogen; halo; acyl; alkyl (e.g., C1-C6
alkyl); substituted alkyl; alkoxy (e.g., C1-C6 alkoxy); substituted
alkoxy; alkenyl; alkynyl; cycloalkyl; hydroxyl; cyano; nitro;
azido; amido; hydrazine; amino; substituted amino (e.g.,
monoalkylamino and dialkylamino); hydroxycarbonyl; alkoxycarbonyl;
alkylcarbonyloxy; alkylcarbonylamino; carbamoyloxy;
arylsulfonyloxy; alkylsulfonyloxy;
--C(R.sub.7).dbd.N--(O).sub.i--R.sub.8 wherein R.sub.7 is H, alkyl,
alkenyl, cycloalkyl, or aryl, i is 0 or 1, and R.sub.8 is H, alkyl,
alkenyl, cycloalkyl, or heterocycle; and R.sub.9C(O)O-- wherein
R.sub.9 is halogen, amino, substituted amino, heterocycle,
substituted heterocycle, or R.sub.10--O--(CH.sub.2).sub.m--where m
is an integer of 1-10 and R.sub.10 is alkyl, phenyl, substituted
phenyl, cycloalkyl, substituted cycloalkyl, heterocycle, or
substituted heterocycle; or
[0031] R.sub.2 together with R.sub.3 or R.sub.3 together with
R.sub.4 form substituted or unsubstituted methylenedioxy,
ethylenedioxy, or ethyleneoxy;
[0032] R.sub.6 is H or OR', wherein R' is alkyl, alkenyl,
cycloalkyl, haloalkyl, or hydroxyalkyl; and
[0033] L is the site of attachment to X.
[0034] In yet another particular embodiment, D is irinotecan.
##STR00002##
[0035] Alternatively, D is a small molecule selected from the group
consisting of platins, oxymorphone analogues, steroids, quinolones,
and nucleosides.
[0036] In one embodiment, D is a platin such as cis-platin,
hydroxyplatin, carboplatin, or oxaliplatin.
[0037] In yet a further embodiment, D is an oxymorphone analogue
such as naloxone, methylnaltrexone, oxymorphone, codeine,
oxycodone, or morphone.
[0038] In yet an additional embodiment, D is a steroid such as
budesonide, triamcinolone, or fluticasone.
[0039] In yet another embodiment, D is a quinolone, isoquinolone or
fluoroquinolone such as ciprofloxacin, moxifloxacin, or
palonosetron.
[0040] In yet an additional embodiment, D is a nucleoside or
nucleotide such as gemcitabine, cladribine, or fludarabine.
[0041] The multi-armed polymer prodrugs of the invention possess
many unique features, particularly in the instance where the small
molecule is an anticancer compound. For example, in one embodiment,
provided is a multi-armed polymer prodrug, which when evaluated in
a suitable animal model for solid tumor-type cancers and
administered in a therapeutically effective amount, is effective to
suppress tumor growth to an extent that is at least 1.5 times that,
or even twice that observed for the unmodified anticancer agent,
when evaluated over a time course of 30 days. In yet another
embodiment, the prodrug is effective to suppress tumor growth to
the above extent or even greater when evaluated over a time course
of 60 days. The small molecule employed is one known to possess
anticancer properties, however, by virtue of its conjugation to a
multi-armed polymer as described herein, possesses significantly
improved efficacy and pharmacokinetics in comparison to the small
molecule, e.g., anticancer compound, itself. Suitable solid tumor
types include malignant sarcomas, carcinomas and lymphomas of the
breast, ovaries, colon, kidney, bile duct, lung and brain.
[0042] In another aspect, the invention encompasses reactive
multi-armed polymers suitable for preparing any of the
above-described prodrug conjugates.
[0043] In another aspect, the invention encompasses a
pharmaceutical composition comprising a multi-arm polymer prodrug
conjugate as described above in combination with a pharmaceutically
acceptable carrier.
[0044] Another aspect of the invention provides a method for
treating various medical conditions in a mammalian subject. More
specifically, the invention encompasses a method of administering
to a mammalian subject in need thereof a therapeutically effective
amount of a multi-arm prodrug conjugate of the invention. In one
embodiment, the drug moiety, D, is an anticancer agent such as a
camptothecin (e.g., irinotecan), and is effective to suppress tumor
growth. In a particularly preferred embodiment, a multi-armed
prodrug conjugate of the invention, particularly one where D is an
anticancer agent, exhibits one or more of the following
characteristics: (i) suppresses tumor growth to an extent greater
than that of unmodified D, (ii) demonstrates a tumor retention time
that is increased over that of unmodified D, (iii) exhibits a rate
of clearance that is reduced in comparison to that of unmodified D,
and/or (iv) produces reduced adverse side effects in comparison to
unmodified D.
[0045] According to yet another aspect, the invention provides a
method of treating cancer or a viral infection by administering a
multi-arm polymer conjugate as described herein.
[0046] In yet another aspect, the invention provides a method of
treating a topoisomerase I inhibitor-related disease in a mammalian
subject by administering a therapeutically effective amount of a
multi-arm polymer prodrug to a mammalian subject in need thereof,
where the small molecule is a camptothecin type molecule.
[0047] According to yet another aspect, provided herein is a method
of targeting a solid tumor in a mammalian subject. The method
includes the step of administering a therapeutically effective
amount of a multi-arm polymer prodrug of an anticancer agent known
to be effective in the treatment of solid tumors to a subject
diagnosed as having one or more cancerous solid tumors. As a result
of said administering, the prodrug is effective to produce an
inhibition of solid tumor growth in the subject that is increased
over the inhibition of solid tumor growth resulting from
administration of the anticancer agent alone.
[0048] In a further aspect, a method for preparing a multi-arm
polymer prodrug conjugate of the invention is provided. In the
method, a small molecule, D, is provided, where the small molecule
comprises a functional group, F, suitable for forming a
hydrolyzable linkage, Z. The small molecule is reacted with a
bifunctional spacer, Y', comprising each a first and a second
functional group, F1 and F2. The functional group F2 is suitable
for reaction with F, and F1 may optionally be in protected form
(F1-Y'-F2). The reaction is carried out under conditions effective
to form a partially modified active agent comprising a hydrolyzable
linkage, Z, resulting from reaction of F and F2, which corresponds
to the structure D-Z-Y'-F1. If necessary, the method includes the
optional step of deprotecting F1 contained in the partially
modified active agent. The method then includes the step of
reacting the partially modified active agent, D-Z-Y'-F1, with a
multi-armed water-soluble polymer comprising the structure,
R(-Q-POLY.sub.1-F3).sub.q, where R, Q, POLY.sub.1, and Q are as
previously defined, and F3 is a functional group that is reactive
with F1. The reaction is carried out under conditions effective to
promote reaction between F3 and F1 to convert Y' to Y, to thereby
form a polymer prodrug having the structure,
R(-Q-POLY.sub.1-Y-Z-D).sub.q, where Y is a spacer fragment, and Z
is a hydrolyzable linkage, which, upon hydrolysis, releases D.
[0049] In one embodiment of the method, a stoichiometric excess in
an amount greater than "q" moles of the partially modified active
agent, D-Z-Y'-F1, is reacted with the multi-armed water-soluble,
R(-Q-POLY.sub.1-F3).sub.q to drive the reaction to completion,
i.e., to covalently attach active agent to each of the reactive
polymer arms.
[0050] In yet another embodiment, where the small molecule D
possesses additional functional groups reactive with F2, the method
further comprises the step of protecting the additional functional
groups with suitable protecting groups prior to reaction with the
bifunctional spacer. These protecting groups are then removed from
the small molecules of the prodrug product,
R(-Q-POLY.sub.1-Y-Z-D).sub.q.
[0051] According to yet another aspect of the invention, provided
is yet another method for preparing a multi-arm polymer prodrug of
the invention. The method includes the step of providing a reactive
multi-arm polymer having the structure, R(-Q-POLY.sub.1-F3).sub.q,
where R, Q, POLY.sub.1, and q are as previously described, and F3
is a reactive functional group. The multi-arm polymer is then
reacted with a bifunctional spacer, Y', comprising each a first and
a second functional group, F1 and F2, wherein F1 is suitable for
reaction with F3, and F1 is optionally in protected form
(F1-Y'-F2). The reaction is carried out under conditions effective
to form an intermediate multi-arm polymer resulting from reaction
of F3 and F1, and having the structure,
R(-Q-POLY.sub.1-Y-F2).sub.q. The method further includes the
optional step of deprotecting F2 in the intermediate multi-arm
polymer, R(-Q-POLY.sub.1-Y-F2).sub.q if such is in protected form.
The intermediate multi-arm polymer, R(-Q-POLY.sub.1-Y-F2).sub.q, is
then reacted with a small molecule, D, comprising a functional
group, F, suitable for forming a hydrolyzable linkage, Z, upon
reaction of F with F2, under conditions effective to thereby form a
prodrug having the structure, R(-Q-POLY.sub.1-Y-Z-D).sub.q, where Z
is a hydrolyzable linkage, which, upon hydrolysis, releases D.
[0052] Reactive functional groups such as those described above as
F1, F2 and F3, are numerous and may be selected from, for example,
hydroxyl, active ester (e.g., N-hydroxysuccinimidyl ester and
1-benzotriazolyl ester), active carbonate (e.g.,
N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate,
p-nitrophenyl carbonate), acid halide, acetal, aldehyde having a
carbon length of 1 to 25 carbons (e.g., acetaldehyde,
propionaldehyde, and butyraldehyde), aldehyde hydrate, alkenyl,
acrylate, methacrylate, acrylamide, active sulfone, amine,
hydrazide, thiol, alkanoic acids having a carbon length (including
the carbonyl carbon) of 1 to about 25 carbon atoms (e.g.,
carboxylic acid, carboxymethyl, propanoic acid, and butanoic acid),
isocyanate, isothiocyanate, maleimide, vinylsulfone,
dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, and
dione.
[0053] In one embodiment, the bifunctional spacer, Y' is an amino
acid or derived from an amino acid. Representative amino acids have
the structure HO--C(O)--CH(R'')--NH-Gp wherein R'' is H, C1-C6
alkyl, or substituted C1-C6 alkyl and Gp is an amino-protecting
group. In an alternative embodiment, the bifunctional spacer, Y'
possesses the structure:
--C(O)--(OCH.sub.2CH.sub.2).sub.1-10--NH-Gp.
[0054] The above methods for preparing a prodrug of the invention
may include the additional steps of purifying the intermediates
and/or the final prodrug products, for example by size exclusion
chromatography or ion exchange chromatography in instances in which
the compounds to be purified contain one or more ionizable groups,
such as carboxyl or amino.
[0055] These and other objects and features of the invention will
become more fully apparent when read in conjunction with the
following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] FIG. 1 is a graph illustrating the effect of an exemplary
multi-arm PEG-irinotecan conjugate on the growth of HT29 human
colon tumors implanted in athymic nude mice in comparison to an
untreated control group and a group treated with irinotecan as
described in detail in Example 2;
[0057] FIG. 2 is a graph illustrating the effects of a variety of
doses (90 mg/kg; 60 mg/kg; and 40 mg/kg) of an exemplary 20
kilodalton (20 K) multi-arm PEG irinotecan conjugate on the growth
of NCl-H460 human lung tumors implanted in athymic nude mice in
comparison to a control group and a group treated with irinotecan
as described in Example 6;
[0058] FIG. 3 is a graph illustrating the effects of a variety of
doses (90 mg/kg; 60 mg/kg; and 40 mg/kg) of an exemplary 40
kilodalton (40 K) multi-arm PEG irinotecan conjugate on the growth
of NCl-H460 human lung tumors implanted in athymic nude mice in
comparison to a control group and a group treated with irinotecan
as described in Example 6;
[0059] FIG. 4 is a graph illustrating the effects of a variety of
doses (90 mg/kg; 60 mg/kg; and 40 mg/kg) of an exemplary 20
kilodalton (20 K) multi-arm PEG-irinotecan conjugate on the growth
of HT29 human colon tumors implanted in athymic nude mice in
comparison to an untreated control group and a group treated with
irinotecan as described in detail in Example 6;
[0060] FIG. 5 is a graph illustrating the effects of a variety of
doses (90 mg/kg; 60 mg/kg; and 40 mg/kg) of an exemplary 40
kilodalton (40 K) multi-arm PEG-irinotecan conjugate on the growth
of HT29 human colon tumors implanted in athymic nude mice in
comparison to an untreated control group and a group treated with
irinotecan as described in detail in Example 6;
[0061] FIG. 6 is a graph illustrating the concentration in venous
plasma over time of (i) an exemplary 20 kilodalton (20 K) multi-arm
PEG irinotecan conjugate, and (ii) a 40 kilodalton multi-arm PEG
irinotecan conjugate, following IV administration as a single dose
in athymic nude mice implanted with either HT29 human colon tumors
or NCl-H460 human lung tumors as described in Example 7.
[0062] FIG. 7 is a graph illustrating the concentration in tumor
tissue over time of (i) an exemplary 20 kilodalton (20 K) multi-arm
PEG irinotecan conjugate, and (ii) a 40 kilodalton multi-arm PEG
irinotecan conjugate, following IV administration as a single dose
in athymic nude mice implanted with either HT29 human colon tumors
or NCl-H460 human lung tumors as described in Example 7.
[0063] FIG. 8 is a graph illustrating the concentration of
PEG-SN-38 in plasma over time following IV administration of (i) an
exemplary 20 kilodalton (20 K) multi-arm PEG irinotecan conjugate,
or (ii) a 40 kilodalton multi-arm PEG irinotecan conjugate, as a
single dose in athymic nude mice implanted with either HT29 human
colon tumors or NCl-H460 human lung tumors as described in Example
7.
[0064] FIG. 9 is a graph illustrating the concentration of PEG
SN-38 in tumor tissue over time following IV administration of (i)
an exemplary 20 kilodalton (20 K) multi-arm PEG irinotecan
conjugate, or (ii) a 40 kilodalton multi-arm PEG irinotecan
conjugate, as a single dose in athymic nude mice implanted with
either HT29 human colon tumors or NCl-H460 human lung tumors as
described in Example 7.
[0065] FIG. 10 is a graph illustrating the concentration of
irinotecan in venous plasma over time following IV administration
of (i) an exemplary 20 kilodalton (20 K) multi-arm PEG irinotecan
conjugate, or (ii) a 40 kilodalton multi-arm PEG irinotecan
conjugate, or (iii) irinotecan itself as a single dose in athymic
nude mice implanted with either HT29 human colon tumors or NCl-H460
human lung tumors as described in Example 7.
[0066] FIG. 11 is a graph illustrating the concentration of
irinotecan in tumor tissue over time following IV administration of
(i) an exemplary 20 kilodalton (20 K) multi-arm PEG irinotecan
conjugate, or (ii) a 40 kilodalton multi-arm PEG irinotecan
conjugate, or (iii) irinotecan itself, as a single dose in athymic
nude mice implanted with either HT29 human colon tumors or NCl-H460
human lung tumors as described in Example 7.
[0067] FIG. 12 is a graph illustrating the concentration of SN-38
in plasma over time following IV administration of (i) an exemplary
20 kilodalton (20 K) multi-arm PEG irinotecan conjugate, or (ii) a
40 kilodalton multi-arm PEG irinotecan conjugate, or (iii)
irinotecan itself, as a single dose in athymic nude mice implanted
with either HT29 human colon tumors or NCl-H460 human lung tumors
as described in Example 7.
[0068] FIG. 13 is a graph illustrating the concentration of SN-38
in tumor tissue over time following IV administration of (i) an
exemplary 20 kilodalton (20 K) multi-arm PEG irinotecan conjugate,
or (ii) a 40 kilodalton multi-arm PEG irinotecan conjugate, or
(iii) irinotecan itself, as a single dose in athymic nude mice
implanted with either HT29 human colon tumors or NCl-H460 human
lung tumors as described in Example 7.
DETAILED DESCRIPTION
[0069] The present invention now will be described more fully
hereinafter. This invention may, however, be embodied in many
different forms and should not be construed as limited to the
embodiments set forth herein; rather, these embodiments are
provided so that this disclosure will be thorough and complete, and
will fully convey the scope of the invention to those skilled in
the art.
DEFINITIONS
[0070] It must be noted that, as used in this specification, the
singular forms "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to a "polymer" includes a single polymer as well as two
or more of the same or different polymers, reference to a
"conjugate" refers to a single conjugate as well as two or more of
the same or different conjugates, reference to an "excipient"
includes a single excipient as well as two or more of the same or
different excipients, and the like.
[0071] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions described below.
[0072] A "functional group" is a group that may be used, under
normal conditions of organic synthesis, to form a covalent linkage
between the structure to which it is attached and another
structure, which typically bears a further functional group. The
functional group generally includes multiple bond(s) and/or
heteroatom(s). Preferred functional groups for use in the polymers
of the invention are described below.
[0073] The term "reactive" refers to a functional group that reacts
readily or at a practical rate under conventional conditions of
organic synthesis. This is in contrast to those groups that either
do not react or require strong catalysts or impractical reaction
conditions in order to react (i.e., a "nonreactive" or "inert"
group).
[0074] "Not readily reactive", with reference to a functional group
present on a molecule in a reaction mixture, indicates that the
group remains largely intact under conditions effective to produce
a desired reaction in the reaction mixture.
[0075] An "activated derivative" of a carboxylic acid refers to a
carboxylic acid derivative which reacts readily with nucleophiles,
generally much more readily than the underivatized carboxylic acid.
Activated carboxylic acids include, for example, acid halides (such
as acid chlorides), anhydrides, carbonates, and esters. Such esters
include, for example, imidazolyl esters, and benzotriazole esters,
and imide esters, such as N-hydroxysuccinimidyl (NHS) esters. An
activated derivative may be formed in situ by reaction of a
carboxylic acid with one of various reagents, e.g.
benzotriazol-1-yloxy tripyrrolidinophosphonium hexafluorophosphate
(PyBOP), preferably used in combination with 1-hydroxy
benzotriazole (HOBT) or 1-hydroxy-7-azabenzotriazole (HOAT);
O-7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU); or bis(2-oxo-3-oxazolidinyl)phosphinic
chloride (BOP-Cl).
[0076] A "protecting group" is a moiety that prevents or blocks
reaction of a particular chemically reactive functional group in a
molecule under certain reaction conditions. The protecting group
will vary depending upon the type of chemically reactive group
being protected as well as the reaction conditions to be employed
and the presence of additional reactive or protecting groups in the
molecule. Functional groups which may be protected include, by way
of example, carboxylic acid groups, amino groups, hydroxyl groups,
thiol groups, carbonyl groups and the like. Representative
protecting groups for carboxylic acids include esters (such as a
p-methoxybenzyl ester), amides and hydrazides; for amino groups,
carbamates (such as tert-butoxycarbonyl) and amides; for hydroxyl
groups, ethers and esters; for thiol groups, thioethers and
thioesters; for carbonyl groups, acetals and ketals; and the like.
Such protecting groups are well-known to those skilled in the art
and are described, for example, in T. W. Greene and G. M. Wuts,
Protecting Groups in Organic Synthesis, Third Edition, Wiley, New
York, 1999, and references cited therein.
[0077] A functional group in "protected form" refers to a
functional group bearing a protecting group. As used herein, the
term "functional group" or any synonym thereof is meant to
encompass protected forms thereof.
[0078] "PEG" or "poly(ethylene glycol)" as used herein, is meant to
encompass any water-soluble poly(ethylene oxide). Typically, PEGs
for use in the present invention will comprise one of the two
following structures: "--(CH.sub.2CH.sub.2O).sub.n--" or
"--(CH.sub.2CH.sub.2O).sub.n-1CH.sub.2CH.sub.2--," depending upon
whether or not the terminal oxygen(s) has been displaced, e.g.,
during a synthetic transformation. The variable (n) is 3 to 3000,
and the terminal groups and architecture of the overall PEG may
vary. When PEG further comprises a spacer as in structure I above
(to be described in greater detail below), the atoms comprising the
spacer (X), when covalently attached to a PEG segment, do not
result in formation of (i) an oxygen-oxygen bond (--O--O--, a
peroxide linkage), or (ii) a nitrogen-oxygen bond (N--O, O--N).
"PEG" means a polymer that contains a majority, that is to say,
greater than 50%, of subunits that are --CH.sub.2CH.sub.2O--. PEGs
for use in the invention include PEGs having a variety of molecular
weights, structures or geometries to be described in greater detail
below.
[0079] "Water-soluble", in the context of a polymer of the
invention or a "water-soluble polymer segment" is any segment or
polymer that is soluble in water at room temperature. Typically, a
water-soluble polymer or segment will transmit at least about 75%,
more preferably at least about 95% of light, transmitted by the
same solution after filtering. On a weight basis, a water-soluble
polymer or segment thereof will preferably be at least about 35%
(by weight) soluble in water, more preferably at least about 50%
(by weight) soluble in water, still more preferably about 70% (by
weight) soluble in water, and still more preferably about 85% (by
weight) soluble in water. It is most preferred, however, that the
water-soluble polymer or segment is about 95% (by weight) soluble
in water or completely soluble in water.
[0080] An "end-capping" or "end-capped" group is an inert group
present on a terminus of a polymer such as PEG. An end-capping
group is one that does not readily undergo chemical transformation
under typical synthetic reaction conditions. An end capping group
is generally an alkoxy group, --OR, where R is an organic radical
comprised of 1-20 carbons and is preferably lower alkyl (e.g.,
methyl, ethyl) or benzyl. "R" may be saturated or unsaturated, and
includes aryl, heteroaryl, cyclo, heterocyclo, and substituted
forms of any of the foregoing. For instance, an end capped PEG will
typically comprise the structure "RO--(CH.sub.2CH.sub.2O).sub.n--",
where R is as defined above. Alternatively, the end-capping group
can also advantageously comprise a detectable label. When the
polymer has an end-capping group comprising a detectable label, the
amount or location of the polymer and/or the moiety (e.g., active
agent) to which the polymer is coupled, can be determined by using
a suitable detector. Such labels include, without limitation,
fluorescers, chemiluminescers, moieties used in enzyme labeling,
colorimetric (e.g., dyes), metal ions, radioactive moieties, and
the like.
[0081] "Non-naturally occurring" with respect to a polymer of the
invention means a polymer that in its entirety is not found in
nature. A non-naturally occurring polymer of the invention may
however contain one or more subunits or segments of subunits that
are naturally occurring, so long as the overall polymer structure
is not found in nature.
[0082] "Molecular mass" in the context of a water-soluble polymer
of the invention such as PEG, refers to the nominal average
molecular mass of a polymer, typically determined by size exclusion
chromatography, light scattering techniques, or intrinsic velocity
determination in 1,2,4-trichlorobenzene. The polymers of the
invention are typically polydisperse, possessing low polydispersity
values of less than about 1.20.
[0083] The term "linker" is used herein to refer to an atom or a
collection of atoms used to link interconnecting moieties, such as
an organic radical core and a polymer segment, POLY.sub.1. A linker
moiety may be hydrolytically stable or may include a
physiologically hydrolyzable or enzymatically degradable linkage. A
linker designated herein as Q is hydrolytically stable.
[0084] The term "spacer" is used herein to refer to a collection of
atoms used to link interconnecting moieties, such as POLY.sub.1 and
the active agent, D. A spacer moiety may be hydrolytically stable
or may include a physiologically hydrolyzable or enzymatically
degradable linkage. A spacer designated herein as X comprises a
hydrolyzable linkage, where the hydrolyzable linkage is attached
directly to the active agent, D, such that upon hydrolysis, the
active agent is released in its parent form.
[0085] A "hydrolyzable" bond is a relatively weak bond that reacts
with water (i.e., is hydrolyzed) under physiological conditions.
The tendency of a bond to hydrolyze in water will depend not only
on the general type of linkage connecting two central atoms but
also on the substituents attached to these central atoms.
Illustrative hydrolytically unstable linkages include carboxylate
ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl
ether, imines, orthoesters, peptides and oligonucleotides.
[0086] An "enzymatically degradable linkage" means a linkage that
is subject to degradation by one or more enzymes. Such a linkage
requires the action of one or more enzymes to effect
degradation.
[0087] A "hydrolytically stable" linkage or bond refers to a
chemical bond, typically a covalent bond, that is substantially
stable in water, that is to say, does not undergo hydrolysis under
physiological conditions to any appreciable extent over an extended
period of time. Examples of hydrolytically stable linkages include
but are not limited to the following: carbon-carbon bonds (e.g., in
aliphatic chains), ethers, amides, urethanes, and the like.
Generally, a hydrolytically stable linkage is one that exhibits a
rate of hydrolysis of less than about 1-2% per day under
physiological conditions. Hydrolysis rates of representative
chemical bonds can be found in most standard chemistry
textbooks.
[0088] "Multi-armed" in reference to the geometry or overall
structure of a polymer refers to polymer having 3 or more
polymer-containing "arms". Thus, a multi-armed polymer may possess
3 polymer arms, 4 polymer arms, 5 polymer arms, 6 polymer arms, 7
polymer arms, 8 polymer arms or more, depending upon its
configuration and core structure. One particular type of highly
branched polymer is a dendritic polymer or dendrimer, that for the
purposes of the invention, is considered to possess a structure
distinct from that of a multi-armed polymer.
[0089] "Branch point" refers to a bifurcation point comprising one
or more atoms at which a polymer splits or branches from a linear
structure into one or more additional polymer arms. A multi-arm
polymer may have one branch point or multiple branch points.
[0090] A "dendrimer" is a globular, size monodisperse polymer in
which all bonds emerge radially from a central focal point or core
with a regular branching pattern and with repeat units that each
contribute a branch point. Dendrimers exhibit certain dendritic
state properties such as core encapsulation, making them unique
from other types of polymers.
[0091] "Substantially" or "essentially" means nearly totally or
completely, for instance, 95% or greater of some given
quantity.
[0092] "Alkyl" refers to a hydrocarbon chain, typically ranging
from about 1 to 20 atoms in length. Such hydrocarbon chains are
preferably but not necessarily saturated and may be branched or
straight chain, although typically straight chain is preferred.
Exemplary alkyl groups include methyl, ethyl, propyl, butyl,
pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like.
As used herein, "alkyl" includes cycloalkyl when three or more
carbon atoms are referenced.
[0093] "Lower alkyl" refers to an alkyl group containing from 1 to
6 carbon atoms, and may be straight chain or branched, as
exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl.
[0094] "Cycloalkyl" refers to a saturated or unsaturated cyclic
hydrocarbon chain, including bridged, fused, or spiro cyclic
compounds, preferably made up of 3 to about 12 carbon atoms, more
preferably 3 to about 8.
[0095] "Non-interfering substituents" are those groups that, when
present in a molecule, are typically non-reactive with other
functional groups contained within the molecule.
[0096] The term "substituted" as in, for example, "substituted
alkyl," refers to a moiety (e.g., an alkyl group) substituted with
one or more non-interfering substituents, such as, but not limited
to: C.sub.3-C.sub.8 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and
the like; halo, e.g., fluoro, chloro, bromo, and iodo; cyano;
alkoxy, lower phenyl; substituted phenyl; and the like. For
substitutions on a phenyl ring, the substituents may be in any
orientation (i.e., ortho, meta, or para).
[0097] "Alkoxy" refers to an --O--R group, wherein R is alkyl or
substituted alkyl, preferably C.sub.1-C.sub.20 alkyl (e.g.,
methoxy, ethoxy, propyloxy, etc.), preferably C.sub.1-C.sub.7.
[0098] As used herein, "alkenyl" refers to a branched or unbranched
hydrocarbon group of 1 to 15 atoms in length, containing at least
one double bond, such as ethenyl, n-propenyl, isopropenyl,
n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, and the
like.
[0099] The term "alkynyl" as used herein refers to a branched or
unbranched hydrocarbon group of 2 to 15 atoms in length, containing
at least one triple bond, ethynyl, n-propynyl, isopropynyl,
n-butynyl, isobutynyl, octynyl, decynyl, and so forth.
[0100] "Aryl" means one or more aromatic rings, each of 5 or 6 core
carbon atoms. Aryl includes multiple aryl rings that may be fused,
as in naphthyl or unfused, as in biphenyl. Aryl rings may also be
fused or unfused with one or more cyclic hydrocarbon, heteroaryl,
or heterocyclic rings. As used herein, "aryl" includes
heteroaryl.
[0101] "Heteroaryl" is an aryl group containing from one to four
heteroatoms, preferably N, O, or S, or a combination thereof.
Heteroaryl rings may also be fused with one or more cyclic
hydrocarbon, heterocyclic, aryl, or heteroaryl rings.
[0102] "Heterocycle" or "heterocyclic" means one or more rings of
5-12 atoms, preferably 5-7 atoms, with or without unsaturation or
aromatic character and having at least one ring atom which is not a
carbon. Preferred heteroatoms include sulfur, oxygen, and
nitrogen.
[0103] "Substituted heteroaryl" is heteroaryl having one or more
non-interfering groups as substituents.
[0104] "Substituted heterocycle" is a heterocycle having one or
more side chains formed from non-interfering substituents.
[0105] "Electrophile" refers to an ion, atom, or collection of
atoms that may be ionic, having an electrophilic center, i.e., a
center that is electron seeking, capable of reacting with a
nucleophile.
[0106] "Nucleophile" refers to an ion or atom or collection of
atoms that may be ionic, having a nucleophilic center, i.e., a
center that is seeking an electrophilic center, and capable of
reacting with an electrophile.
[0107] "Active agent" as used herein includes any agent, drug,
compound, composition of matter or mixture which provides some
pharmacologic, often beneficial, effect that can be demonstrated
in-vivo or in vitro. This includes foods, food supplements,
nutrients, nutraceuticals, drugs, vaccines, antibodies, vitamins,
and other beneficial agents. As used herein, these terms further
include any physiologically or pharmacologically active substance
that produces a localized or systemic effect in a patient.
[0108] "Pharmaceutically acceptable excipient" or "pharmaceutically
acceptable carrier" refers to an excipient that can be included in
the compositions of the invention and that causes no significant
adverse toxicological effects to the patient.
[0109] "Pharmacologically effective amount," "physiologically
effective amount," and "therapeutically effective amount" are used
interchangeably herein to mean the amount of a PEG-active agent
conjugate present in a pharmaceutical preparation that is needed to
provide a desired level of active agent and/or conjugate in the
bloodstream or in a target tissue. The precise amount will depend
upon numerous factors, e.g., the particular active agent, the
components and physical characteristics of pharmaceutical
preparation, intended patient population, patient considerations,
and the like, and can readily be determined by one skilled in the
art, based upon the information provided herein and available in
the relevant literature.
[0110] "Multi-functional" in the context of a polymer of the
invention means a polymer having 3 or more functional groups, where
the functional groups may be the same or different, and are
typically present on the polymer termini. Multi-functional polymers
of the invention will typically contain from about 3-100 functional
groups, or from 3-50 functional groups, or from 3-25 functional
groups, or from 3-15 functional groups, or from 3 to 10 functional
groups, i.e., contains 3, 4, 5, 6, 7, 8, 9 or 10 functional groups.
Typically, in reference to a polymer precursor used to prepare a
polymer prodrug of the invention, the polymer possesses 3 or more
polymer arms having at the terminus of each arm a functional group
suitable for coupling to an active agent moiety via a hydrolyzable
linkage.
[0111] "Difunctional" or "bifunctional" as used interchangeable
herein means an entity such as a polymer having two functional
groups contained therein, typically at the polymer termini. When
the functional groups are the same, the entity is said to be
homodifunctional or homobifunctional. When the functional groups
are different, the polymer is said to be heterodifunctional or
heterobifunctional
[0112] A basic or acidic reactant described herein includes
neutral, charged, and any corresponding salt forms thereof.
[0113] "Polyolefinic alcohol" refers to a polymer comprising an
olefin polymer backbone, such as polyethylene, having multiple
pendant hydroxyl groups attached to the polymer backbone. An
exemplary polyolefinic alcohol is polyvinyl alcohol.
[0114] As used herein, "non-peptidic" refers to a polymer backbone
substantially free of peptide linkages. However, the polymer may
include a minor number of peptide linkages spaced along the repeat
monomer subunits, such as, for example, no more than about 1
peptide linkage per about 50 monomer units.
[0115] The term "patient," refers to a living organism suffering
from or prone to a condition that can be prevented or treated by
administration of a polymer of the invention, typically but not
necessarily in the form of a polymer-active agent conjugate, and
includes both humans and animals.
[0116] "Optional" or "optionally" means that the subsequently
described circumstance may or may not occur, so that the
description includes instances where the circumstance occurs and
instances where it does not.
[0117] A "small molecule" may be defined broadly as an organic,
inorganic, or organometallic compound typically having a molecular
weight of less than about 1000. Small molecules of the invention
encompass oligopeptides and other biomolecules having a molecular
weight of less than about 1000.
[0118] An "active agent moiety" in reference to a prodrug conjugate
of the invention, refers to the portion or residue of the
unmodified parent active agent up to the covalent linkage resulting
from covalent attachment of the drug (or an activated or chemically
modified form thereof) to a polymer of the invention. Upon
hydrolysis of the hydrolyzable linkage between the active agent
moiety and the multi-armed polymer, the active agent per se is
released.
Multi-Arm Polymer Prodrug Conjugates--Overview
[0119] As described generally above, the polymer conjugates of the
invention comprise a multi-arm water-soluble and non-peptidic
polymer covalently attached to at least three active agent
compounds. The conjugates of the invention are typically prodrugs,
meaning that the active agent, attached to the polymer via a
hydrolytically degradable linkage, is released over time following
administration of the conjugate to a subject. Moreover, the
conjugates of the invention are well-characterized, isolable, and
purifiable compositions, in comparison to, for example, a
degradable polymer-matrix having molecules of drug encapsulated
therein. The conjugates of the invention exhibit higher drug
loading characteristics when compared to their linear polymer-based
counterparts, thus lowering the total dosage weight needed to treat
a particular disease state. That is to say, the polymer scaffold of
the invention is effective to covalently attach multiple active
agent molecules thereto, thereby allowing a greater amount of
therapeutic agent (i.e., active agent) to be administered per given
weight of polymer when compared to a linear monofunctional or
bifunctional polymer of about the same size but having only one or
two active agent molecules attached thereto. The polymers used in
the invention are hydrophilic in nature, thereby imparting
hydrophilicity to the resulting conjugates, which, particularly in
the case of water-insoluble active agents, facilitates their
formulation into useful pharmaceutical compositions.
[0120] Typically, the total number average molecular weight of the
overall multi-arm polymer portion of a polymer conjugate of the
invention is about 1,000 daltons (Da) to about 100,000 Da, more
preferably about 10,000 Da to about 60,000 Da, most preferably
about 15,000 to about 60,000 Da. Multi-armed polymers having a
number average molecular weight of about 5,000 Da, about 8,000 Da,
about 10,000 Da, about 12,000 Da, about 15,000 Da, about 20,000 Da,
about 25,000 Da, about 30,000 Da, about 35,000 Da, about 40,000 Da,
about 45,000 Da, about 50,000 Da, and about 60,000 Da are
particularly preferred. Multi-armed polymers having a molecular
weight of 20,000 Da or greater, i.e., of about 20,000 Da, or 25,000
Da, or 30,000 Da, or 40,000 Da or 50,000 Da, or 60,000 Da, are
particularly preferred for tumor-targeting applications. The actual
molecular weight of the multi-armed polymer will depend, of course,
on the number of polymer arms and the molecular weight of each
polymer arm in the overall multi-armed polymer.
[0121] The linkage between the multi-armed polymer portion and the
active agent is preferably hydrolytically degradable for in vivo
release of the parent drug molecule over time. Representative
hydrolytically degradable linkages corresponding to X in structure
I include carboxylate ester, carbonate ester, phosphate ester,
anhydride, acetal, ketal, acyloxyalkyl ether, imine, orthoester,
and oligonucleotides. Esters such as carboxylate and carbonate
esters are particularly preferred linkages. The particular linkage
and linkage chemistry employed will depend upon the particular
active agent, the presence of additional functional groups within
the active agent, and the like, and can be readily determined by
one skilled in the art based upon the guidance presented
herein.
[0122] With respect to the multi-arm prodrug conjugates of the
invention, it is not necessary for the polymer conjugate itself to
exhibit biological activity, since the parent drug is released upon
hydrolysis. However, in certain embodiments, the polymer conjugate
maintains at least a measurable degree of activity. That is to say,
in some instances, a multi-armed polymer conjugate possesses
anywhere from about 1% to about 100% or more of the specific
activity of the unmodified parent compound. That is to say, a
multi-armed polymer prodrug of the invention will possess from
about 1% to about 100% bioactivity relative to the unmodified
parent active agent, prior to conjugation. Such activity may be
determined using a suitable in-vivo or in-vitro model, depending
upon the known activity of the particular parent compound. For
anticancer drugs, in vivo anticancer activity is typically
evaluated by comparison of growth rates of tumor implants in drug
treated and control groups of athymic mice using well-established
animal models (See for example, Examples 2 and 6). Anticancer
activity is indicated by slower tumor growth rates in the treated
group relative to the control group (J. W. Singer, et al., Ann.
N.Y. Acad. Sci., 922: 136-150, 2000). In general, certain polymer
conjugates of the invention will possess a specific activity of at
least about 2%, 5%, 10%, 15%, 25%, 30%, 40%, 50%, 60%, 80%, 90% or
more relative to that of the unmodified parent drug when measured
in a suitable model.
[0123] As demonstrated in Examples 2, 6, and 7, preferred polymer
prodrug conjugates of the invention exhibit enhanced properties in
comparison to their unmodified parent drug counterparts. The
polymer conjugates of the invention exhibit enhanced permeation and
retention (EPR) in target tissues by passively accumulating in such
tissues, to provide targeted delivery of the drug to desired sites
in the body (See Matsumara Y, Maeda H. "A NEW CONCEPT FOR
MACROMOLECULAR THERAPEUTICS IN CANCER THERAPY; MECHANISM OF
TUMORITROPIC ACCUMULATION OF PROTEINS AND THE ANTITUMOUR AGENT
SMANCS", Cancer Res 1986; 46:6387-92).
[0124] Additionally, the severity of the side effects associated
with administration of the polymer conjugates of the invention is
preferably comparable to, or even more preferably, is less than,
the side effects associated with administration of the parent
compound. In particular, preferred conjugates, particularly those
comprising 3 or more molecules of an anticancer agent such as
irinotecan, when administered to a patient, result in reduced
leukopenia and diarrhea when compared to the unmodified parent drug
molecule. The severity of side effects of anticancer agents such as
camptothecin and camptothecin-like compounds can be readily
assessed (See, for example, Kado, et al., Cancer Chemotherapy and
Pharmacology, Aug. 6, 2003). The polymer conjugates of the
invention are believed to exhibit reduced side effects as compared
to the unconjugated parent drug, in part, due to the accumulation
of the conjugate molecules in the target tissue and away from other
sites of likely toxicity. Each of these features of the prodrugs of
the invention will now be discussed in greater detail below.
[0125] Structural Features of the Polymer Prodrug
[0126] As described above, a prodrug of the invention comprises a
multi-arm polymer, i.e., having three or more arms, where the
conjugate comprises the following generalized structure:
R(-Q-POLY.sub.1-X-D).sub.q
[0127] Each arm of the multi-armed prodrug is independent from the
other. That is to say, each of the "q" arms of the prodrug may be
composed of a different Q, POLY.sub.1, X, D and so forth. Typical
of such embodiments, a generalized structure corresponds to:
R[(-Q.sub.1-POLY.sub.1A-X.sub.1-D.sub.1)(Q.sub.2-POLY.sub.1B-X.sub.2-D.su-
b.2)(Q.sub.3-POLY.sub.1C-X.sub.3-D.sub.3)] and so forth for each of
the arms emanating from the central organic core. Generally,
however, each arm of the multi-armed prodrug is the same.
[0128] Each of the variable components of structure I will now be
described in detail.
[0129] Organic Core, "R"
[0130] In structure I, R is an organic core radical possessing from
about 3 to about 150 carbon atoms. Preferably, R contains from
about 3 to about 50 carbon atoms, and even more preferably, R
contains from about 3 to about 10 carbon atoms. That is to say, R
may possess a number of carbon atoms selected from the group
consisting of 3, 4, 5, 6, 7, 8, 9, and 10. The organic core may
optionally contain one or more heteroatoms (e.g., O, S, or N),
depending of course on the particular core molecule employed. R may
be linear or cyclic, and typically, emanating therefrom are at
least 3 independent polymer arms, three or more of which have at
least one active agent moiety covalently attached thereto. Looking
at Structure I, "q" corresponds to the number of polymer arms
emanating from "R". In some instances one or more of the polymer
arms may not have an active agent covalently attached thereto, but
rather may have a relatively unreactive or unreacted functional
group at its terminus, resulting from a synthesis that failed to go
to completion. In this instance, D is absent and the individual
structure of at least one of the polymer arms is in its precursor
form (or is a derivative thereof), i.e., having at its terminus not
an active agent, D, but rather an unreacted functional group.
[0131] The central core organic radical, R, is derived from a
molecule that provides a number of polymer attachment sites
approximately equal to the desired number of water soluble and
non-peptidic polymer arms. Preferably, the central core molecule of
the multi-arm polymer structure is the residue of a polyol,
polythiol, or a polyamine bearing at least three hydroxyl, thiol,
or amino groups available for polymer attachment. A "polyol" is a
molecule comprising a plurality (greater than 2) of available
hydroxyl groups. A "polythiol" is a molecule that possesses a
plurality (greater than 2) thiol groups. A "polyamine" is a
molecule comprising a plurality (greater than 2) of available amino
groups. Depending on the desired number of polymer arms, the
precursor polyol, polyamine or polythiol, (prior to covalent
attachment of POLY.sub.1) will typically contain 3 to about 25
hydroxyl, or amino groups or orthiol groups, respectively,
preferably from 3 to about 10 hydroxyl, amino groups or thiol
groups, (i.e., 3, 4, 5, 6, 7, 8, 9, 10), most preferably, will
contain from 3 to about 8 (e.g., 3, 4, 5, 6, 7, or 8) hydroxyl,
amino groups or thiol groups suitable for covalent attachment of
POLY.sub.1. The polyol, polyamine or polythiol may also include
other protected or unprotected functional groups. Focusing on
organic cores derived from polyols or polyamines, although the
number of intervening atoms between each hydroxyl or amino group
will vary, preferred cores are those having a length of from about
1 to about 20 intervening core atoms, such as carbon atoms, between
each hydroxyl or amino group, preferably from about 1 to about 5.
In referring to intervening core atoms and lengths, --CH.sub.2--,
for example, is considered as having a length of one intervening
atom, although the methylene group itself contains three atoms
total, since the Hs are substituents on the carbon, and
--CH.sub.2CH.sub.2--, for instance, is considered as having a
length of two carbon atoms, etc. The particular polyol or polyamine
precursor depends on the desired number of polymer arms in the
final conjugate. For example, a polyol or polyamine core molecule
having 4 functional groups, Q, is suitable for preparing a prodrug
in accordance with structure I having four polymer arms extending
therefrom and covalently attached to active agent.
[0132] The precursor polyol or polyamine core will typically
possess a structure R--(OH).sub.p or R--(NH.sub.2).sub.p prior to
functionalization with a polymer. The value of p corresponds to the
value of q in structure I, since each functional group, typically
--OH or --NH.sub.2, in the parent core organic molecule, if
sterically accessible and reactive, is covalently attached to a
polymer arm, POLY.sub.1. Note that in structure I, the variable
"Q", when taken together with R, typically represents a residue of
the core organic radical as described herein. That is to say, when
describing preferred organic core molecules, particularly by name,
the core molecules are described in their precursor form, rather
than in their radical form after removal of, for example, a proton.
So, if for example, the organic core radical is derived from
pentaerythritol, the precursor polyol possesses the structure
C(CH.sub.2OH).sub.4, and the organic core radical, together with Q,
corresponds to C(CH.sub.2O--).sub.4, where Q is O.
[0133] Illustrative polyols that are preferred for use as the
polymer core include aliphatic polyols having from 1 to 10 carbon
atoms and from 1 to 10 hydroxyl groups, including for example,
ethylene glycol, alkane diols, alkyl glycols, alkylidene alkyl
diols, alkyl cycloalkane diols, 1,5-decalindiol,
4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidene diols,
dihydroxyalkanes, trihydroxyalkanes, and the like. Cycloaliphatic
polyols include straight chained or closed-ring sugars and sugar
alcohols, such as mannitol, sorbitol, inositol, xylitol,
quebrachitol, threitol, arabitol, erythritol, adonitol, dulcitol,
facose, ribose, arabinose, xylose, lyxose, rhamnose, galactose,
glucose, fructose, sorbose, mannose, pyranose, altrose, talose,
tagitose, pyranosides, sucrose, lactose, maltose, and the like.
Additional examples of aliphatic polyols include derivatives of
glyceraldehyde, glucose, ribose, mannose, galactose, and related
stereoisomers. Aromatic polyols may also be used, such as
1,1,1-tris(4'-hydroxyphenyl) alkanes, such as
1,1,1-tris(4-hydroxyphenyl)ethane, (1,3-adamantanediyl)diphenol,
2,6-bis(hydroxyalkyl)cresols,
2,2'alkylene-bis(6-t-butyl-4-alkylphenols),
2,2'-alkylene-bis(t-butylphenols), catechol, alkylcatechols,
pyrogallol, fluoroglycinol, 1,2,4-benzenetriol, resorcinol,
alkylresorcinols, dialkylresorcinols, orcinol monohydrate,
olivetol, hydroquinone, alkylhydroquinones, 1,1-bi-2-naphthol,
phenyl hydroquinones, dihydroxynaphthalenes,
4,4'-(9-fluorenylidene)-diphenol, anthrarobin, dithranol,
bis(hydroxyphenyl)methane biphenols, dialkylstilbesterols,
bis(hydroxyphenyl)alkanes, bisphenol-A and derivatives thereof,
meso-hexesterol, nordihydroguairetic acid, calixarenes and
derivatives thereof, tannic acid, and the like. Other core polyols
that may be used include crown ethers, cyclodextrins, dextrins and
other carbohydrates (e.g., monosaccharides, oligosaccharides, and
polysaccharides, starches and amylase).
[0134] Preferred polyols include glycerol, trimethylolpropane,
reducing sugars such as sorbitol or pentaerythritol, and glycerol
oligomers, such as hexaglycerol. A 21-arm polymer can be
synthesized using hydroxypropyl-.beta.-cyclodextrin, which has 21
available hydroxyl groups.
[0135] Exemplary polyamines include aliphatic polyamines such as
diethylene triamine, N,N',N''-trimethyldiethylene triamine,
pentamethyl diethylene triamine, triethylene tetramine,
tetraethylene pentamine, pentaethylene hexamine, dipropylene
triamine, tripropylene tetramine, bis-(3-aminopropyl)-amine,
bis-(3-aminopropyl)-methylamine, and
N,N-dimethyl-dipropylene-triamine. Naturally occurring polyamines
that can be used in the present invention include putrescine,
spermidine, and spermine. Numerous suitable pentamines, tetramines,
oligoamines, and pentamidine analogs suitable for use in the
present invention are described in Bacchi et al., Antimicrobial
Agents and Chemotherapy, January 2002, p. 55-61, Vol. 46, No. 1,
which is incorporated by reference herein.
[0136] Provided below are illustrative structures corresponding to
the organic radical portion of the conjugate, R, and the
corresponding conjugate, assuming that each of the hydroxyls in the
parent polyol has been transformed to a polymer arm. Note that the
organic radicals shown below, derived from polyols, include the
oxygens, which, in the context of structure I, for the arms that
are polymer arms, are considered as part of Q. It is not necessary
that all hydroxyls in, for example, a polyol-derived organic
radical, form part of a polymer arm. In the illustrative examples
below, Q is shown as O, but can equally be considered as
corresponding to S, --NH--, or --NH--C(O)--.
##STR00003##
[0137] Linkages, Q and X.
[0138] The linkages between the organic radical, R, and the polymer
segment, POLY.sub.1, or between POLY.sub.1 and the active agent, D,
result from the reaction of various reactive groups contained
within R, POLY.sub.1, and D. The particular coupling chemistry
employed will depend upon the structure of the active agent, the
potential presence of multiple functional groups within the active
molecule, the need for protection/deprotection steps, the chemical
stability of the active agent, and the like, and will be readily
determined by one skilled in the art based upon the guidance
herein. Illustrative linking chemistry useful for preparing the
polymer conjugates of the invention can be found, for example, in
Wong, S. H., (1991), "Chemistry of Protein Conjugation and
Crosslinking", CRC Press, Boca Raton, Fla. and in Brinkley, M.
(1992) "A Brief Survey of Methods for Preparing Protein Conjugates
with Dyes, Haptens, and Crosslinking Reagent"s, in Bioconjug.
Chem., 3, 2013. As noted above, the overall linkage between the
multi-armed polymer core and each drug molecule preferably
comprises a hydrolytically degradable portion, such as an ester
linkage, so that the active agent is released over time from the
multi-armed polymer core.
[0139] The multi-arm polymeric conjugates provided herein (as well
as the corresponding reactive polymer precursor molecules, and so
forth) comprise a linker segment, Q, and a spacer segment, X.
Exemplary spacers or linkers can include segments such as those
independently selected from the group consisting of --O--, --S--,
--NH--, --C(O)--, --O--C(O)--, --C(O)--O--, --C(O)--NH--,
--NH--C(O)--NH--, --O--C(O)--NH--, --C(S)--, --CH.sub.2--,
--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, --O--CH.sub.2--,
--CH.sub.2--O--, --O--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--CH.sub.2--O--,
--O--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--O--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--O--,
--O--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--O--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--O--,
--C(O)--NH--CH.sub.2--, --C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--NH--CH.sub.2--, --CH.sub.2--CH.sub.2--C(O)--NH--,
--C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--,
--C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--,
--C(O)--O--CH.sub.2--, --CH.sub.2--C(O)--O--CH.sub.2--,
--CH.sub.2--CH.sub.2--, C(O)--O--CH.sub.2--,
--C(O)--O--CH.sub.2--CH.sub.2--, --NH--C(O)--CH.sub.2--,
--CH.sub.2--NH--C(O)--CH.sub.2--,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.2--,
--NH--C(O)--CH.sub.2--CH.sub.2--,
--CH.sub.2--NH--C(O)--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.2--CH.sub.2--,
--C(O)--NH--CH.sub.2--, --C(O)--NH--CH.sub.2--CH.sub.2--,
--O--C(O)--NH--CH.sub.2--, --O--C(O)--NH--CH.sub.2--CH.sub.2--,
--O--C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--, --NH--CH.sub.2--,
--NH--CH.sub.2--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--,
--CH.sub.2--CH.sub.2--NH--CH.sub.2--, --C(O)--CH.sub.2--,
--C(O)--CH.sub.2--CH.sub.2--, --CH.sub.2--C(O)--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--C(O)--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--C(O)--C-
H.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--N-
H--C(O)--CH.sub.2--CH.sub.2--,
--O--C(O)--NH--[CH.sub.2].sub.0-6--(OCH.sub.2CH.sub.2).sub.0-2--,
--C(O)--NH--(CH.sub.2).sub.1-6--NH--C(O)--,
--NH--C(O)--NH--(CH.sub.2).sub.1-6--NH--C(O)--,
--O--C(O)--CH.sub.2--, --O--C(O)--CH.sub.2--CH.sub.2--, and
--O--C(O)--CH.sub.2--CH.sub.2--CH.sub.2--.
[0140] In any of the above examples, a simple cycloalkylene group,
e.g. 1,3- or 1,4-cyclohexylene, may replace any two, three or four
carbon alkylene group. For purposes of the present disclosure,
however, a series of atoms is not a spacer moiety when the series
of atoms is immediately adjacent to a water-soluble polymer segment
and the series of atoms is but another monomer, such that the
proposed spacer moiety would represent a mere extension of the
polymer chain. A spacer or linker as described herein may also
comprise a combination of any two or more of the above groups, in
any orientation.
[0141] Referring to structure I, Q is a linker, preferably one that
is hydrolytically stable. Typically, Q contains at least one
heteroatom such as O, or S, or NH, where the atom proximal to R in
Q, when taken together with R, typically represents a residue of
the core organic radical R. Generally, Q contains from 1 to about
10 atoms, or from 1 to about 5 atoms. Q typically contains one of
the following number of atoms: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
Illustrative Qs include O, S, or --NH--C(O)--.
[0142] Again in reference to structure I, X is a spacer that
comprises a hydrolyzable linkage, where the hydrolyzable linkage is
attached directly to the active agent, D. Typically, at least one
atom of the hydrolyzable linkage is contained in the active agent
in its unmodified form, such that upon hydrolysis of the
hydrolyzable linkage comprised within X, the active agent, D, is
released. Generally speaking, the spacer has an atom length of from
about 4 atoms to about 50 atoms, or more preferably from about 5
atoms to about 25 atoms, or even more preferably from about 5 atoms
to about 20 atoms. Typically, the spacer is of an atom length
selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, and 20. When considering atom chain
length, only atoms contributing to the overall distance are
considered. For example, a spacer having the structure,
--CH.sub.2--C(O)--NH--CH.sub.2CH.sub.2O--CH.sub.2CH.sub.2O--C(O)--C--
has a chain length of 11 atoms, since substituents are not
considered to contribute significantly to the length of the
spacer.
[0143] In yet another particular embodiment, X possesses the
structure: Y-Z, where Y is a spacer fragment covalently attached to
Z, a hydrolytically degradable linkage. In certain embodiments, Z
itself may not constitute a hydrolytically degradable linkage,
however, when taken together with Y, or at least a portion of Y,
forms a linkage that is hydrolytically degradable.
[0144] In yet a more particular embodiment of the spacer, X, Y has
the structure:
--(CR.sub.xR.sub.y).sub.a--K--(CR.sub.xR.sub.y).sub.b--(CH.sub.2CH.sub.2O-
).sub.c--, wherein each R.sub.1 and R.sub.2, in each occurrence, is
independently H or an organic radical selected from the group
consisting of alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl,
a ranges from 0 to 12 (i.e., can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12), b ranges from 0 to 12 (i.e., can be 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, or 12), K is selected from --C(O)--,
--C(O)NH--, --NH--C(O)--, --O--, --S--, O--C(O)--, C(O)--O--,
O--C(O)--O--, O--C(O)--NH--, NH--C(O)--O--, c ranges from 0 to 25,
and Z is selected from C(O)--O--, O--C(O)--O--, --O--C(O)--NH--,
and NH--C(O)--O--. The particular structure of K and of Z will
depend upon the values of each of a, b, and c, such that none of
the following linkages result in the overall structure of spacer X:
--O--O--, NH--O--, NH--NH--.
[0145] Preferably, Y comprises
(--CH.sub.2).sub.a--C(O)NH--(CH.sub.2).sub.0,1--(CH.sub.2CH.sub.2O).sub.0-
-10.
[0146] In yet another embodiment of the spacer, X, Y has the
structure:
--(CR.sub.xR.sub.y).sub.a--K--(CR.sub.xR.sub.y).sub.b--(CH.sub.2CH.sub.2
NH).sub.c--, where the variables have the values previously
described. In certain instances, the presence of the short ethylene
oxide or ethyl amino fragments in spacer, X, can be useful in
achieving good yields during preparation of the prodrug conjugate,
since the presence of the linker can help to circumvent problems
associated with steric hindrance, due to the multi-armed reactive
polymer, the structure of the active agent, or a combination of
both. Preferably, c is selected from the group consisting of 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, and 10.
[0147] Preferably, R.sub.x and R.sub.y in each occurrence are
independently H or lower alkyl. In one embodiment, R.sub.x and
R.sub.y are in each occurrence H. In yet another embodiment, "a"
ranges from 0 to 5, i.e., is selected from 0, 1, 2, 3, 4, or 5. In
yet another embodiment, b ranges from 0 to 5, i.e., is selected
from 0, 1, 2, 3, 4, or 5. In yet another embodiment, c ranges from
0 to 10. In yet another embodiment, K is --C(O)--NH. Any of the
embodiments described herein is meant to apply not only to
generalized structure I, but also extend to particular combinations
of embodiments.
[0148] In yet another embodiment, R.sub.x and R.sub.y in each
occurrence are H, a is 1, K is --C(O)--NH, and b is 0 or 1.
[0149] Particular examples of X include
--CH.sub.2--C(O)--NH--CH.sub.2--C(O)O-- (here, Y corresponds to
--CH.sub.2--C(O)--NH--CH.sub.2-- and Z corresponds to --C(O)--O--),
and --CH.sub.2--C(O)--NH--(CH.sub.2CH.sub.2O).sub.2--C(O)--O--
(here, Y corresponds to
--CH.sub.2--C(O)--NH--(CH.sub.2CH.sub.2O).sub.2-- and Z corresponds
to --C(O)--O--).
[0150] The Polymer, Poly.sub.1
[0151] In structure I, POLY.sub.1 represents a water-soluble and
non-peptidic polymer. POLY.sub.1 in each polymer arm of structure I
is independently selected, although preferably, each polymer arm
will comprise the same polymer. Preferably, each of the arms (i.e.,
each "(-Q-POLY.sub.1-X-D) of structure I is identical. Any of a
variety of polymers that are non-peptidic and water-soluble can be
used to form a conjugate in accordance with the present invention.
Examples of suitable polymers include, but are not limited to,
poly(alkylene glycols), copolymers of ethylene glycol and propylene
glycol, poly(olefinic alcohol), poly(vinylpyrrolidone),
poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),
poly(saccharides), poly(.alpha.-hydroxy acid), poly(acrylic acid),
poly(vinyl alcohol), polyphosphazene, polyoxazoline,
poly(N-acryloylmorpholine), such as described in U.S. Pat. No.
5,629,384, which is incorporated by reference herein in its
entirety, and copolymers, terpolymers, and mixtures of any one or
more of the above.
[0152] Preferably, POLY.sub.1 is a polyethylene glycol or PEG.
POLY.sub.1 can be in any of a number of geometries or forms,
including linear chains, branched, forked, etc., although
preferably POLY.sub.1 is linear (i.e., in each arm of the overall
multi-arm structure) or forked. A preferred structure for a
multi-armed polymer prodrug having a "forked" polymer configuration
is as follows:
##STR00004##
[0153] F represents a forking group, and the remaining variables
are as previously described. Preferably, the fork point in the
forking group, F, comprises or is (--CH), though it may also be a
nitrogen atom (N). In this way, each polymer arm is forked to
possess two active agent moieties releasably covalently attached
thereto, rather than one.
[0154] Illustrative forked polymers useful for preparing a
multi-armed polymer of the type shown in Fig. XII are described in
U.S. Pat. No. 6,362,254.
[0155] When POLY.sub.1 is PEG, its structure typically comprises
--(CH.sub.2CH.sub.2O).sub.n--, where n ranges from about 5 to about
400, preferably from about 10 to about 350, or from about 20 to
about 300.
[0156] In the multi-arm embodiments described here, each polymer
arm, POLY.sub.1, typically has a molecular weight corresponding to
one of the following: 200, 250, 300, 400, 500, 600, 700, 800, 900,
1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 7500, 8000, 9000,
10000, 12,000, 15000, 17,500, 18,000, 19,000, 20,000 daltons or
greater. Overall molecular weights for the multi-armed polymer
configurations described herein (that is to say, the molecular
weight of the multi-armed polymer as a whole) generally correspond
to one of the following: 800, 1000, 1200, 1600, 2000, 2400, 2800,
3200, 3600, 4000, 6000, 8000, 12,000, 16,000, 20,000, 24,000,
28,000, 30,000, 32,000, 36,000, 40,000, 48,000, 60,000 or greater.
Typically, the overall molecular weight for a multi-armed polymer
of the invention ranges from about 800 to about 60,000 daltons.
[0157] Active Agent, D.
[0158] Returning now to structure I, D is an active agent moiety,
and q (the number of independent polymer arms) ranges from about 3
to about 50. Preferably, q ranges from about 3 to about 25. More
preferably, q is from 3 to about 10, and possesses a value of 3, 4,
5, 6, 7, 8, 9, or 10. The active agent moiety, D contains at least
one functional group suitable for covalent attachment to the
multi-armed polymer described herein to form a hydrolyzable
linkage, such that upon hydrolysis, the active agent is released in
its unmodified form.
[0159] In accordance with one embodiment of the invention, a
prodrug conjugate is characterized as a polymer having from about 3
to about 25 active agent molecules covalently attached thereto.
More particularly, the conjugate is characterized as a water
soluble polymer having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 active agent molecules
covalently attached thereto. In a further embodiment, the conjugate
of the invention has from about 3 to about 8 active agent molecules
covalently attached to the water-soluble polymer. Typically,
although not necessarily, the number of polymer arms will
correspond to the number of active agents covalently attached to
the water soluble polymer.
[0160] In yet another embodiment, rather than having multiple
polymer arms emanating from a central organic radical core, a
conjugate of the invention is characterized as a water-soluble
polymer having pendant active agent moieties covalently attached
thereto, each preferably covalently attached by a degradable
linkage. In such an embodiment, the structure of the polymer
prodrug conjugate is described generally as POLY.sub.1(X-D).sub.q,
where and POLY.sub.1, X, D, and q are as set forth above, and the
polymer, typically a linear polymer, possesses "q" active agent
moieties attached thereto, typically at discrete lengths along the
polymer chain, via the spacer X which contains a hydrolyzable
linkage.
[0161] In a specific embodiment, the active agent moiety is a small
molecule possessing a molecular weight of less than about 1000. In
yet additional embodiments, the small molecule drug possesses a
molecular weight of less than about 800, or even less than about
750. In yet another embodiment, the small molecule drug possesses a
molecular weight of less than about 500 or, in some instances, even
less than about 300.
[0162] Preferred active agent moieties include anticancer agents.
Particularly preferred are oncolytics having at least one hydroxyl
group.
[0163] One preferred class of active agents are the camptothecins.
In one embodiment, a camptothecin for use in the invention
corresponds to the structure:
##STR00005##
wherein R.sub.1-R.sub.5 are each independently selected from the
group consisting of hydrogen; halo; acyl; alkyl (e.g., C1-C6
alkyl); substituted alkyl; alkoxy (e.g., C1-C6 alkoxy); substituted
alkoxy; alkenyl; alkynyl; cycloalkyl; hydroxyl; cyano; nitro;
azido; amido; hydrazine; amino; substituted amino (e.g.,
monoalkylamino and dialkylamino); hydroxycarbonyl; alkoxycarbonyl;
alkylcarbonyloxy; alkylcarbonylamino; carbamoyloxy;
arylsulfonyloxy; alkylsulfonyloxy;
--C(R.sub.7).dbd.N--(O).sub.i--R.sub.8 wherein R.sub.7 is H, alkyl,
alkenyl, cycloalkyl, or aryl, i is 0 or 1, and R.sub.8 is H, alkyl,
alkenyl, cycloalkyl, or heterocycle; and R.sub.9C(O)O-- wherein
R.sub.9 is halogen, amino, substituted amino, heterocycle,
substituted heterocycle, or R.sub.10--O--(CH.sub.2).sub.m-- where m
is an integer of 1-10 and R.sub.10 is alkyl, phenyl, substituted
phenyl, cycloalkyl, substituted cycloalkyl, heterocycle, or
substituted heterocycle; or
[0164] R.sub.2 together with R.sub.3 or R.sub.3 together with
R.sub.4 form substituted or unsubstituted methylenedioxy,
ethylenedioxy, or ethyleneoxy;
[0165] R.sub.6 is H or OR', wherein R' is alkyl, alkenyl,
cycloalkyl, haloalkyl, or hydroxyalkyl; and
[0166] L is the site of attachment to X.
[0167] In one particular embodiment, D is irinotecan, where the H
on the 20-position hydroxyl is absent in the final multi-armed
prodrug conjugate.
##STR00006##
[0168] Active agents for use in the invention include hypnotics and
sedatives, psychic energizers, tranquilizers, respiratory drugs,
anticonvulsants, muscle relaxants, antiparkinson agents (dopamine
antagnonists), analgesics, anti-inflammatories, antianxiety drugs
(anxiolytics), appetite suppressants, antimigraine agents, muscle
contractants, anti-infectives (antibiotics, antivirals,
antifungals, vaccines) antiarthritics, antimalarials, antiemetics,
anepileptics, bronchodilators, cytokines, growth factors,
anti-cancer agents, antithrombotic agents, antihypertensives,
cardiovascular drugs, antiarrhythmics, antioxidants, anti-asthma
agents, hormonal agents including contraceptives, sympathomimetics,
diuretics, lipid regulating agents, antiandrogenic agents,
antiparasitics, anticoagulants, neoplastics, antineoplastics,
hypoglycemics, nutritional agents and supplements, growth
supplements, antienteritis agents, vaccines, antibodies, diagnostic
agents, and contrasting agents.
[0169] More particularly, the active agent may fall into one of a
number of structural classes, including but not limited to small
molecules, oligopeptides, polypeptides or protein mimetics,
fragments, or analogues, steroids, nucleotides, oligonucleotides,
electrolytes, and the like. Preferably, an active agent for use in
the invention possesses a free hydroxyl, carboxyl, thio, amino
group, or the like (i.e., "handle") suitable for covalent
attachment to the polymer. Preferably, an active agent possesses at
least one functional group suitable for forming a hydrolyzable
linkage when reacted with a multi-armed polymer precursor suitable
for forming a prodrug conjugate of the invention.
[0170] Alternatively, the drug is modified by introduction of a
suitable "handle", preferably by conversion of one of its existing
functional groups to a functional group suitable for formation of a
hydrolyzable covalent linkage between the multi-armed polymer and
the drug. Ideally, such a modification should not adversely impact
the therapeutic effect or activity of the active agent to a
significant degree. That is to say, any modification of an active
agent to facilitate its attachment to a multi-armed polymer of the
invention should result in no greater than about a 30% reduction of
its bioactivity relative to the known parent active agent prior to
modification. More preferably, any modification of an active agent
to facilitate its attachment to a multi-armed polymer of the
invention preferably results in a reduction of its activity
relative to the known parent active agent prior to modification of
no greater than about 25%, 20%, 15%, 10% or 5%.
[0171] Specific examples of active agents include proteins, small
molecule mimetics thereof, and active fragments (including
variants) of the following: asparaginase, amdoxovir (DAPD), antide,
becaplermin, calcitonins, cyanovirin, denileukin diftitox,
erythropoietin (EPO), EPO agonists (e.g., peptides from about 10-40
amino acids in length and comprising a particular core sequence as
described in WO 96/40749), dornase alpha, erythropoiesis
stimulating protein (NESP), coagulation factors such as Factor V,
Factor VII, Factor VIIa, Factor VIII, Factor IX, Factor X, Factor
XII, Factor XIII, von Willebrand factor; ceredase, cerezyme,
alpha-glucosidase, collagen, cyclosporin, alpha defensins, beta
defensins, exedin-4, granulocyte colony stimulating factor (GCSF),
thrombopoietin (TPO), alpha-1 proteinase inhibitor, elcatonin,
granulocyte macrophage colony stimulating factor (GMCSF),
fibrinogen, filgrastim, growth hormones human growth hormone (hGH),
growth hormone releasing hormone (GHRH), GRO-beta, GRO-beta
antibody, bone morphogenic proteins such as bone morphogenic
protein-2, bone morphogenic protein-6, OP-1; acidic fibroblast
growth factor, basic fibroblast growth factor, CD-40 ligand,
heparin, human serum albumin, low molecular weight heparin (LMWH),
interferons such as interferon alpha, interferon beta, interferon
gamma, interferon omega, interferon tau, consensus interferon;
interleukins and interleukin receptors such as interleukin-1
receptor, interleukin-2, interleukin-2 fusion proteins,
interleukin-1 receptor antagonist, interleukin-3, interleukin-4,
interleukin-4 receptor, interleukin-6, interleukin-8,
interleukin-12, interleukin-13 receptor, interleukin-17 receptor;
lactoferrin and lactoferrin fragments, luteinizing hormone
releasing hormone (LHRH), insulin, pro-insulin, insulin analogues
(e.g., mono-acylated insulin as described in U.S. Pat. No.
5,922,675), amylin, C-peptide, somatostatin, somatostatin analogs
including ocreotide, vasopressin, follicle stimulating hormone
(FSH), influenza vaccine, insulin-like growth factor (IGF),
insulintropin, macrophage colony stimulating factor (M-CSF),
plasminogen activators such as alteplase, urokinase, reteplase,
streptokinase, pamiteplase, lanoteplase, and teneteplase; nerve
growth factor (NGF), osteoprotegerin, platelet-derived growth
factor, tissue growth factors, transforming growth factor-1,
vascular endothelial growth factor, leukemia inhibiting factor,
keratinocyte growth factor (KGF), glial growth factor (GGF), T Cell
receptors, CD molecules/antigens, tumor necrosis factor (TNF),
monocyte chemoattractant protein-1, endothelial growth factors,
parathyroid hormone (PTH), glucagon-like peptide, somatotropin,
thymosin alpha 1, thymosin alpha 1 IIb/IIIa inhibitor, thymosin
beta 10, thymosin beta 9, thymosin beta 4, alpha-1 antitrypsin,
phosphodiesterase (PDE) compounds, VLA-4 (very late antigen-4),
VLA-4 inhibitors, bisphosphonates, respiratory syncytial virus
antibody, cystic fibrosis transmembrane regulator (CFTR) gene,
deoxyreibonuclease (Dnase), bactericidal/permeability increasing
protein (BPI), and anti-CMV antibody. Exemplary monoclonal
antibodies include etanercept (a dimeric fusion protein consisting
of the extracellular ligand-binding portion of the human 75 kD TNF
receptor linked to the Fc portion of IgG1), abciximab, afeliomomab,
basiliximab, daclizumab, infliximab, ibritumomab tiuexetan,
mitumomab, muromonab-CD3, iodine 131 tositumomab conjugate,
olizumab, rituximab, and trastuzumab (herceptin).
[0172] Additional agents suitable include but are not limited to
amifostine, amiodarone, aminocaproic acid, aminohippurate sodium,
aminoglutethimide, aminolevulinic acid, aminosalicylic acid,
amsacrine, anagrelide, anastrozole, asparaginase, anthracyclines,
bexarotene, bicalutamide, bleomycin, buserelin, busulfan,
cabergoline, capecitabine, carboplatin, carmustine, chlorambucin,
cilastatin sodium, cisplatin, cladribine, clodronate,
cyclophosphamide, cyproterone, cytarabine, camptothecins, 13-cis
retinoic acid, all trans retinoic acid; dacarbazine, dactinomycin,
daunorubicin, deferoxamine, dexamethasone, diclofenac,
diethylstilbestrol, docetaxel, doxorubicin, epirubicin,
estramustine, etoposide, exemestane, fexofenadine, fludarabine,
fludrocortisone, fluorouracil, fluoxymesterone, flutamide,
gemcitabine, epinephrine, L-Dopa, hydroxyurea, idarubicin,
ifosfamide, imatinib, irinotecan, itraconazole, goserelin,
letrozole, leucovorin, levamisole, lisinopril, lovothyroxine
sodium, lomustine, mechlorethamine, medroxyprogesterone, megestrol,
melphalan, mercaptopurine, metaraminol bitartrate, methotrexate,
metoclopramide, mexiletine, mitomycin, mitotane, mitoxantrone,
naloxone, nicotine, nilutamide, octreotide, oxaliplatin,
pamidronate, pentostatin, pilcamycin, porfimer, prednisone,
procarbazine, prochlorperazine, ondansetron, raltitrexed,
sirolimus, streptozocin, tacrolimus, tamoxifen, temozolomide,
teniposide, testosterone, tetrahydrocannabinol, thalidomide,
thioguanine, thiotepa, topotecan, tretinoin, valrubicin,
vinblastine, vincristine, vindesine, vinorelbine, dolasetron,
granisetron; formoterol, fluticasone, leuprolide, midazolam,
alprazolam, amphotericin B, podophyllotoxins, nucleoside
antivirals, aroyl hydrazones, sumatriptan; macrolides such as
erythromycin, oleandomycin, troleandomycin, roxithromycin,
clarithromycin, davercin, azithromycin, flurithromycin,
dirithromycin, josamycin, spiromycin, midecamycin, leucomycin,
miocamycin, rokitamycin, andazithromycin, and swinolide A;
fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin,
trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin,
grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin,
temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin,
prulifloxacin, irloxacin, pazufloxacin, clinafloxacin, and
sitafloxacin; aminoglycosides such as gentamicin, netilmicin,
paramecin, tobramycin, amikacin, kanamycin, neomycin, and
streptomycin, vancomycin, teicoplanin, rampolanin, mideplanin,
colistin, daptomycin, gramicidin, colistimethate; polymixins such
as polymixin B, capreomycin, bacitracin, penems; penicillins
including penicillinase-sensitive agents like penicillin G,
penicillin V; penicillinase-resistant agents like methicillin,
oxacillin, cloxacillin, dicloxacillin, floxacillin, nafcillin; gram
negative microorganism active agents like ampicillin, amoxicillin,
and hetacillin, cillin, and galampicillin; antipseudomonal
penicillins like carbenicillin, ticarcillin, azlocillin,
meziocillin, and piperacillin; cephalosporins like cefpodoxime,
cefprozil, ceftbuten, ceftizoxime, ceftriaxone, cephalothin,
cephapirin, cephalexin, cephradrine, cefoxitin, cefamandole,
cefazolin, cephaloridine, cefaclor, cefadroxil, cephaloglycin,
cefuroxime, ceforanide, cefotaxime, cefatrizine, cephacetrile,
cefepime, cefixime, cefonicid, cefoperazone, cefotetan,
cefinetazole, ceftazidime, loracarbef, and moxalactam, monobactams
like aztreonam; and carbapenems such as imipenem, meropenem,
pentamidine isethiouate, albuterol sulfate, lidocaine,
metaproterenol sulfate, beclomethasone diprepionate, triamcinolone
acetamide, budesonide acetonide, fluticasone, ipratropium bromide,
flunisolide, cromolyn sodium, and ergotamine tartrate; taxanes such
as paclitaxel; SN-38, and tyrphostines.
[0173] The above exemplary drugs are meant to encompass, where
applicable, analogues, agonists, antagonists, inhibitors, isomers,
polymorphs, and pharmaceutically acceptable salt forms thereof.
[0174] As described previously, one preferred class of active
agents is the camptothecins. The term "camptothecin compound" as
used herein includes the plant alkaloid 20(S)-camptothecin, as well
as pharmaceutically active derivatives, analogues and metabolites
thereof. Examples of camptothecin derivatives include, but are not
limited to, 9-nitro-20(S)-camptothecin, 9-amino-20(S)-camptothecin,
9-methyl-camptothecin, 9-chloro-camptothecin,
9-fluoro-camptothecin, 7-ethyl camptothecin,
10-methyl-camptothecin, 10-chloro-camptothecin,
10-bromo-camptothecin, 10-fluoro-camptothecin,
9-methoxy-camptothecin, 11-fluoro-camptothecin, 7-ethyl-10-hydroxy
camptothecin (SN38), 10,11-methylenedioxy camptothecin, and
10,11-ethylenedioxy camptothecin, and
7-(4-methylpiperazinomethylene)-10,11-methylenedioxy camptothecin,
7-ethyl-10-(4-(1-piperidino)-1-piperidino)-carbonyloxy-camptothecin,
9-hydroxy-camptothecin, and 11-hydroxy-camptothecin. Particularly
preferred camptothecin compounds include camptothecin, irinotecan,
and topotecan.
[0175] Native and unsubstituted, the plant alkaloid camptothecin
can be obtained by purification of the natural extract, or may be
obtained from the Stehlin Foundation for Cancer Research (Houston,
Tex.). Substituted camptothecins can be obtained using methods
known in the literature or can be obtained from commercial
suppliers. For example, 9-nitro-camptothecin may be obtained from
SuperGen, Inc. (San Ramon, Calif.), and 9-amino-camptothecin may be
obtained from Idec Pharmaceuticals (San Diego, Calif.).
Camptothecin and various analogues and derivatives may also be
obtained from standard fine chemical supply houses, such as Sigma
Chemicals.
[0176] Preferred camptothecin compounds are illustrated below in
Formula XI.
##STR00007##
wherein R.sub.1-R.sub.5 are each independently selected from the
group consisting of hydrogen; halo; acyl; alkyl (e.g., C1-C6
alkyl); substituted alkyl; alkoxy (e.g., C1-C6 alkoxy); substituted
alkoxy; alkenyl; alkynyl; cycloalkyl; hydroxyl; cyano; nitro;
azido; amido; hydrazine; amino; substituted amino (e.g.,
monoalkylamino and dialkylamino); hydroxycarbonyl; alkoxycarbonyl;
alkylcarbonyloxy; alkylcarbonylamino; carbamoyloxy;
arylsulfonyloxy; alkylsulfonyloxy;
--C(R.sub.7).dbd.N--(O).sub.i--R.sub.8 wherein R.sub.7 is H, alkyl,
alkenyl, cycloalkyl, or aryl, i is 0 or 1, and R.sub.8 is H, alkyl,
alkenyl, cycloalkyl, or heterocycle; and R.sub.9C(O)O-- wherein
R.sub.9 is halogen, amino, substituted amino, heterocycle,
substituted heterocycle, or R.sub.10--O--(CH.sub.2).sub.m-- where m
is an integer of 1-10 and R.sub.10 is alkyl, phenyl, substituted
phenyl, cycloalkyl, substituted cycloalkyl, heterocycle, or
substituted heterocycle; or
[0177] R.sub.2 together with R.sub.3 or R.sub.3 together with
R.sub.4 form substituted or unsubstituted methylenedioxy,
ethylenedioxy, or ethyleneoxy; and
[0178] R.sub.6 is H or OR', wherein R' is alkyl, alkenyl,
cycloalkyl, haloalkyl, or hydroxyalkyl.
[0179] Exemplary substituting groups include hydroxyl, amino,
substituted amino, halo, alkoxy, alkyl, cyano, nitro,
hydroxycarbonyl, alkoxycarbonyl, alkylcarbonyloxy,
alkylcarbonylamino, aryl (e.g., phenyl), heterocycle, and glycosyl
groups.
[0180] In one embodiment of the invention, the small molecule is
not taxol, or is not taxane-based.
[0181] Other preferred active agents for preparing a multi-armed
polymer prodrug conjugate as described herein include platins,
oxymorphone analogues, steroids, quinolones, isoquinolones, and
fluoroquinolones, and nucleosides and nucleotides. Structures of
illustrative compounds belonging to each of the above structural
classes are provided below.
##STR00008## ##STR00009## ##STR00010##
[0182] Method of Forming a Multi-Armed Polymer Prodrug
Conjugate
[0183] Multi-armed reactive polymers, such as those for preparing a
prodrug of the invention can be readily prepared from commercially
available starting materials in view of the guidance presented
herein, coupled with what is known in the art of chemical
synthesis.
[0184] Hydroxyl-terminated multi-armed PEGs having either a
pentaerythritol core or a glycerol core are available from Nektar,
Huntsville Ala. Such multi-armed PEGs can be used directly for
coupling to active agents having, e.g., a carboxyl group in a
position suitable for coupling, e.g., to provide a polymer prodrug
having a hydrolyzable carboxyl ester bond. Alternatively, terminal
hydroxyls present on a multi-armed polymer precursor can be
oxidized to terminal carboxyl groups, e.g., for coupling to
hydroxyls present on an active agent.
[0185] Alternatively, a multi-armed reactive polymer for preparing
a prodrug of the invention may be synthetically prepared. For
instance, any of a number of suitable polyol core materials can be
purchased from a chemical supplier such as Aldrich (St. Louis,
Mo.). The terminal hydroxyls of the polyol are first converted to
their anionic form, using, for example, a strong base, to provide a
site suitable for initiating polymerization, followed by direct
polymerization of monomer subunits, e.g., ethylene oxide, onto the
core. Chain building is allowed to continue until a desired length
of polymer chain is reached in each of the arms, followed by
terminating the reaction, e.g., by quenching.
[0186] In an alternative approach, an activated multi-armed polymer
precursor to the prodrugs of the invention can be synthetically
prepared by first providing a desired polyol core material, and
reacting the polyol under suitable conditions with a
heterobifunctional PEG mesylate of a desired length, where the
non-mesylate PEG terminus is optionally protected to prevent
reaction with the polyol core. The resulting multi-armed polymer
precursor is then suitable for additional transformations or direct
coupling to an active agent, following deprotection if
necessary.
[0187] Multi-armed polymer precursors based on polyamino cores can
be prepared, for example, by direct coupling to a polymer reagent
activated with an acylating agent such as an NHS ester, a
succinimidyl carbonate, a BTC ester or the like, to provide
multi-armed polymer precursors having an amide linker, Q.
Alternatively, a core molecule having multiple amino groups can be
coupled with an aldehyde terminated polymer, such as a PEG, by
reductive amination (using, for example, a reducing agent such as
sodium cyanoborohydride) to provide a multi-armed polymer precursor
having an internal amine linker, Q.
[0188] Although the polymer PEG is described as a representative
polymer in the synthetic descriptions above, such approaches apply
equally as well to other water-soluble polymers described
herein.
[0189] The prodrugs of the invention can be formed using known
chemical coupling techniques for covalent attachment of activated
polymers, such as an activated PEG, to a biologically active agent
(See, for example, Poly(Ethylene Glycol) Chemistry and Biological
Applications, American Chemical Society, Washington, D.C. (1997)).
Selection of suitable functional groups, linkers, protecting
groups, and the like to achieve a multi-arm polymer prodrug in
accordance with the invention, will depend, in part, on the
functional groups on the active agent and on the multi-armed
polymer starting material and will be apparent to one skilled in
the art, based upon the contents of the present disclosure.
[0190] A multi-armed polymer of the invention suitable for coupling
to an active agent or derivatized active agent will typically have
a terminal functional group such as the following: N-succinimidyl
carbonate (see e.g., U.S. Pat. Nos. 5,281,698, 5,468,478), amine
(see, e.g., Buckmann et al. Makromol. Chem. 182:1379 (1981),
Zalipsky et al. Eur. Polym. J. 19:1177 (1983)), hydrazide (See,
e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidyl
propionate and succinimidyl butanoate (see, e.g., Olson et al. in
Poly(ethylene glycol) Chemistry & Biological Applications, pp
170-181, Harris & Zalipsky Eds., ACS, Washington, D.C., 1997;
see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See,
e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) and
Joppich et al., Makromol. Chem. 180:1381 (1979), succinimidyl ester
(see, e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see,
e.g., U.S. Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et
al. Eur. J. Biochem. 94:11 (1979), Elling et al., Biotech. Appl.
Biochem. 13:354 (1991), oxycarbonylimidazole (see, e.g., Beauchamp,
et al., Anal. Biochem. 131:25 (1983), Tondelli et al. J. Controlled
Release 1:251 (1985)), p-nitrophenyl carbonate (see, e.g.,
Veronese, et al., Appl. Biochem. Biotech., 11:141 (1985); and
Sartore et al., Appl. Biochem. Biotech., 27:45 (1991)), aldehyde
(see, e.g., Harris et al. J. Polym. Sci. Chem. Ed. 22:341 (1984),
U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714), maleimide (see,
e.g., Goodson et al. Bio/Technology 8:343 (1990), Romani et al. in
Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan,
Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g.,
Woghiren, et al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g.,
Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see,
e.g., U.S. Pat. No. 5,900,461).
[0191] In turning now to one of the preferred classes of active
agents, the camptothecins, since the 20-hydroxyl group of the
camptothecin compound is sterically hindered, a single step
conjugation reaction is difficult to accomplish in significant
yields. As a result, a preferred method is to react the 20-hydroxyl
group with a short linker or spacer moiety carrying a functional
group suitable for reaction with a multi-arm polymer. Such an
approach is applicable to many small molecules, particularly those
having a site of covalent attachment that is inaccessible to an
incoming reactive polymer. Preferred linkers include t-BOC-glycine
or other amino acids having a protected amino group and an
available carboxylic acid group (See Zalipsky et al., "Attachment
of Drugs to Polyethylene Glycols", Eur. Polym. J., Vol. 19, No. 12,
pp. 1177-1183 (1983)). The carboxylic acid group reacts readily
with the 20-hydroxyl group in the presence of a coupling agent
(e.g., dicyclohexylcarbodiimide (DCC)) and a base catalyst (e.g.,
dimethylaminopyridine (DMAP)). Thereafter, the amino protecting
group, such as t-BOC(N-tert-butoxycarbonyl), is removed by
treatment with the appropriate deprotecting agent (e.g.,
trifluoroacetic acid (TFA) in the case of t-BOC). The free amino
group is then reacted with a multi-arm or forked polymer bearing
carboxylic acid groups in the presence of a coupling agent (e.g.,
hydroxybenzyltriazole (HOBT)) and a base (e.g., DMAP).
[0192] In a preferred embodiment, the spacer moiety is derived from
and comprises an amino acid and has the structure
HO--C(O)--CH(R'')--NH-Gp wherein R'' is H, C1-C6 alkyl, or
substituted C1-C6alkyl and Gp is a protecting group protecting the
alpha-amino group of the amino acid. Typical labile protecting
groups include t-BOC and FMOC (9-fluorenylmethloxycarbonyl). t-BOC
is stable at room temperature and easily removed with dilute
solutions of TFA and dichloromethane. FMOC is a base labile
protecting group that is easily removed by concentrated solutions
of amines (usually 20-55% piperidine in N-methylpyrrolidone).
Preferred amino acids include alanine, glycine, isoleucine,
leucine, phenylalanine, and valine.
[0193] Other spacer moieties having an available carboxylic acid
group or other functional group reactive with a hydroxyl group and
a protected amino group can also be used in lieu of the amino acids
described above. For example, a spacer moiety having the structure
HOOC-alkylene-NH-Gp may be employed, where Gp is as described above
and the alkylene chain is, for example, about 1 to about 20 carbon
atoms in length. Spacers comprising short
--(CH.sub.2CH.sub.2O).sub.c-- groups or (CH.sub.2CH.sub.2NH).sub.c
groups are also preferred, where c varies from about 0 to about 25.
More particularly, c possesses a value selected from 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, and 12.
[0194] In a particular embodiment exemplified in Example 1,
conjugation is accomplished by first reacting the camptothecin
compound with t-BOC-glycine, followed by deprotection of the
glycine amino group and coupling of the glycine-modified
camptothecin to a 4-arm PEG molecule comprising a pentaerythritol
core.
[0195] In an alternative approach exemplified in Example 8, a
bifunctional spacer comprising a number of --(CH.sub.2CH.sub.2O)--
subunits is provided. One terminal functional group of the spacer
is an acid chloride (--O--C(O)--Cl) suitable for reaction with an
active agent hydroxyl group to form a carbonate ester (i.e., a
hydrolyzable linkage), while the other terminal functional group is
a protected amine. The bifunctional spacer is coupled to
irinotecan, in particular to the 20-position hydroxyl thereof, in
the presence of a coupling agent such as DMAP to provide a
partially modified active agent. In the partially modified active
agent, a hydrolyzable bond, Z, has been introduced, coupled to a
spacer, Y' having a protected terminus, which upon deprotection, is
suitable for reaction with an activated multi-armed polymer. The
partially modified active agent is then reacted with a multi-armed
polymer precursor having a reactive terminus suitable for coupling
to an amine, to provide a stable amide linkage as part of the
overall linkage, X.
[0196] The prodrug product may be further purified. Methods of
purification and isolation include precipitation followed by
filtration and drying, as well as chromatography. Suitable
chromatographic methods include gel filtration chromatography and
ion exchange chromatography.
Pharmaceutical Compositions
[0197] The invention provides pharmaceutical formulations or
compositions, both for veterinary and for human medical use, which
comprise one or more polymer prodrugs of the invention or a
pharmaceutically acceptable salt thereof, with one or more
pharmaceutically acceptable carriers, and optionally any other
therapeutic ingredients, stabilizers, or the like. The carrier(s)
must be pharmaceutically acceptable in the sense of being
compatible with the other ingredients of the formulation and not
unduly deleterious to the recipient thereof. The compositions of
the invention may also include polymeric excipients/additives or
carriers, e.g., polyvinylpyrrolidones, derivatized celluloses such
as hydroxymethylcellulose, hydroxyethylcellulose, and
hydroxypropylmethylcellulose, Ficolls (a polymeric sugar),
hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as
2-hydroxypropyl-.beta.-cyclodextrin and
sulfobutylether-.beta.-cyclodextrin), polyethylene glycols, and
pectin. The compositions may further include diluents, buffers,
binders, disintegrants, thickeners, lubricants, preservatives
(including antioxidants), flavoring agents, taste-masking agents,
inorganic salts (e.g., sodium chloride), antimicrobial agents
(e.g., benzalkonium chloride), sweeteners, antistatic agents,
surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80",
and pluronics such as F68 and F88, available from BASF), sorbitan
esters, lipids (e.g., phospholipids such as lecithin and other
phosphatidylcholines, phosphatidylethanolamines, fatty acids and
fatty esters, steroids (e.g., cholesterol)), and chelating agents
(e.g., EDTA, zinc and other such suitable cations). Other
pharmaceutical excipients and/or additives suitable for use in the
compositions according to the invention are listed in "Remington:
The Science & Practice of Pharmacy", 19.sup.th ed., Williams
& Williams, (1995), and in the "Physician's Desk Reference",
52.sup.nd ed., Medical Economics, Montvale, N.J. (1998), and in
"Handbook of Pharmaceutical Excipients", Third Ed., Ed. A. H.
Kibbe, Pharmaceutical Press, 2000.
[0198] The prodrugs of the invention may be formulated in
compositions including those suitable for oral, rectal, topical,
nasal, ophthalmic, or parenteral (including intraperitoneal,
intravenous, subcutaneous, or intramuscular injection)
administration. The compositions may conveniently be presented in
unit dosage form and may be prepared by any of the methods well
known in the art of pharmacy. All methods include the step of
bringing the active agent or compound (i.e., the prodrug) into
association with a carrier that constitutes one or more accessory
ingredients. In general, the compositions are prepared by bringing
the active compound into association with a liquid carrier to form
a solution or a suspension, or alternatively, bring the active
compound into association with formulation components suitable for
forming a solid, optionally a particulate product, and then, if
warranted, shaping the product into a desired delivery form. Solid
formulations of the invention, when particulate, will typically
comprise particles with sizes ranging from about 1 nanometer to
about 500 microns. In general, for solid formulations intended for
intravenous administration, particles will typically range from
about 1 nm to about 10 microns in diameter. Particularly preferred
are sterile, lyophilized compositions that are reconstituted in an
aqueous vehicle prior to injection.
[0199] A preferred formulation is a solid formulation comprising
the multi-arm polymer prodrug where the active agent, D, is
irinotecan. The solid formulation comprises sorbitol and lactic
acid, and is typically diluted with 5% dextrose injection or 0.9%
sodium chloride injection prior to intravenous infusion.
[0200] The amount of polymer conjugate in the formulation will vary
depending upon the specific opioid antagonist employed, its
activity in conjugated form, the molecular weight of the conjugate,
and other factors such as dosage form, target patient population,
and other considerations, and will generally be readily determined
by one skilled in the art. The amount of conjugate in the
formulation will be that amount necessary to deliver a
therapeutically effective amount of camptothecin compound to a
patient in need thereof to achieve at least one of the therapeutic
effects associated with the camptothecin compound, e.g., treatment
of cancer. In practice, this will vary widely depending upon the
particular conjugate, its activity, the severity of the condition
to be treated, the patient population, the stability of the
formulation, and the like. Compositions will generally contain
anywhere from about 1% by weight to about 99% by weight prodrug,
typically from about 2% to about 95% by weight prodrug, and more
typically from about 5% to 85% by weight prodrug, and will also
depend upon the relative amounts of excipients/additives contained
in the composition. More specifically, the composition will
typically contain at least about one of the following percentages
of prodrug: 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, or more by
weight.
[0201] Compositions of the present invention suitable for oral
administration may be presented as discrete units such as capsules,
cachets, tablets, lozenges, and the like, each containing a
predetermined amount of the active agent as a powder or granules;
or a suspension in an aqueous liquor or non-aqueous liquid such as
a syrup, an elixir, an emulsion, a draught, and the like.
[0202] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine, with the active
compound being in a free-flowing form such as a powder or granules
which is optionally mixed with a binder, disintegrant, lubricant,
inert diluent, surface active agent or dispersing agent. Molded
tablets comprised with a suitable carrier may be made by molding in
a suitable machine.
[0203] A syrup may be made by adding the active compound to a
concentrated aqueous solution of a sugar, for example sucrose, to
which may also be added any accessory ingredient(s). Such accessory
ingredients may include flavorings, suitable preservatives, an
agent to retard crystallization of the sugar, and an agent to
increase the solubility of any other ingredient, such as polyhydric
alcohol, for example, glycerol or sorbitol.
[0204] Formulations suitable for parenteral administration
conveniently comprise a sterile aqueous preparation of the prodrug
conjugate, which can be formulated to be isotonic with the blood of
the recipient.
[0205] Nasal spray formulations comprise purified aqueous solutions
of the active agent with preservative agents and isotonic agents.
Such formulations are preferably adjusted to a pH and isotonic
state compatible with the nasal mucous membranes.
[0206] Formulations for rectal administration may be presented as a
suppository with a suitable carrier such as cocoa butter, or
hydrogenated fats or hydrogenated fatty carboxylic acids.
[0207] Ophthalmic formulations are prepared by a similar method to
the nasal spray, except that the pH and isotonic factors are
preferably adjusted to match that of the eye.
[0208] Topical formulations comprise the active compound dissolved
or suspended in one or more media such as mineral oil, petroleum,
polyhydroxy alcohols or other bases used for topical formulations.
The addition of other accessory ingredients as noted above may be
desirable.
[0209] Pharmaceutical formulations are also provided which are
suitable for administration as an aerosol, by inhalation. These
formulations comprise a solution or suspension of the desired
polymer conjugate or a salt thereof. The desired formulation may be
placed in a small chamber and nebulized. Nebulization may be
accomplished by compressed air or by ultrasonic energy to form a
plurality of liquid droplets or solid particles comprising the
conjugates or salts thereof.
Methods of Use
[0210] The multi-armed polymer prodrugs of the invention can be
used to treat or prevent any condition responsive to the unmodified
active agent in any animal, particularly in mammals, including
humans.
[0211] The prodrugs of the invention are particularly useful as
anticancer agents, i.e., have been shown to be effective in
significantly reducing the growth of certain representative lung
and colon cancers in in-vivo studies. In particular, the prodrugs
of the invention have been shown to be nearly five times more
effective at preventing the growth of human lung cancer tumors and
human colon cancer tumors than the corresponding anticancer agent
per se, when administered at comparable doses over illustrative
time periods ranging from 30 to 80 days.
[0212] The multi-armed polymer prodrugs of the invention, in
particular, those where the small molecule drug is an anticancer
agent such as a camptothecin compound as described herein or other
oncolytic, are useful in treating breast cancer, ovarian cancer,
colon cancer, gastric cancer, malignant melanoma, small cell lung
cancer, thyroid cancers, kidney cancer, cancer of the bile duct,
brain cancer, lymphomas, leukemias, rhabdomyosarcoma,
neuroblastoma, and the like. The prodrugs of the invention are
particularly effective in targeting and accumulating in solid
tumors. The prodrugs are also useful in the treatment of HIV and
other viruses.
[0213] Methods of treatment comprise administering to a mammal in
need thereof a therapeutically effective amount of a composition or
formulation containing a polymer prodrug of the invention.
[0214] A therapeutically effective dosage amount of any specific
prodrug will vary from conjugate to conjugate, patient to patient,
and will depend upon factors such as the condition of the patient,
the activity of the particular active agent employed, and the route
of delivery.
[0215] For camptothecin-type active agents, dosages from about 0.5
to about 100 mg camptothecin/kg body weight, preferably from about
10.0 to about 60 mg/kg, are preferred. When administered conjointly
with other pharmaceutically active agents, even less of the prodrug
may be therapeutically effective.
[0216] Methods of treatment also include administering a
therapeutically effective amount of a composition or formulation
containing a multi-arm polymer prodrug of a camptothecin compound
as described herein, in conjunction with a second anticancer agent.
Preferably, such camptothecin type prodrugs are administered in
combination with 5-fluorouracil and folinic acid, as described in
U.S. Pat. No. 6,403,569.
[0217] The prodrug of the invention may be administered once or
several times a day, preferably once a day or less. The duration of
the treatment may be once per day for a period of from two to three
weeks and may continue for a period of months or even years. The
daily dose can be administered either by a single dose in the form
of an individual dosage unit or several smaller dosage units or by
multiple administration of subdivided dosages at certain
intervals.
EXAMPLES
[0218] It is to be understood that while the invention has been
described in conjunction with certain preferred specific
embodiments thereof, the foregoing description as well as the
examples that follow are intended to illustrate and not limit the
scope of the invention. Other aspects, advantages and modifications
within the scope of the invention will be apparent to those skilled
in the art to which the invention pertains.
[0219] All PEG reagents referred to in the appended examples are
available from Nektar Therapeutics, Huntsville, Ala. All .sup.1HNMR
data was generated by a 300 or 400 MHz NMR spectrometer
manufactured by Bruker.
ABBREVIATIONS
[0220] DCM: dichloromethane [0221] DCC dicyclohexylcarbodiimide
[0222] DMAP dimethylaminopyridine [0223] HCl hydrochloric acid
[0224] MeOH methanol [0225] CM carboxymethylene [0226] HOBT
hydroxybenzyltriazole [0227] TFA trifluoroacetic acid [0228] RT
room temperature [0229] SCM succinimidyl
Example 1
Synthesis of Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2
Oxo-Vinylamino Acetate Linked-Irinotecan)-20 K
[0230] A. Synthesis of t-Boc-Glycine-Irinotecan
##STR00011##
[0231] In a flask, 0.1 g Irinotecan (0.1704 mmoles), 0.059 g
t-Boc-Glycine (0.3408 mmoles), and 0.021 g DMAP (0.1704 mmoles)
were dissolved in 13 mL of anhydrous dichloromethane (DCM). To the
solution was added 0.070 g DCC (0.3408 mmoles) dissolved in 2 mL of
anhydrous DCM. The solution was stirred overnight at room
temperature. The solid was removed through a coarse frit, and the
solution was washed with 10 mL of 0.1N HCL in a separatory funnel.
The organic phase was further washed with 10 mL of deionized
H.sub.2O in a separatory funnel and then dried with
Na.sub.2SO.sub.4. The solvent was removed using rotary evaporation
and the product was further dried under vacuum. .sup.1H NMR (DMSO):
.delta. 0.919 (t, CH.sub.2CH.sub.3), 1.34 (s, C(CH.sub.3).sub.3),
3.83 (m, CH.sub.2), 7.66 (d, aromatic H).
B. Deprotection of t-Boc-Glycine-Irinotecan
##STR00012##
[0232] 0.1 g t-Boc-Glycine-Irinotecan (0.137 mmoles) was dissolved
in 7 mL of anhydrous DCM. To the solution was added 0.53 mL
trifluoroacetic acid (6.85 mmoles). The solution was stirred at
room temperature for 1 hour. The solvent was removed using rotary
evaporation. The crude product was dissolved in 0.1 mL MeOH and
then precipitated in 25 mL of ether. The suspension was stirred in
an ice bath for 30 minutes. The product was collected by filtration
and dried under vacuum. .sup.1H NMR (DMSO): .delta. 0.92 (t,
CH.sub.2CH.sub.3), 1.29 (t, CH.sub.2CH.sub.3), 5.55 (s, 2H), 7.25
(s, aromatic H).
C. Covalent Attachment of a Multi-Armed Activated Polymer to
Glycine Irinotecan.
##STR00013##
[0234] 0.516 g Glycine-Irinotecan (0.976 mmoles), 3.904 g
4arm-PEG(20 K)-CM (0.1952 mmoles), 0.0596 g
4-(dimethylamino)pyridine (DMAP, 0.488 mmoles), and 0.0658 g
2-hydroxybenzyltriazole (HOBT, 0.488 mmoles) were dissolved in 60
mL anhydrous methylene chloride. To the resulting solution was
added 0.282 g 1,3-dicyclohexylcarbodiimide (DCC, 1.3664 mmoles).
The reaction mixture was stirred overnight at room temperature. The
mixture was filtered through a coarse frit and the solvent was
removed using rotary evaporation. The syrup was precipitated in 200
mL of cold isopropanol over an ice bath. The solid was filtered and
then dried under vacuum. Yield: 4.08 g. .sup.1H NMR (DMSO):
.quadrature.0.909 (t, CH.sub.2CH.sub.3), 1.28 (m,
CH.sub.2CH.sub.3), 3.5 (br m, PEG), 3.92 (s, CH.sub.2), 5.50 (s,
2H).
Example 2
Anti-Tumor Activity of Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2
Oxo-Vinylamino Acetate Linked-Irinotecan)-20 K,
"4-Arm-Peg-Gly-Irino-20 K" in a Colon Cancer Mouse Xenograft
Model
[0235] Human HT29 colon tumor xenografts were subcutaneously
implanted in athymic nude mice. After about two weeks of adequate
tumor growth (100 to 250 mg), these animals were divided into
different groups of ten mice each. One group was dosed with normal
saline (control), a second group was dosed with 60 mg/kg of
irinotecan, and the third group was dosed with 60 mg/kg of the
4-arm PEG-GLY-Irino-20 K (dose calculated per irinotecan content).
Doses were administered intravenously, with one dose administered
every 4 days for a total of 3 administered doses. The mice were
observed daily and the tumors were measured with calipers twice a
week. FIG. 1 shows the effect of irinotecan and PEG-irinotecan
treatment on HT29 colon tumors in athymic nude mice.
[0236] As can be seen from the results depicted in FIG. 1, mice
treated with both irinotecan and 4-arm-PEG-GLY-Irino-20 K exhibited
a delay in tumor growth (anti-tumor activity) that was
significantly improved when compared to the control. Moreover, the
delay in tumor growth was significantly better for the
4-arm-PEG-GLY-Irino-20 K group of mice when compared to the group
of animals administered unconjugated irinotecan.
Example 3
Synthesis of Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2
Oxo-Vinylamino Acetate Linked-Irinotecan)-40 K,
"4-Arm-Peg-Gly-Irino-40 K"
[0237] 4-arm-PEG-GLY-IRINO-40 K was prepared in an identical
fashion to that described for the 20 K compound in Example 1, with
the exception that in step C, the multi-armed activated PEG reagent
employed was 4 arm-PEG(40 K)-CM rather than the 20 K material.
Example 4
Synthesis of Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2
Oxo-Vinylamino Acetate Linked-SN-38)-20 K, "4-Arm-Peg-Gly-SN-38-20
k"
[0238] 4-arm PEG-GLY-SN-38-20 K was prepared in a similar fashion
to its irinotecan counterpart as described in Example 1, with the
exception that the active agent employed was SN-38, an active
metabolite of camptothecin, rather than irinotecan, where the
phenolic-OH of SN-38 was protected with MEMCl
(2-methoxyethoxymethyl chloride) during the chemical
transformations, followed by deprotection with TEA to provide the
desired multi-armed conjugate.
Example 5
Synthesis of Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2
Oxo-Vinylamino Acetate Linked-SN-38)-40 K, "4-Arm-Peg-Gly-SN-38-40
k"
[0239] 4-arm PEG-GLY-SN-38-40 K was prepared in a similar fashion
to the 20 K version described above, with the exception that the
multi-armed activated PEG reagent employed was 4 arm-PEG(40 K)-CM
rather than the 20 K material.
Example 6
Additional Xenograft Studies
[0240] Additional mouse xenograft studies were conducted to further
examine the efficacy of exemplary multi-armed polymer conjugates of
the invention.
[0241] Athymic nude mice were implanted subcutaneously with human
cancer cell lines (lung cancer cell line NCl-H460, and colon cancer
cell line HT-29) and the tumors allowed to grow to approximately
150 mg in size. The animals were divided into groups of ten mice
each.
[0242] Various compounds and doses were evaluated as follows:
irinotecan (40, 60 and 90 mg/kg); 4-arm-PEG-GLY-IRINO-20 K (40, 60,
and 90 mg/kg); 4-arm-PEG-GLY-IRINO-40 K ((40, 60, and 90 mg/kg);
4-arm-PEG-GLY-SN-38-20 K (7.5, 15, 30 mg/kg), and PEG-GLY-SN-38-40
K (7.5, 15, 30 mg/kg). Doses were administered intravenously, with
one dose administered every 4 days for a total of 3 administered
doses.
[0243] Tumor volume measurements were taken over a period of 60-80
days; tumor volumes were converted to tumor weight. Body weights
were also measured over the same period to provide an indication of
weight loss. The results are presented graphically in FIGS.
2-5.
Example 7
PK Study
Colon Tumor Xenograft in Mice
[0244] A comparative single dose pharmacokinetic (PK) study of a
multi-armed PEG-irinotecan versus unmodified irinotecan in nude
mice was conducted to assess tumor distribution of parent and
metabolite drug.
[0245] The study employed 108 nude mice, 36 mice per group, 4
animals per sample point. Drug was administered intravenously as a
single dose. Drug form and doses were as follows: irinotecan (40
mg/kg); 4-arm-PEG-GLY-IRINO-20 K (40 mg/kg equivalents);
4-arm-PEG-GLY-IRINO-40 K ((40 mg/kg equivalents). Venous plasma and
tumor tissue samples were taken at the following time points: 20
minutes, 40 minutes, and 1, 2, 4, 12, 24, 48, and 72 hours, and
evaluated for concentrations of the following species:
4-arm-PEG-GLY-IRINO-20 K, 4-arm-PEG-GLY-IRINO-40 K, irinotecan and
SN-38. The results are plotted in FIGS. 6 to 13.
[0246] As can be seen in FIGS. 6-13, based upon the rate of decline
of the multi-armed PEGylated species in tumor tissue in comparison
to plasma, the PEGylated species demonstrate a notable increase in
tumor retention time when compared to unmodified parent drug.
[0247] In looking at the metabolite results, the concentrations of
SN-38 derived from the PEGylated compounds appear to be increasing
at the end of the 72 hour period, while in contrast, SN-38 derived
from irinotecan is essentially cleared in 12 hours. In sum, the
tumor exposure to SN38 following administration of either of the
PEGylated compounds is approximately five times greater than for
irinotecan over the same 72 hour sampling period. In sum, both
multi-arm PEGylated compounds provide an increased inhibition of
tumor growth (colon and lung) for both in-vivo tumor models
investigated in comparison to unmodified drug. More specifically,
both multi-arm PEGylated compounds demonstrated a marked
suppression of tumor growth when compared to unmodified drug in
mouse xenograft models, indicating the effectiveness of such
compounds as anti-cancer agents. Lastly, administration of the
multi-arm PEGylated irinotecan compounds described herein appears
to cause less diarrhea in rats than irinotecan itself.
Example 8
Synthesis of
Pentaerythritolyl-4-Arm-(PEG-2-{2-[2-1-Hydroxy-2-Oxo-Vinyloxy)-Ethoxy]-Et-
hylamino}-Propen-1-One Linked-Irinotecan)-20 K and -40 K
##STR00014## ##STR00015##
[0248] A. 2-(2-t-Boc-aminoethoxy)ethanol (1)
[0249] 2-(2-Aminoethoxy)ethanol (10.5 g, 0.1 mol) and NaHCO.sub.3
(12.6 g, 0.15 mol) were added to 100 mL CH.sub.2Cl.sub.2 and 100 mL
H.sub.2O. The solution was stirred at RT for 10 minutes, then
di-tert-butyl dicarbonate (21.8 g, 0.1 mol) was added. The
resulting solution was stirred at RT overnight, then extracted with
CH.sub.2Cl.sub.2 (3.times.100 mL). The organic phases were combined
and dried over anhydrous sodium sulfate and evaporated under
vacuum. The residue was subjected to silica gel column
chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH=50:1.about.10:1) to
afford 2-(2-t-Boc-aminoethoxy)ethanol (1) (16.0 g, 78 mmol, yield
78%)
B. 2-(2-t-Boc-aminoethoxy)ethoxycarbonyl-Irinotecan (2)
[0250] 2-(2-t-Boc-aminoethoxy)ethanol (1) (12.3 g, 60 mmol) and
4-dimethylaminopyridine (DMAP) (14.6 g, 120 mmol) were dissolved in
200 ml anhydrous CH.sub.2Cl.sub.2. Triphosgene (5.91 g, 20 mmol)
was added to the solution while stirring at room temperature. After
20 minutes, the solution was added to a solution of irinotecan (6.0
g, 10.2 mmol) and DMAP (12.2 g, 100 mmol) in anhydrous
CH.sub.2Cl.sub.2 (200 mL). The reaction was stirred at RT for 2
hrs, then washed with HCl solution (pH=3, 2 L) to remove DMAP. The
organic phases were combined and dried over anhydrous sodium
sulfate. The dried solution was evaporated under vacuum and
subjected to silica gel column chromatography
(CH.sub.2Cl.sub.2:CH.sub.3OH=40:1.about.10:1) to afford
2-(2-t-Boc-aminoethoxy)ethoxycarbonyl-irinotecan (2) (4.9 g, 6.0
mmol, yield 59%).
C. 2-(2-aminoethoxy)ethoxycarbonyl-irinotecan TFA salt (3)
[0251] 2-(2-t-Boc-aminoethoxy)ethoxycarbonyl-irinotecan (2) (4.7 g,
5.75 mmol) was dissolved in 60 mL CH.sub.2Cl.sub.2, and
trifluoroacetic acid (TFA) (20 mL) was added at RT. The reaction
solution was stirred for 2 hours. The solvents were removed under
vacuum and the residue was added to ethyl ether and filtered to
give a yellow solid as product 3 (4.3 g, yield 90%).
D. 4-arm-PEG.sub.20k-carbonate-inotecan (4)
[0252] 4-arm-PEG.sub.20k-SCM (16.0 g) was dissolved in 200 mL
CH.sub.2Cl.sub.2. 2-(2-aminoethoxy)ethoxycarbonyl-irinotecan TFA
salt (3) (2.85 g, 3.44 mmol) was dissolved in 12 mL DMF and treated
with 0.6 mL TEA, then added to a solution of 4-arm-PEG.sub.20k-SCM.
The reaction was stirred at RT for 12 hrs then precipitated in
Et.sub.2O to yield a solid product, which was dissolved in 500 mL
IPA at 50.degree. C. The solution was cooled to RT and the
resulting precipitate collected by filtration to give
4-arm-PEG.sub.20k-glycine-irinotecan (4) (16.2 g, drug content 7.5%
based on HPLC analysis). Yield: 60%.
E. 4-arm-PEG.sub.40k-carbonate-irinotecan (5)
[0253] 4-arm-PEG.sub.40k-SCM (32.0 g) was dissolved in 400 mL
CH.sub.2Cl.sub.2. 2-(2-aminoethoxy)ethoxycarbonyl-irinotecan TFA
salt (3) (2.85 g, 3.44 mmol) was dissolved in 12 mL DMF and treated
with 0.6 mL TEA, then added to the solution of
4-arm-PEG.sub.40k-SCM. The reaction was stirred at RT for 12 hrs
and then precipitated in Et.sub.2O to get solid product, which was
dissolved in 1000 mL isopropyl alcohol (IPA) at 50.degree. C. The
solution was cooled to RT and the precipitate collected by
filtration to gave 4-arm-PEG.sub.40k-glycine-irinotecan (4) (g,
drug content 3.7% based on HPLC analysis). Yield: 59%.
[0254] Many modifications and other embodiments of the invention
will come to mind to one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Therefore, it is to be understood that the
invention is not to be limited to the specific embodiments
disclosed and that modifications and other embodiments are intended
to be included within the scope of the appended claims. Although
specific terms are employed herein, they are used in a generic and
descriptive sense only and not for purposes of limitation.
* * * * *