WO2005112999A2

WO2005112999A2 - Cancer treatment with inhibitors of collagen synthesis

Info

Publication number: WO2005112999A2
Application number: PCT/US2005/017732
Authority: WO
Inventors: Paul M. Simon
Original assignee: Vectramed, Inc.
Priority date: 2004-05-20
Filing date: 2005-05-20
Publication date: 2005-12-01
Also published as: WO2005112999A3

Abstract

Compositions and methods are provided for the treatment of tumors with a combination of anti-collagen agents and anti-neoplastic agents. In some embodiments one or more of the anti-collagen agents and anti-neoplastic agents may be provided in a biocompatible polymer matrix. The invention also provides prodrugs comprising anti-collagen and anti-tumor agent conjugates. The inventive compositions and methods can be useful in shrinking or reducing tumor size by abrogating drug diffusion barriers that are often the result of collagenous extracellular matrix of solid tumors.

Description

Cancer Treatment with Inhibitors of Collagen Synthesis

FIELD OF THE INVENTION [0001] The invention relates generally to anti -tumor agents and, more

particularly, to drugs and methods of treating tumors with anti-fϊbrotic agents in conjunction with chemotherapy.

BACKGROUND OF THE INVENTION [0002] Despite recent advances in anti-tumor agents and drug delivery, limitations in cancer treatment remain. With particular relevance to solid tumors,

therapeutic macromolecules, such as, for example, monoclonal antibodies ("Mabs") have not fared well in clinical trials. Indeed, while there are a number of approved

anti-tumor Mabs, with the exception of HERCEPTIN^® and ERBITUX^®, available therapeutic Mabs target - tumor cells that reside in the vasculature or are well vascularized, or molecular targets that reside on vascular endothelial cells. Therefore, the promise of biological and other macromolecular therapeutic agents against solid

tumors remains to fully materialize.

[0003] The presence of an excessively collagenous extracellular matrix in the milieu of solid tumors has been reported, which suggests that the collagen matrix may

produce a diffusion barrier to macromolecules and thereby limit their access to

cancerous cells in a solid tumor mass (Brown E, et al., 2003, Nature Medicine 9: 796-

801.) Accordingly, there is a need for anti-tumor treatment that provides an anti- tumor agent in conjunction with an anti-fϊbrotic agent that inhibits collagen

production thereby enhancing diffusion of the anti-tumor agent into the tumor.

SUMMARY OF THE INVENTION [0004] The foregoing needs are met, to an extent, by the present invention,

wherein in one aspect an anti-tumor composition for shrinking or retarding the growth of a tumor, which may be a solid tumor, is provided, comprising: a biocompatible polymer matrix, an anti-tumor agent, and an anti-fibrotic agent, wherein the anti-

fibrotic agent is free or incorporated into and released from the polymer matrix by degradation of the matrix or diffusion of the agent out of the matrix over a period of time in an amount effective to shrink or retard growth of the tumor.

[0005] The anti-tumor agent may comprise an anti-tumor antibody, an anti- angiogenic agent, a monoclonal antibody, a micellar or liposomal preparation, a fusion protein, or a combination thereof. The anti-tumor agent may further comprise a

protein, liposome, or a polymer conjugate. The anti-tumor agent may further yet comprise a taxane, such as paclitaxel or docetaxel; and, the anti-fibrotic agent may comprise a proline analog or a lathyrogen. The anti-fibrotic agent can be selected from the group consisting of ra-hydroxyproline, dehydroproline, thiaproline,

azetidinecarboxylic acid, and β-aminopropionitrile.

[0006] The polymer matrix may be biodegradable, formed of a polymer

selected from the group consisting of poly(ethylene glycol), polymethylmethacrylate, polyamino acids, oligosaccharides, polysaccharides, polyvinyl alcohol, polylactic acid,

polyglycolic acid and copolymers and blends thereof, or formed of poly(lactide-co- glycolide). The composition may further comprise additional biologically active compounds selected from the group consisting of chemotherapeutics, immunoglobulins, antibiotics, antivirals, antiinflammatories, cytokines,

immunotoxins, anti-tumor antibodies, anti-angiogenic agents, anti-edema agents,

radiosensitizers, monoclonal antibodies, cytokine receptors or their cognate ligands, toxins, nucleic acids, oligosaccharides, polysaccharides, synthetic polymers, lipids, micellar or liposomal preparations, chemical conjugates of the above with cytotoxic or

cytostatic or other drugs, fusion proteins, and combinations thereof.

[0007] In another embodiment of the present invention a method of treating a patient in need of shrinking or retarding the growth of a tumor, which may be a solid tumor, is provided, comprising: administering to the patient an effective amount of an anti-tumor agent and an effective amount of an anti-fibrotic agent, wherein the anti- fibrotic agent is incorporated into and released from a polymer matrix by degradation of the polymer matrix or diffusion of the anti-fibrotic agent out of the matrix over a

period of time, to shrink or retard growth of the tumor.

[0008] The anti-tumor agent may comprise an anti-tumor antibody, an anti- angiogenic agent, a monoclonal antibody, a micellar or liposomal preparation, a fusion protein, or a combination thereof. The anti-tumor agent may further comprise a

protein, liposome, or a polymer conjugate. The anti-tumor agent may further yet comprise a taxane, such as paclitaxel or docetaxel; and, the anti-fibrotic agent may

comprise a proline analog or a lathyrogen. The anti-fibrotic agent can be selected

from the group consisting of cz^'s-hydroxyproline, dehydroproline, thiaproline,

azetidinecarboxylic acid, and β-aminopropionitrile. [0009] The anti-tumor agent may be administered systemically or locally delivered by direct infusion to the tumor, and/or the anti-fibrotic agent may be

administered systemically or locally delivered by implantation of the biocompatible

polymer matrix incorporating the anti-fibrotic and/or the anti-tumor agent. The polymer matrix may be biodegradable, formed of a polymer selected from the group

consisting of poly(ethylene glycol), polymethylmethacrylate, polyamino acids, oligosaccharides, polysaccharides, polyvinyl alcohol, polylactic acid, polyglycolic acid

and copolymers and blends thereof, or formed of poly(lactide-co-glycolide). [0010] The method may further comprise administering radiation in

combination with the anti-tumor agent and the anti-fibrotic agent. In some embodiments, the method may also comprise administering an additional biologically active compounds selected from the group consisting of chemotherapeutics, immunoglobulins, antibiotics, antivirals, anti-inflammatories, cytokines, immunotoxins, anti-tumor antibodies, anti-angiogenic agents, anti-edema agents, radiosensitizers, monoclonal antibodies, cytokine receptors or their cognate ligands, toxins, nucleic acids, oligosaccharides, polysaccharides, synthetic polymers, lipids, micellar or liposomal preparations, chemical conjugates of the above with cytotoxic or cytostatic or other drugs, fusion proteins, and combinations thereof.

[0011] The compositions may be in the form of micro-implants and may be administered by injection or infusion. The method may involve the substantially

simultaneous administration of the anti-tumor agent with the anti-fibrotic agent. In

some embodiments, at least one of the anti-tumor agent and anti-fibrotic agent can be

administered intravenously, intraperitonealy, or intramuscularly, and in other embodiments, both of the anti-tumor agent and the anti-fibrotic agent is administered intravenously. [0012] In yet another embodiment of the present invention, a prodrug

composition is provided, comprising: at least one anti-fibrotic agent; at least one anti-

tumor agent; and wherein the at least one anti-fibrotic agent and the at least one anti- tumor agent are coupled by one or more hydrolysable bonds. The hydrolysable bond

may be selected from an ester, diester, urethane, amide, secondary or tertiary amine, and an ether bond. The hydrolysable bond may also include any enzyme-cleavable

group such as a peptide bond, a phosphodiester bond, oligosaccharide bonds, and other chemical groups. The composition may further include a biodegradable polymer, and the biodegradable polymer may be coupled to the at least one anti- fibrotic agent and the at least one anti-tumor agent. The prodrug composition may be

water-soluble. [0013] In still yet another embodiment of the present invention, a method of treating a patient in need of shrinking or retarding the growth of tumors is provided, comprising: administering a prodrug composition comprising: at least one anti-fibrotic agent; at least one anti-tumor agent; and wherein the at least one anti-fibrotic agent

and the at least one anti -tumor agent are joined by one or more hydrolysable bonds. The hydrolysable bond may be selected from an ester, diester, urethane, amide, secondary or tertiary amine, and an ether bond. The hydrolysable bond may also

include any enzyme-cleavable group such as a peptide bond, a phosphodiester bond,

oligosaccharide bonds, and other chemical groups. The composition may further include a biodegradable polymer, and the biodegradable polymer may be coupled to

the at least one anti-fibrotic agent and the at least one anti-tumor agent.

[0014] In further still yet another embodiment of the present invention, a drug- polymer composition having the general formula D1-X-P-Y-D2 is provided wherein P is a polymer; wherein the drugs Dl and D2 are separately an anti-tumor agent and an

anti-fibrotic agent; and the drugs and polymer are linked by the covalent linkages X and Y. P may be a water-soluble polymer, optionally cross-linked to form an

insoluble gel, and/or selected from the group consisting of poly(ethylene glycol),

polymethylmethacrylate, polyamino acids, oligosaccharides, polysaccharides, polyvinyl alcohol, polylactic acid, polyglycolic acid and copolymers and blends thereof. X and Y may be individually an ester, diester, urethane, amide, secondary or tertiary amine or ether linking groups. Alternatively, X any Y may also include any enzyme-cleavable group such as a peptide bond, a phosphodiester bond, oligosaccharide bonds, and other chemical groups.

[0015] In still yet another embodiment of the present invention, a method of treating a patient in need of shrinking or retarding the growth of tumors, is provided, comprising: administering a drug-polymer composition having the general formula D1-X-P-Y-D2, wherein P is a polymer; wherein the drugs Dl and D2 are separately an anti-tumor agent and an anti-fibrotic agent; and the drugs and polymer are linked by the covalent linkages X and Y. P may be a water-soluble polymer, optionally cross-linked to form an insoluble gel, and/or selected from the group consisting of

poly(ethylene glycol), polymethylmethacrylate, polyamino acids, oligosaccharides,

polysaccharides, polyvinyl alcohol, polylactic acid, polyglycolic acid and copolymers and blends thereof. X and Y may be individually an ester, diester, urethane, amide,

secondary or tertiary amine or ether linking groups. Alternatively, X any Y may also

include any enzyme-cleavable group such as a peptide bond, a phosphodiester bond, oligosaccharide bonds, and other chemical groups. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is a graph depicting the molar therapeutic efficacy of free CHOP

and conjugated CHOP-PEG. Anti-hypertensive effects of free CHOP (D) and CHOP- PEG (■) administered in 7-day s.c. miniosmotic pumps from a representative dose-

response experiment is shown. Each data point represents the mean value for groups

of 4-6 rats. The regression lines fit to the data have r² values of 0.55 for free CHOP and 0.92 for CHOP-PEG, and the half-maximal response intercepts occur at 170 mM for free CHOP and at 0.17 mM for conjugated CHOP-PEG.

[0017] FIG. 2 shows the treatment of Balb/c female mice bearing the mammary carcinoma EMT-6 s.c. with the polymer Pt prodrug VEO-063 in the presence and absence of the collagen inhibitory conjugate CHOP-PEG.

[0018] FIG. 3 depicts the survival curve of EMT-6 bearing mice treated with the regimen described in Fig. 1.

[0019] FIG. 4 is the treatment profile of Balb/c female mice bearing the mammary carcinoma EMT-6 s.c. with the polymer Pt prodrug VEO-067 in the presence and absence of the collagen inhibitory conjugate CHOP-PEG, and also in the presence of the control analog prodrug conjugate THOP-PEG.

[0020] FIG. 5 is a schematic of one preparation of poly(PEG2000-Lys-c/,s-

hydroxy-L-4-proline).

[0021] FIG. 6 is a schematic of one preparation of VEO-063. DETAILED DESCRIPTION

[0022] In accordance with the instant invention, compositions and methods are provided for the treatment of tumors with a combination of anti-collagen agents and

anti-tumor agents. Anti-tumor agents are defined herein broadly to encompass both

naturally occurring and synthetic pharmaceuticals, chemotherapeutics, as well as so- called biologies, including, for example, antibodies, nucleic acids, proteins, lipids, and

other biological molecules. [0023] Before proceeding with one application of the present invention in treating tumors, it will be apparent to one of ordinary skill in the art from the teachings herein, that the agents and methods provided may be equally suitable for any and all applications where fibrosis is an impediment to therapy. Such applications may include, without limitations to, inflammation therapy, such as, for example,

arthritis. [0024] Among the anti-inflammatory Abs that have anti-fibrotic effects are the

following: anti-TGFβ, anti-ILl, and anti-TNFα. Neutralization of the CXC

chemokine also has anti-inflammatory and anti-fibrotic effects. In addition, thalidomide has anti-inflammatory and anti-collagen effects. Abs that inhibit

angiogenic factors also have anti-inflammatory activity and include anti-FGFβ and

anti-VEGF are such Mabs that have been cited for anti-fibrotic effects.

[0025] Without being limited to or bound by theory, treatment with an

inhibitor of collagen biosynthesis would be expected to have a selective effect on sites of fibrosis. Collagen, a ubiquitous protein that provides the "glue" of most tissues, as well as many other biological functions, turns over rather slowly during normal tissue maintenance; but in fibrotic sites, such as in inflammation, wound healing, and also tumors, collagen turnover is relatively rapid.

[0026] Accordingly, one method of inhibiting collagen synthesis is by the use of proline analogues that interfere with the posttranslational processing of collagen

precursors. Such interference can result in enhanced degradation and reduced production of mature collagen molecules. Proline analogs may be incorporated into

pro-α-collagen chains in lieu of endogenous proline and impair folding of pro- collagen into a stable triple helix by steric hindrance. In this way, "flawed" collagen molecules (i.e., molecules incorporating proline analogues) are produced, which are then targetted for intracellular degradation.

[0027] Table 1 lists a variety of known anti-fibrotic agents that may be used in accordance with the present invention. Cw-hydroxyproline ("CHOP") is an analog of trα -hydroxyproline, the natural form of the imino acid which is abundant in

collagen. In the presence of CHOP, which is incorporated into the nascent chains, collagen is degraded, rather than secreted. However, the efficacy of treatment with

free CHOP can be limited by its very rapid renal elimination. Aggressive dosing has been used to overcome this problem, which in turn, has been shown to generate systemic toxic side effects.

[0028] To overcome, at least in part, some of these limitations, a prodrug

conjugate of CHOP has been synthesized and a co-polymer backbone consisting of

poly[(ethylene glycol)-lysine], or CHOP-PEG. In one embodiment, this water-soluble

conjugate can release free CHOP at fibrotic sites undergoing elevated collagen biosynthesis in a sustained manner. [0029] The conjugate can be administered by any parenteral route, and even a single dose given subcutaneously (s.c.) or intravenously (i.v.). Administration of the conjugate provides about a week-long anti-fibrotic activity in animal models of

pulmonary hypertension and fibrosis, as well as surgical adhesions. The polymer

version can exhibit a 1, 000-fold enhanced molar potency of CHOP over its free form

in some embodiments. A partial list of polymer supports that may be used in conjugate or in conjunction with the aforementioned anti-fibrotic agents is listed in Table 1.

[0030] A comparison of free CHOP to a conjugate, CHOP-PEG, was examined in acute pulmonary hypertension models using continuous infusion with a s.c.-implanted mini-pump (FIG. 1). Rats treated with graded doses of free CHOP exhibited a reduction in right ventricular pressure (RVP); but, even near the limit of

solubility (50 mg, or 382 μmol, in a 100 μl reservoir of a 7-day osmotic pump) the

mean reduction in RVP achieved on day 7 of hypoxia was about 57 ± 8%. By

contrast, use of CHOP-PEG resulted in more than about a 70% reduction in RVP at a

dose of 2.5 mg conjugate (1.05 μmol CHOP). A comparison of the therapeutic effects of free and polymeric forms of CHOP suggests that free CHOP exhibits approximately a half-maximal molar potency of 170 μM CHOP, which is about 3 orders of magnitude lower than that of CHOP-PEG (0.17 μM CHOP).

[0031] Again, without being bound by or limited to theory, it is probable that a

carboxypeptidase (not yet identified) is upregulated at the site of inflammatory fibrosis and causes the local release of free CHOP from the carrier backbone. Solid tumor

environments are also characterized by the presence of inflammatory cytokines and cells, and it is probable that free CHOP can be similarly liberated, with the result of inhibiting collagenous fibrotic activity.

[0032] Since the heavily collagenous matrix surrounding tumors is thought to

limit tumor growth, the activity of a collagen-inhibitory agent can facilitate tumor

growth in some models. Treatment with CHOP alone has resulted in accelerated growth of mammary carcinoma in rats. Consistent with this mechanism, expression of particular extracellular matrix degrading metalloproteinases (MMP) provides a growth

advantage to tumors in 3-D matrices (Hotary KB, et al., 2003, Cell 114:33).

[0033] However, in accordance with the present invention, anti-fibrosis treatment has been combined with the administration of an anti-tumor agent, which may result in enhanced anti-tumor activity. The combination of agents suppressing collagen biosynthesis can enhance the therapeutic efficacy of anti-tumor agents, especially macromolecular ones, by facilitating their diffusional access to tumor cells. Table 2 is a partial list of anti-cancer Mabs and other anti-tumor macromolecular agents that may be used in some embodiments of the inventive method.

[0034] This strategy was tested by treating mice with a growing mammary carcinoma (EMT-6) with a combination of CHOP-PEG and a macromolecular conjugate of Platinum (Pt) (VEO-063). This conjugate bears a Pt-chelating group

held to the PEG backbone through a peptide (SSSGPQG-IFGN) designed to be

cleaved by the enzyme MMP-2 (at the site maked by "~"), which is upregulated in tumors. CHOP-PEG treatment was daily x 4 s.c, and a single dose of VEO-063 was

administered on day 3 of this regimen. Control animals received either agent alone or

vehicle.

[0035] The results indicate that, while neither agent alone inhibited tumor growth very much, the combination of CHOP-PEG with the anti-tumor conjugate VEO-063 was at least additive. This effect was seen in reduced tumor size at the time the drug-free control group's reached endpoint (2,000 mg), delayed growth of tumor

by several days in mice treated with the combination at the midpoint of control tumor

growth (1,000 mg) (FIG. 2), and also in the survival of the mice treated with the drug combination (FIG. 3). Addition of CHOP-PEG to VEO-063 did not significantly alter

the Pt-conjugate's toxicity to the mice.

[0036] This strategy was repeated with a different Pt-containing polymer prodrug, VEO-067, also designed to be cleaved by MMP-2, but bearing a different peptide sequence (SSSLIPVS~LIS). In this experiment we also included a polymer prodrug that bears the trans- analog of CHOP (THOP), or THOP-PEG (FIG. 4). Treatment of Balb/c female mice bearing the mammary carcinoma EMT-6 s.c. with the polymer Pt prodrug VEO-067 in the presence and absence of the collagen inhibitory conjugate CHOP-PEG, and also in the presence of the control analog prodrug conjugate THOP-PEG.

[0037] In this experiment CHOP-PEG alone caused substantially enhanced tumor growth, but the combination of CHOP-PEG and the macromolecular Pt- containing conjugate VEO-067, just as in the previous experiment, resulted in

significant inhibition of tumor growth. The combination of CHOP-PEG + VEO-067

caused a maximum reduction of tumor growth of about 64% (on day 26) and a delay to reach 1,000 mg of 7 days. This delay corresponded to a tumor log kill of 5.0. Free

VEO-067 had more negligible effects against this tumor. The use of the conjugate

THOP-PEG bearing the trans analog of CHOP, had no effect by itself or in

combination with VEO-067. There was no net survival benefit for the mice treated with any of the drugs, when compared to vehicle control animals.

[0038] These results indicate that disruption of the fibrous matrix surrounding

solid tumors by the treatment with collagen-inhibitory prodrugs augments the potency of macromolecular anti-tumor agents by facilitating their diffusion from the vascular

compartment. The anti-neoplastic agents used were two different constructs consisting of PEG-Lys copolymer backbones bearing repeating units of a Pt-chelate

complex linked to these backbones with a peptide designed to be cleaved by MMP-2, a proteinase up-regulated in tumor microenvironments. The single dose regimen of these Pt-containing regimens were minimally active. However, in combination with the anti-fibrotic agent CHOP-PEG their anti-tumor potency was dramatically increased.

[0039] The potentiating effect of the collagenolytic agent is likely to be useful in the treatment with other macromolecular therapeutic agents such as monoclonal antibodies, cytokines, toxins, and other proteins, and their various conjugates, fragments, constructs, and fusion proteins. The Pt-containing polymer prodrugs used in this study had MW in the range of about 25 - about 35 kDa, which is sufficient to retard and concentrate them in the tumor milieu, according to the EPR effect (Greish K, et al., 2003, Clin Pharmacokinet 42:1089-1105). The combination of an anti-

fibrotic and an anti-neoplastic agent should be even more effective in the case of anti-

tumor Mabs, because their greater MW (-150 kDa) would hinder their diffusional access across the collagenous matrix to the tumor even more.

Chemistry

[0040] The following chemicals were purchased and used without further

purification: poly(PEG2000-Lys-CHOP), or CHOP-PEG, and PEG2000 bis(succinimidyl) carbonate (BSC-PEG2000) (NJ Center for Biomaterials), and Na,Nε-di-Boc-L-lysine hydroxysuccinimide ester. Η NMR spectra were recorded on

a Varian Gemini-300 spectrometer. FT-IR spectra were obtained on a Perkin-Elmer Spectrum One spectrometer.

[0041] HPLC grade solvents (acetonitrile, dichloromethane, tetrahydrofuran

(THF), water, isopropyl alcohol (IP A)) were used for synthesis and gel permeation chromatography (GPC). Molecular weights were determined, relative to PEG standards, by GPC on an Agilent 1100 chromatography system with Shodex GPC

KD-G and Shodex GPC KD-803 columns (JM Science, Inc.) connected in series using DMF (0.1% LiBr) as eluent.

Preparation of polv(PEG2000-Lvs-c .y-hvdroxy-L-4-proline\ or CHOP-PEG.

[0042] The poly(PEG-Lys-CHOP) polymer was synthesized as previously described (Greco MJ, et al., 1997, Am. J. Respir. Crit. Care. Med. 155:1391-1397). Briefly, Lys-CHOP dipeptides were synthesized, and chain extension was

accomplished by copolymerizing BSC-PEG2000 to the α- and e-amines of the Lys- CHOP dipeptides (FIG. 5). Two batches of CHOP-PEG were used in the experiments reported here. Their respective characteristics were GPC: MW = 8,000 and 11,000. Preparation of poly(PEG2000-Lys-tram'-hvdroxy-L-4-proline), or THOP-PEG.

[0043] This conjugate was prepared identically to CHOP-PEG but using Lys- THOP instead of Lys-CHOP.

Preparation of VEO-063. VEO-067.

[0044] The polymer constructs can be described using the general formula:

where in P is a short segment of poly(ethylene glycol) with an average molecular

weight of approximately 2000 Daltons, M is a trifunctional monomer such as L-lysine

or l,3-diamino-2-propanol, L is an enzymatically labile linker functions (in most cases a peptide), D is a biologically active pharmaceutical agent, and n is the number of repeating units of the co-polymer backbone. Although these systems can be prepared using several pathways, the preferred route to assemble the desired products is illustrated in Figure 5. General Synthetic Procedure.

[0045] The linker-monomer conjugates, M-L, were assembled on an automated peptide synthesizer using Fmoc protected amino acids and standard coupling reagents. In the case of VEO-063, L was SSSGPQG-IFGN, and in the case of VEO-067, L was SSSLIPVS-LIS. The presumptive MMP-2 cleavage site is indicated by "~" (Netzel-Arnet, et al. 1993, Biochemistry 32:6427-32; Turk, et al., 2001, Nat Biotechnol 19:661-7, respectively). PEG 6w-(Ν-hydroxysuccinimidyl) carbonate was reacted with the M-L to give the regular repeating block co-polymer

with 8-12 pendant linker groups, L, on each backbone, terminating in Asp. The

pharmaceutical agent, D, was then attached to the linker functions on the polymer

system. D was Pt, added as PtC14, for chelation by the terminal Asp at the C-terminus of the L peptide group. It was stabilized as the diaminochclohexyl (DACH) adduct,

which was prepared by the direct addition of diaminocyclohexylplatinum diiodide/Ag SO₄ H O to the terminal aspartic acid residue of the linker yielding the desired product.

Biology

Tumor growth.

[0046] Balb/c female mice were implanted with l-2xl0⁶ EMT-6 mammary

carcinoma cells (obtained from the American Type Culture Collection) s.c. in the

flank. CHOP-PEG or THOP-PEG, dissolved in PBS, was inoculated s.c. on days 4-7 at 2 mg/kg. On day 6 a single i.p. inoculation of VEO-063 (20 mg Pt/kg) or VEO-067

(20 mg Pt/kg) dissolved in distilled water was administered. These were the previously determined maximum-tolerated doses of the Pt-containing conjugates.

[0047] Tumor growth was monitored by measuring two orthogonal diameters of the s.c. mass with calipers. Tumor volume was calculated by the formula ab²/2, where a is the larger and b is the smaller tumor diameter in mm, and expressed as mg,

assuming a density of 1 mg/mm³. Tumors were measured until mice died or until tumors reached a volume of 2,000 mg, at which time mice were euthanized.

[0048] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or alterations of the

invention following. In general, the principles of the invention and including such

departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential

features hereinbefore set forth and as follows in the scope of the appended claims. Table 1. Anti-fibrotic agents and polymeric supports anti-fibrotic agent polymeric support Reference cis-hydroxyproline (CHOP) none

CHOP poly(lactide-co-glycoli e) Yasukawa T, et al., 2002, Graefes Arch Clin Exp Ophthalmol 240:672-678

CHOP albumin microparticles, magnetic lanotti JP, et al., 1991 , J Orthoped Res 9:423-444

CHOP-PEG poly(ethylene glycol)

CHOP-PEG polyethylene glycol) polymethylmethacrylate polyamino acids oligosaccharides polysaccharides polyvinyl alcohol polylactic acid polyglycolic acid dehydroproline (DHP) none

DHP-PEG PEG thiaproline (THP) none

THP-PEG PEG azetidinecarboxylic acid (ACA) none ACA-PEG PEG beta-aminopropionitrile (BAPN) none

Table 2. Anti-tumor agents (for use in combination with anti-fibrotic agents) Approved Antl-Cancer Mabs Solid

Trade name Alias Mab form Payload TTaarrgαeett Indication tumor Reference

Atemtuzumab Campath humanized CD52 CLL, CML Ross JS, et al., 2003, Am J Clin Pathol 119:472-485 cancer, .

Oacllzumab Zenapax chimeric CD25 leukemia Ross JS, et al., 2003, Am J din Pathol 119:472-485 NHL, BCL, Rituximab Rituxan chimeric CD20 CLL Ross JS. et al..2003. Am J Clin Pathol 119:472-485 Breast Ca, NSCLC, pancreas Ca

Trastuzumab Herceptin humanized pβδneu + Ross JS, et al., 2003, Am J Clin Pathol 119:472-485 Gemtuzumab ylotarg humanized calicheamycin CCDD3333 AML Ross JS. et al., 2003, Am J Clin Pathol 119:472-185 Lymphoma, NHL, follicular

Ibrttumomab Zevalin munne Y-90 CD20 lymphoma Ross JS, et al., 2003, Am J Clin Pathol 119:472-485 epithelial Colon Edrecolomab Panorex murinβ adhesion + Ross JS, et al., 2003, Am J Clin Pathol 119:472-485 Ross JS, et al., 2003, Am J Clin Pathol 119:472-485 Ross JS, et al., 2003, Am J Clin Pathol 119:472-485

Mabs in Development Ross JS, et al., 2003, Am J Clin Pathol 119:472-485 Phase 3 Ross JS, et al., 2003, Am J Clin Pathol 119:472-485 Ross JS. et al., 2003, Am J Clin Pathol 119:472-485

Tositumomab Bexxar 1-131 CD2Θ NHL Ross JS. et al., 2003, Am J Clin Pathol 119:472-485 Colon, NSCLC,

CβaVac muππe CEA breast, liver Ross JS. et al., 2003, Am J Clin Pathol 119:472-485 Epratuzumab LymphoCide chimeric NHL Ross JS, et al., 2003, Am J Clin Pathol 119:472-485 SCCL,

Mitumomab muriπe melanoma Ross JS, et al..2003. Am J Clin Pathol 19:472-485 Colon, breast,

Bevaclzumab Avastin humanized VEGF

NSCLC. colon, breast,

Cetuximab Erbitux chimeric EGFR pancreas Ross JS, et al., 2003, Am J Clin Pathol 119:472-485

Colon,

Edrecolomab murine breast Ross JS, et al., 2003. Am J Clin Pathol 119:472-485 Lintuzumab Zamyl chimeric AML Ross JS, et al., 2003, Am J Clin Pathol 119:472-485 bi-specitlc Ovary,

chimeric prostate, colon, renal,

MDX-210 HER-2/neu Fc breast Ross JS, et al..2003. Am J Clin Pathol 119:472-485 NSCLC, liver, colon, esophagus,

IGN-101 muπne stomach Ross JS. et al., 2003. Am J Clin Pathol 119:472-485

Phase 2 humanized Prostate,

MDX-010 HER-2 melanoma Ross JS. et al., 2003, Am J Clin Pathol 119:472-*85 Sarcoma, MabAME chimeric colon Ross JS, et al., 2003. Am J Clin Pathol 119:472-485 Renal, NSCLC.

ABX-EGF human EGF H&N, ovary Ross JS, etal., 2003, Am J Clin Pathol 119:472-485

EMD 72,000 Ross JS, etal., 2003. Am J Clin Pathol 119:472-485

Apolizumab Ross JS, et al..2003, Am J Clin Pathol 119:472-485

Labetuzomab Ross JS, etal., 2003, Am J Clin Pathol 119:472-485 lor-rl Ross JS, et al., 2003, Am J Clin Pathol 119:472-485

MDX-220 Ross JS. et al., 2003, Am J Clin Pathol 119:472-485

MRA Ross JS, et al., 2003, Am J din Pathol 119:472-485

H-11 scFv Ross JS, et al., 2003, Am J Clin Pathol 119:472-485

Oregvomab Ross JS, et al., 2003. Am J Clin Pathol 119:472-485 huJ591 Mab BZL RossJS, et al., 2003, Am J Clin Pathol 119:472-485

Vlsllizumab Ross JS, et al., 2003, Am J Clin Pathol 119:472-485

TriGe Ross S, et al., 2003, Am J Clin Pathol 119:472-485

TrlAb Ross S, etal., 2003, Am J Clin Pathol 119:472-485

R3 Ross S, et al., 2003, Am J Clin Pathol 119:472-485

MT-201 Ross S, et al..2003, Am J Clin Pathol 119:472-485

G-250 Ross JS, et al., 2003, Am J Clin Pathol 119:472-485

ACA-125 Ross JS, et al., 2003, Am J Clin Pathol 119:472-485

)πyvax-105 Ross JS, et al., 2003, Am J Clin Pathol 119:472-485

Phase 1

CDP-860 Ross JS, et al , 2003. Am J din Pathol 119:472-485

BrevaRex Ross JS, et al, , 2003, Am J Clin Pathol 119:472-485

AR54 Ross JS, et al, , 2003, Am J din Pathol 119:472-485

IMC-1C11 Ross JS, et al. , 2003, Am J Clin Pathol 119:472-485

GlioMab-H Ross JS, et al. , 2003, Am J Clin Pathol 119:472-485

ING-1 Ross JS, et al, , 2003, Am J Clin Pathol 119:472-485

Antl-LCG Ross JS, et al. , 2003, Am J Clin Pathol 119:472-485

MT- 03 Ross JS, et al, , 2003, Am J din Pathol 119:472-485

KSB-303 Ross JS, et al, , 2003, Am J Clin Pathol 119:472-485

Therex Ross JS, et al. , 2003, Am J din Pathol 119:472-485 W-2871 Ross JS, et al. , 2003, Am J din Pathol 119:472-485

Antl-HMI.24 Ross JS, et al. , 2003, Am J din Pathol 119:472-485

Antl-PTHrP Ross JS, et al..2003, Am J Clin Pathol 119:472-485

2C4 Ross JS, etal. , 2003, Am J Clin Pathol 119:472-485

SGN-30 CD30 Ross JS, etal. , 2003, Am J din Pathol 119:472-485

TRAIL-RI Ross JS, etal. , 2003, Am J Clin Pathol 119:472-485

ABX-MA1 Ross JS, etal. , 2003. Am J Clin Pathol 119:472-485

Imuteran Ross JS, et al. , 2003. Am J Clin Pathol 119:472-485

Liposomes

Doxil Doxorubicln PELT Doxorublcin Cathepsin B Duncan R, et al., 2001, J Contr Rel 74:135-146

Polymeric Pro-druαs Active Enzymatic Company Backbone aαent linker

VEO-063 VectraMed PEG Pt MMP-2 + VEO-0β7 VectraMed PEG Pt MMP-2 + VEO-020 VectraMed PEG Pt MMP-2 + VEO-035 VectraMed PEG Pt MMP-2 VEO-028 VectraMed PEG Pt MMP-2 VEO-061 VectraMed PEG Pt MMP-2 VEO-037 VectraMed PEG Pt plasmin VEO-039 VectraMed PEG Pt plasmin VEO-066 VectraMed PEG Pt uPA VEO-069 VectraMed PEG Doxorubicin MMP-2 VEO-002 VectraMed PEG 5FU Cathepsin B VEO-003 VectraMed PEG Doxorubicin Cathepsin B

+ +

I ω m ω m m

ABBREVIATIONS

CLL chronic lymphocytic leukemia

CML chronic myelogenous leukemia

NHL non-Hodgkins lymphoma

BCL B-cell lymphoma

Ca cancer/ carcinoma

NSCCL non-small cell carcinoma of the lung

AML acute myelogenous leukemia

SCCL small cell cacrinoma of the lung

H&N head and neck

CEA carcinoembryonic antigen

VEGF vascular-endothβlial growth factor

EGF epidermal growth factor

EGFR EGF-receptor

PEG polyethylene glycol)

HPMA poly(hydrosypropylmethacrylate)

Pt platinum

MMP matrix metalloproteinase uPA urokinase-type plasminogen activator

SMA poly(styrene-co-maleic acid) half-n-butyl ester

ADEPT Ab-directed enzyme prodrug therapy

Claims

What is claimed is:

1. An anti-tumor composition for retarding the growth of a tumor or causing its shrinkage, comprising:

a biocompatible polymer matrix, an anti-tumor agent, and an anti-fibrotic agent,

wherein the anti-fibrotic agent is free or incorporated into and released from the polymer matrix by degradation of the matrix or diffusion of the anti-fibrotic agent out of the matrix over a period of time in an amount effective to facilitate the retardation of the growth of a tumor or facilitate its shrinkage.

2. The composition of claim 1 wherein the anti-tumor agent comprises an anti-tumor antibody, an anti-angiogenic agent, a monoclonal antibody, a micellar or liposomal preparation, a fusion protein, or a combination thereof.

3. The composition of claim 1 wherein the anti-tumor agent comprises a protein, liposome, or a polymer conjugate.

4. The composition of claim 1 wherein the anti-tumor agent comprises one or more taxanes.

5. The composition of claim 4 wherein the taxane is paclitaxel, docetaxel, or a combination thereof.

6. The composition of claim 1 wherein the anti-fibrotic agent comprises a proline analog, a lathyrogen, or a combination thereof.

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7. The composition of claim 6 wherein the anti-fibrotic agent is selected from the group consisting of c/s-hydroxyproline, dehydroproline, thiaproline, azetidinecarboxylic acid, β- aminopropionitrile, or combinations thereof.

8. The composition of claim 1 wherein the polymer matrix is biodegradable.

9. The composition of claim 1 wherein the polymer matrix is formed of a polymer selected from the group consisting of poly(ethylene glycol), polymethylmethacrylate, polyamino acids, oligosaccharides, polysaccharides, polyvinyl alcohol, polylactic acid, polyglycolic acid and copolymers and blends thereof.

10. The composition of claim 1 wherein the polymer matrix is formed of poly(lactide-co- glycolide).

11. The composition of claim 1 further comprising additional biologically active compounds selected from the group consisting of chemotherapeutics, immunoglobulins, antibiotics, antivirals, antiinflammatories, cytokines, immunotoxins, anti-tumor antibodies, anti-angiogenic agents, anti-edema agents, radiosensitizers, monoclonal antibodies, cytokine receptors or their cognate ligands, toxins, nucleic acids, oligosaccharides, polysaccharides, synthetic polymers, lipids, micellar or liposomal preparations, chemical conjugates of the above with cytotoxic or cytostatic or other drugs, fusion proteins, and combinations thereof.

12. The composition of claim 1 wherein the tumor is a solid tumor.

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13. A method of treating a patient suffering from the negative effects of tumor, comprising:

administering to the patient an effective amount of an anti-tumor agent and an effective amount of an anti-fibrotic agent,

wherein the anti-fibrotic agent is free or incorporated into and released from a polymer matrix by degradation of the polymer matrix or diffusion of the anti-fibrotic agent out of the matrix over a period of time, to facilitate the retardation of the growth of a tumor or facilitate its shrinkage.

14. The method of claim 13 wherein the tumor is a solid tumor.

15. The method of claim 13 wherein the anti-tumor agent comprises an anti -tumor antibody, an anti-angiogenic agent, a monoclonal antibody, a micellar or liposomal preparation, a fusion protein, or a combination thereof.

16. The method of claim 13 wherein the anti-tumor agent comprises a protein, liposome, or a polymer conjugate.

17. The method of claim 13 wherein the anti-tumor agent comprises one or more taxanes.

18. The method of claim 17 wherein the taxane is paclitaxel, docetaxel, or a combination thereof.

19. The method of claim 13 wherein the anti-fibrotic agent comprises a proline analog, a lathyrogen, or a combination thereof.

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20. The method of claim 19 wherein the anti-fibrotic agent is selected from the group consisting of c/s-hydroxyproline, dehydroproline, thiaproline, azetidinecarboxylic acid, β- aminopropionitrile, or a combination thereof.

21. The method of claim 13 wherein the anti-tumor agent is locally delivered by direct infusion to the tumor.

22. The method of claim 13 wherein the anti-fibrotic agent is locally delivered by implantation of the biocompatible polymer matrix incorporating the anti -tumor agent.

23. The method of claim 13 wherein the polymer matrix is biodegradable.

24. The method of claim 13 wherein the polymer matrix is formed of a polymer selected from the group consisting of poly(ethylene glycol), polymethylmethacrylate, polyamino acids, oligosaccharides, polysaccharides, polyvinyl alcohol, polylactic acid, polyglycolic acid and copolymers and blends thereof.

25. The method of claim 24 wherein the polymer matrix is formed of poly(lactide-co- glycolide).

26. The method of claim 13 further comprising administering radiation in combination with the anti-tumor agent and/or the anti-fibrotic agent.

27. The method of claim 13 further comprising administering an additional biologically active compounds selected from the group consisting of chemotherapeutics, immunoglobulins, antibiotics, antivirals, anti-inflammatories, cytokines, immunotoxins,

20 anti-tumor antibodies, anti-angiogenic agents, anti-edema agents, radiosensitizers, monoclonal antibodies, cytokine receptors or their cognate ligands, toxins, nucleic acids, oligosaccharides, polysaccharides, synthetic polymers, lipids, micellar or liposomal preparations, chemical conjugates of the above with cytotoxic or cytostatic or other drugs, fusion proteins, and combinations thereof.

28. The method of claim 13 wherein the composition is in the form of micro-implants and is administered by injection or infusion.

29. The method of claim 13 wherein the anti-tumor agent is administered substantially simultaneously with the anti-fibrotic agent.

30. The method of claim 13 wherein at least one of the anti-tumor agent and anti-fibrotic agent is administered intravenously, intraperitonealy, or intramuscularly.

31. The method of claim 30 wherein both of the anti-tumor agent and the anti-fibrotic agent is administered intravenously.

32. A prodrug composition comprising:

at least one anti-fibrotic agent;

at least one anti-tumor agent; and

wherein the at least one anti-fibrotic agent and the at least one anti-tumor agent are coupled by one or more hydrolysable bonds.

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33. The composition of claim 32 wherein the hydrolysable bond is selected from an ester, diester, urethane, amide, secondary or tertiary amine, or ether bond.

34. The composition of claim 33 further including a biodegradable polymer.

35. The composition of claim 34 wherein the biodegradable polymer is coupled to the at least one anti-fibrotic agent and/or the at least one anti-tumor agent.

36. The composition of claim 32 wherein the prodrug composition is water-soluble.

37. A method of treating a patient in need of shrinking or retarding the growth of tumors, comprising:

administering a prodrug composition comprising:

at least one anti-fibrotic agent;

at least one anti-tumor agent; and

wherein the at least one anti-fibrotic agent and the at least one anti-tumor agent are joined by one or more hydrolysable bonds.

38. The method of claim 37 wherein the hydrolysable bond is selected from an ester, diester, urethane, amide, secondary or tertiary amine, or ether bond.

39. A method of claim 37 further including a biodegradable polymer.

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40. A composition of claim 39 wherein the biodegradable polymer is couple to the at least one anti-fibrotic agent and the at least one anti-tumor agent.

41. A drug-polymer composition having the general formula D1-X-P-Y-D2:

wherein P is a polymer;

wherein the drugs Dl and D2 are separately an anti-tumor agent and an anti-fibrotic agent;

and the drugs and polymer are linked by the covalent linkages X and Y.

42. The composition of claim 41 wherein P is a water-soluble polymer.

43. The composition of claim 41 wherein P is a polymer selected from the group consisting of poly(ethylene glycol), polymethylmethacrylate, polyamino acids, oligosaccharides, polysaccharides, polyvinyl alcohol, polylactic acid, polyglycolic acid and copolymers and blends thereof.

44. The composition of claim 41 wherein X and Y are individually an ester, diester, urethane, amide, secondary or tertiary amine or ether linking groups.

45. The composition of claim 42 in which said water-soluble polymer is cross-linked to form an insoluble gel.

46. A method of treating a patient in need of shrinking or retarding the growth of tumors, comprising:

23 administering a drug-polymer composition having the general formula D1-X-P-Y-D2,

wherein P is a polymer;

and the drugs and polymer are linked by the covalent linkages X and Y.

47. The composition of claim 46 wherein P is a water-soluble polymer.

48. The composition of claim 46 wherein P is a polymer selected from the group consisting of poly(ethylene glycol), polymethylmethacrylate, polyamino acids, oligosaccharides, polysaccharides, polyvinyl alcohol, polylactic acid, polyglycolic acid and copolymers and blends thereof.

49. The composition of claim 46 wherein X and Y are individually an ester, diester, urethane, amide, secondary or tertiary amine or ether linking groups.

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