WO2004089311A2

WO2004089311A2 - Polymeric drug agents for the treatment of fibrotic disorders

Info

Publication number: WO2004089311A2
Application number: PCT/US2004/009919
Authority: WO
Inventors: James M. Pachence; Benjamin A. Belinka; Charles L. Putnam
Original assignee: Vectramed, Inc.
Priority date: 2003-03-31
Filing date: 2004-03-30
Publication date: 2004-10-21
Also published as: CA2521407A1; JP2006522140A; WO2004089311A3; EP1608380A2

Abstract

Agents and methods for treatment of adhesions and fibrotic diseases, through the release of drugs that retard or inhibit fibrotic tissue production. A method for releasing fibrotic tissue-inhibiting agents from a polymer is provided. The polymer/drug combination can be applied directly to affected site as a liquid, gel, or paste. Alternatively, the polymer/drug combination can be injected ( intravenous, intraperitoneal, or subcutaneous) in an appropriate vehicle.

Description

POLYMERIC DRUG AGENTS FOR THE TREATMENT OF FIBROTIC

DISORDERS

FIELD OF THE INVENTION [0001] The present invention relates generally to agents and methods for treating and/or preventing fibrosis. More particularly, the present invention relates to agents and methods for treating fibrotic disorders through the release of drugs from within a polymeric matrix.

BACKGROUND OF THE INVENTION [0002] Fibrosis, also known as scarring, is manifest in many clinical diseases, conditions,

and disorders (the terms have been used interchangeably herein). Hepatic fibrosis, for

example, usually results from chronic liver disease, and is often complicated by severe

distortion (cirrhosis). Cardiovascular fibrosis leads to the loss of flexibility in the ventriculo-

arterial system, which is often accompanied by isolated systolic hypertension and congestive

heart failure.

[0003] As well, surgical intervention leads to the formation of fibrotic adhesions between

incision sites of organs, an undesirable side effect often requiring corrective surgery

thereafter. By way of example, surgical intervention for correcting bowel obstruction which

occurs in 60% of all laparotomy cases has a 5% to 30% mortality rate (Ellis, 1997). Pelvic

adhesions involving ovaries or the fallopian tubes following gynecological operations result

in infertility for up to 45% of the patient population (Stangel et al., 1984). Thus, there is a

significant need for methods of treating fibrosis.

[0004] Of the numerous extracellular matrix proteins that comprise a fibrotic adhesion, collagens are believed to contribute significantly to clinical fibrosis. In addition to their relative abundance at fibrotic adhesions, the proteolysis of collagen appears to be a rate- limiting step for extracellular matrix removal. Accordingly, a number of approaches have

been attempted to retard and/or prevent the accumulation of extracellular matrix proteins, and in particular, collagen.

[0005] In an attempt to minimize surgical trauma, for example, a wide range of resorbable and nonresorbable barrier materials have been investigated (Sawhney, et al, 1994; Edwards, et

al, 1997; Urman, et al, 1991). Despite the development in this field, the few products available for clinical use (principally barrier materials such as oxidized regenerated cellulose

or hyaluronic acid) remain only marginally effective.

[0006] An alternative, molecular, approach has been to perturb the functionally requisite

three-dimensional structure of collagen, which structure contains a high percentage of trans-

hydroxylated proline and lysine residues. Indeed, hydroxylation is a prerequisite for collagen

fiber formation, and proline analogues such as cw-4-hydroxy- (cHyp), fluoro-, or bromo-

proline or 3,4-L-dehydroproline (DHP), that incorporate directly into nascent chains of

procollagen, have been effective (Uitto and Prockop, 1974; Uitto and Prockop, 1977; Kao and

Prockop, 1976; Liotta et al., 1978). Administration of monomeric cHyp for limited periods of time has been shown to prevent collagen accumulation in animals in a variety of experimental

models, including abdominal adhesions, cirrhosis, and pulmonary fibrosis (Giri, 1990; Tan et

al, 1983).

[0007] Although short-term studies have demonstrated the efficacy of some anti-fribrotic

drugs, there are at least two limitations to the potential long-term use of such drugs: (1)

systemic toxicity resulting from interference with general protein synthesis (Eldridge, et al,

1988), and (2) rapid resorption, diffusion, and/or excretion of the drug. Thus, there is a need for treatments that may target anti-fibrotic agents to the fibrotic tissues and/or increase localized retention of the therapeutic agent.

SUMMARY OF THE INVENTION [0008] The foregoing needs are met, to an extent, by the present invention, wherein in one

aspect a fibrotic tissue inhibiting agent is provided, comprising: a drug/polymer conjugate of formula:

-[P-M]- n or Q-(P-L-D)k or (D-L)m-PP

L

D

wherein, P is a water-soluble polymer segment; M is a multifunctional moiety joining water

soluble polymer segments P into a co-polymer backbone and providing attachment for groups -L-D to the backbone; L is a linker or a chemical bond; D is a fibrotic tissue inhibiting

compound; Q is a multivalent coupler; n is an integer greater than 2; k is an integer of 2 to

1000; PP is a polymer with one or more functional groups to attach the -L-D groups; and, m

is an integer. D may be an anti-fibrotic agent, an anti-proliferative agent, and/or a fibrotic tissue inhibiting proline analogue: ct5^,-4-hydroxy-L-proline (cHyp), cts-4-hydroxy-D-proline,

cw-3-hydroxy-DL-proline, 3, 4-dehydro-L-proline(DHP), cw-4-fluoro-L-proline, cts-4-chloro-

L-proline, cts-4-bromo-L-proline, L-azetidine-2-carboxylic acid (AZA), or L-thiazolidine-4-

carboxylic acid (THP). Where D is an anti-fibrotic agent, D may be pirfenidone, tranilast, halofuginone, pentoxifylline, relaxin, estradiol, interleukin 10, pyridine-2, or 4-dicaboxylic-

di(2-methoxyethyl) amide. Where D is an anti-proliferative agent, D may be 5-flurouracil,

mitomycin-C, or paclitaxel. D may also be retinoic acid, a retinoic acid analogue, a retinoic

acid antagonist, a plasminogen activator, a vastatin, or a non-steroidal anti-inflammatory compound. D may be released by enzymatic action or metabolic activity on linker L. Polymer segment P may be of average molecular weight 400-25,000Da, derived from compounds having at least two functionalities for covalent attachment to M, selected from the group consisting of hydroxyl, amino, thiol, alkyl disulfide, aryl disulfide, isothiocyanate,

thiocarbonylimidazole, thiocarbonylchloride, aldehyde, ketone, carboxylic acid, carboxylic acid ester, sulfonic acid, sulfonic acid ester, sulfonyl chloride, phosphoric acid, alkyl succinimidylcarbonate, aryl succinimidyl- carbonate, alkyl chlorocarbonate, aryl

chlorocarbonate, alkyl succinimidylthiocarbonate, aryl succinimidylthiocarbonate, alkyl

chlorothiocarbonate, aryl chlorothiocarbonate, halide, and thioester. hi some embodiments, P

may be a polyethylene glycol, polyvinyl alcohol, poly(2-hydroxyethyl methacrylate),

poly(acrylic acid), poly(methacrylic acid), poly(maleic acid), polylysine, or a mixture thereof.

In other embodiments, P may be a poly(carboxylic acid), poly(orthoester), poly(anhydride),

pluronic polyol, poly(vinylpyrrolidone), poly(acrylate), polyamide, polyphosphazine,

poly(amino acid),polypeptide, pseudo-poly (amino acid), linear or branched polymer

containing PEG, copolymers of PEG, a dendrimer, or a PEG-dendrimer. PP may be a

polyethylene glycol, polyvinyl alcohol, poly(2-hydroxyethyl methacrylate), poly(acrylic acid), poly(methacrylic acid), poly(maleic acid), polylysine, or a mixture thereof, or a poly(carboxylic acid), poly(orthoester), poly(anhydride), pluronic polyol,

poly(vinylpyrrolidone), poly(acrylate), polyamide, polyphosphazine, poly(amino

acid),polypeptide, pseudo-poly (amino acid), linear or branched polymer containing PEG,

copolymers of PEG, a dendrimer, a PEG-dendrimer, collagen, hyaluronic acid, fatty acid

lipid, polyhydroxyalkanoate, chondrotin sulfate, glycosaminoglycan, chitosan, alginate, starch, dextran, a carbohydrate-based polymer, cellulose, or cellulose derivative, n may be an

integer from 3 to 100 inclusive, k may be an integer from 2 to 100 inclusive, and m may be an

integer from 1 to 100 inclusive. [0009] In another embodiment of the present invention, a method for treatment of tissue

adhesions is provided, comprising: administering to a tissue in need of treatment for surgically-induced adhesions or fibrotic disease, an effective amount of a fibrotic tissue inhibiting agent, comprising: a drug/polymer conjugate of formula:

-[P-M]- n or Q-(P-L-D)k or (D-L)m-PP

I

L

D

wherein, P is a water-soluble polymer segment; M is a multifunctional moiety joining water soluble polymer segments P into a co-polymer backbone and providing attachment for groups

-L-D to the backbone;L is a linker or a chemical bond; D is a fibrotic tissue inhibiting

is an integer, hi some embodiments, m is an integer greater than 1 and in other embodiments,

m is an integer of 1 to 1000. The agent may be administered topically by inhalation, or by

injection, and may be encapsulated in an eroding polymer or entrapped in a polymer matrix.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 shows the growth of rat lung fibroblasts on uncoated plates. All treatment

began at day 0 and at 2.0 mg/ml.

[0011] Figure 2 depicts the cytotoxicity of free CHYP and Poly(PEG-Lys-cHyp)

constructs (2 synthesis batches) on RFL-6 rat fibroblast cells. [0012] Figure 3 shows the effects of 0.1 mg/ml free proline analogs on RFL-6 cell growth in regular or collagen-precoated wells (day 4). CHYP (0.76 mM), THP (0.75 mM), DHP (0.90 mM).

[0013] Figure 4 shows the effect of four anti-fibrotic formulations on the return of

adhesions after adhesiolysis and treatment. The percent of returning adhesions (number of adhesions present/number of adhesions at the initial observation).

[0014] Figure 5 depicts a comparison of the adhesion grade (as graded according to Table

4) at the initial observation versus three weeks after adhesiolysis and treatment.

DETAILED DESCRIPTION [0015] The compositions and methods provided herein can enhance delivery of a fibrotic tissue inhibiting (also referred to as "anti-fibrotic") drug and may do so by extending the half-

life of the drug, sustaining its release, releasing the drug by metabolic activity and/or releasing same at the site of inflammation. Such enhanced delivery of the fibrotic tissue inhibiting drug

is accomplished by combining the drug with a polymer. Any anti-fibrotic agent may be

incorporated into the compositions and methods of the invention for the prevention and

treatment of fibrotic tissue and/or adhesions.

[0016] By way of example, fibrotic tissue inhibiting drugs can include, but are not limited

to, proline analogues such as, for example, cw-4-hydroxy-L-proline, cw-4-hydroxy-D-proline,

ct_r-3-hydroxy-DL-proline, 3,4-dehydro-L-proline, cw-4-fluoro-L-proline, c/.s-4-chloro-L-

proline, cw-4-bromo-L-proline, L-azetidine-2-carboxylic acid (AZA), and L-thiazolidine-4-

carboxylic acid (THP). Anti-fibrotic drags including pirfenidone, tranilast, halofuginone, pentoxifylline, relaxin, estradiol, interleukin 10, pyridine-2,4-dicarboxylic-di(2-

methoxyethyl)amide may also be useful as drugs incorporated into the delivery format disclosed herein. In addition, anti-proliferative drugs, such as 5-fluorouracil, mitomycin-C,

and paclitaxel may be employed. Drags showing anti-fibrotic effects such as retinoic acid, retinoic acid analogues, retinoic acid antagonists, plasminogen activator, vastatins, and/or non-steroidal anti-inflammatory drags may also be used.

[0017] In one embodiment of the invention, a fibrotic tissue inhibiting drag can be

covalently linked to synthetic or naturally occurring polymers. Attachment of the drug to a polymer confers a number of properties to the drag/polymer conjugate, including, increased

accumulation at the site of inflammation, which has been referred to as "enhanced

permeability and retention" (Maeda, H, 2001); enhanced solubilization of highly hydrophobic

drugs (if the polymer itself is soluble); increased half-life and/or sustained delivery of the

drag. The macromolecular size of the drag/polymer conjugates can effect the drug half-life

and biodistribution.

[0018] A linker that attaches the drag to the polymer may contain one or more chemical

bonds that may be cleaved by enzymes; alternatively, chemical bonds between the drag and

linker may be used that can release the drag by local metabolic and/or chemical activity such as changes in pH, ionic, or redox conditions, or released by reaction with the in situ

environment such as hydrolysis, reduction reactions, oxidative reactions, pH shifts,

photolysis, or combinations thereof, thus releasing the active drag. The drag from the drag/polymer conjugates can be released by a combination of enzymatic and non-enzymatic

activity. In a preferred embodiment, the anti-fibrotic drag may be released from the polymer

by a condition related to the formation of adhesions.

[0019] In some embodiments of the present invention, the drag/polymer conjugates comprise polymers of the type poly[D-L-M-P], in which M is a multifunctional chemical moiety that is used to join water soluble polymer segments P to form a regular repeating

linear co-polymer backbone and additionally to provide the chemical substituents for the

attachment of the fibrotic tissue inhibiting drag D via a linker L, and in which n is the number of repeats of D-L-M-P. The fibrotic tissue inhibiting drag D can be covalently attached to linker L or can be directly attached to M, with the polymer then being poly[D-M-P] . The

water-soluble polymer segment P consists of a relatively short, water-soluble polymeric

system (for example, average M.W. 400-25,000 Da) which contains at least two homogenous

chemical functionalities (for example, including but not limited to, hydroxyl, amino, thiol, alkyl or aryl disulfide, isothiocyanate, thiocarbonylimidazole, thiocarbonylchloride, aldehyde,

ketone, carboxylic acid, carboxylic acid ester, sulfonic acid, sulfonic acid ester, sulfonyl

chloride, phosphoric acid, alkyl or aryl succinimidyl carbonate, alkyl or aryl chlorocarbonate,

alkyl or aryl succinimidylthiocarbonate, alkyl or aryl chlorothiocarbonate, halide, or thioester)

that can be used for covalent attachment to the multifunctional chemical moiety M.

[0020] P can be, but is not restricted to, poly(ethylene glycol), poly(vinyl alcohol), poly(2-

hydroxyethyl methacrylate), poly(acrylic acid), poly(methacrylic acid), poly(maleic acid), poly(lysine), and the like or a copolymer consisting of a mixture of these or other polymeric

entities possibly substituted with organic functional groups. Similarly, polymers of the type

Q-(P-L-D) can be used for this invention, in which Q is designed to couple to k number of

short water soluble polymer segments, P. For this polymer, the fibrotic tissue inhibiting drag D can be covalently attached to linker L or can be directly attached to P, with the polymer

then being Q-(P-D)_k.

[0021] Other suitable polymers for covalent linkage of the fibrotic tissue inhibiting drug

according to the invention include, but are not limited to, synthetic polymers such as

poly(carboxylic acids), poly(ortho esters), poly(anhydrides), poly(vinyl alcohol), pluronic

polyols, poly(vinylpyrrolidone), poly(acrylates), poly(amides), polyphosphazenes, poly(amino acids), branched polypeptides, pseudo-poly(amino acids), poly(ethylene glycol), branched

polymers containing PEG, co-polymers of PEG and other mentioned polymers, PEG-

dendrimers, and other denrimers; natural polymers such as collagens, hyaluronic acid, fatty acids, lipids, polyhydroxyalkanoates, chondrotin sulfates, glycosaminoglycans, chitosans,

alginates, starches, dextran, cellulose and its derivatives, and other carbohydrate based

polymers.

[0022] The drag/polymer composite may be applied by injection or administered

topically. The drug/polymer composite can be encapsulated within an eroding polymer, and

then applied by injection or administered topically. The drug/polymer composite can be

entrapped into a polymer matrix using synthetic or naturally occurring polymers, and then

applied topically, by injection, or by implantation.

[0023] The anti-fibrotic drag D or the linking group/drag D-L can be covalently attached to many of the above mentioned polymers by previously described methods, forming a system

such as PP-(D-L)_n, whereby PP is one of the polymers listed above having a molecular weight between 1,000 and 1,000,000 Daltons and chemical groups that can be used to attach n copies of the linking group/drag conjugate D-L.

[0024] The polymer used for the covalent attachment of the fibrotic tissue inhibiting drag may also have a moiety that can be targeted to the tissue or cell types associated with the formation of adhesions or fibrotic diseases. In a preferred embodiment, the polymers may be of the type T-R-P-L-D, (T-R) _q-poly[D-L-M-P] , (T-R) _q-poly[D-M-P] , (T-R) _q-Q-(P-L-D)_k,

(T-R) _q-Q-(P-D) _ki poly[D-L-M]-poly[T-R-M] (with either random or sequential repeats of the

pendant chains), and poly[D-M]-poly[T-R-M] (with either random or sequential repeats of

the pendant chains) in which T is a targeting agent (such as an antibody, receptor binder, or enzyme binder), R is a chemical group which links T to the polymer, and q is the number of

repeats of T-R.

= (T-R)_q-poly[D-L-M-P]

T-ΓM-P]— T

I n = (T-R)_q-poly[D-M-P]

D

poly[D-L-M]-poly[T-R-M]

-[M-P] — [M-P]— ^L i n ^L i ^J _n poly[D-M]-poly[T-R-M]

T D [0025] In some embodiments, the fibrotic tissue inhibiting drag may be encapsulated using synthetic or naturally occurring polymers. The polymer composite may surround the core of drag, and can be in the form of particles such as microspheres that can be used for

injection, implantation, or topical delivery. The encapsulating polymer(s) may be degraded by enzymes, by local metabolic activity such as changes in pH, ionic, or redox conditions, or

the drug may be released by reaction with the in situ environment such as hydrolysis,

reduction reactions, oxidative reactions, pH shifts, photolysis, or combinations thereof. In a

preferred embodiment, the drag could be released from the polymer by a condition related to the formation of adhesions.

[0026] hi other embodiments, the fibrotic tissue inhibiting drag can be entrapped into a

polymer matrix using synthetic or naturally occurring polymers. The drag may be uniformly distributed throughout the polymer. The fibrotic tissue inhibiting drag may be crosslinked

onto the polymer within the matrix (resulting in covalent attachment between the drug and the

polymer), or held in place by non-covalent forces. The polymer/drag composite may be in the

form of particles (such as microspheres, applied by injection or administered topically), gels

(used as an implant or applied topically), or films (used as an implant or applied topically), hi particular, entrapping polymer(s) may be degraded by enzymes, released by local metabolic

activity such as changes in pH, ionic, or redox conditions, or the drug may be released by

reaction with the in situ environment such as hydrolysis, reduction reactions, oxidative

reactions, pH shifts, photolysis, or combinations thereof. In a preferred embodiment, the drag would be released from the polymer by a condition related to the formation of adhesions.

A. Synthesis of polymers

[0027] A variety of polymeric structures of the type poly[D-M-P]_n have been synthesized and tested for the controlled release of antifibrotic agents. These have included alternating polyethylene glycol (PEG) and lysine (PEG-lysine) co-polymers, using PEGs of varying

subunit lengths (PEG1000, PEG2000, PEG4000, and PEG8000), as well as PEG2000 co- polymerized with des-amino tyrosyl-tyrosine (PEG2000-TT).

[0028] In one example, a poly[Poly(PEG-Lys-cHyp)] conjugate was synthesized and

tested in vitro and in limited in vivo experiments, and found to be effective, where P is a

segment of poly(ethylene glycol) with and average molecular weight of 2000 Daltons (PEG-

2000), L-Lysine (Lys) is M, and cw-4-hydroxy-L-proline (cHyp) is D. The starting material was Lys-CHYP dipeptides, and chain extension was accomplished by copolymerizing with

bis(succinimidyl) poly(ethylene glycol)(BSC-PEG) to α- and ε-amines of the cHyp-Lys

dipeptide. For more efficient chain extension, CHYP construct using the bis(succinimidyl)

poly(ethylene glycol)(BSC-PEG) is preferrably greater than 99.99% bi-functional; mono-

functional contaminant of the PEG can act as a chain terminator. It was found that some

commercial sources of bis(succinimidyl) poly(ethylene glycol)(BSC-PEG) MW=1000 had

more than 0.1% mono-functional contaminant. Supplies of bis(succinimidyl) poly(ethylene

glycol)(BSC-PEG) MW=2000 did not appear to pose such a problem. Longer PEG building

blocks (MW = 4000 Da and 8000 Da) have more limited CHYP loading capacity, and did not demonstrate appreciably improved drug efficacy.

poly[PEG-Lys-cHyp] [0029] Proline analogs were linked through their imino group, forming a peptide bond

with the lysine carboxylic group. The final constructs ranged between 20 to 35 kDa in molecular weight, as determined by gel permeation chromatography on columns calibrated with PEGs of different length, and detected by refractive index. The molecular weight, however, can range from about 10 to about 200 kDa. The amino acid content and the ratio of

Lys to proline analog were determined by amino acid analysis. The co-polymers typically

contained 8-12 pendent amino acids per construct, as estimated from the mean conjugate

molecular weight and the known size of the PEG subunits. For example, Poly(PEG-Lys-

cHyp) with PEG (molecular weight of 2000 "PEG2000") has a mean molecular weight of 25,000 Da, and therefore has an average of 11 PEG units and 11 pendent CHYPs. The

average polydispersity for the Poly(PEG-Lys-cHyp) is 1.6.

[0030] In vitro studies have shown that Poly(PEG-Lys-cHyp) formulations of varying

PEG chain lengths (MW = 1000, 2000, and 4000 Da) can be effective in inhibition of collagen production. Figure 1 shows the inhibition of rat lung fibroblast growth for the

various forms of Poly(PEG-Lys-cHyp). This design provides delivery of 4 times more cHyp

for the PEG1000 polymer conjugate than the PEG4000 agent, and two times more cHyp for

the PEG2000 polymer conjugate than the PEG4000 agent.

B. Stability of polymers

[0031] The stability of poly(PEG-[¹⁴C]-Lys) over a period of 48 h was determined by

incubation in human plasma and physiological phosphate buffer at 37 °C, followed by GPC

analysis of the polymer molecular weight at predetermined time intervals. There were no noticeable changes in the molecular weight and the radioactivity per molecular weight

(specific activity) over 48 h. HPLC analysis of Poly(PEG-Lys-cHyp) after incubation in human plasma, physiological phosphate buffer at 37 °C, trypsin, thrombin, plasmin, or collagenase showed no release of CHYP from the polymer.

C. Cell Growth Inhibition and Cytotoxicity In Vitro

[0032] Various proline analogs were tested alone, as peptidic prodrugs, and as polymer- bound prodrug constracts on rat fibroblast RFL-6 cells grown as monolayers in microtiter

plates. Test compounds were added in serial dilutions from the outset of the culture.

Viability was assessed on day 4 using a tetrazolium viability indicator dye (Dojindo).

[0033] A representative IC₅₀ experiment is shown in Figure 2. In this test, two different

synthesis batches of c/s-hydroxyprorine (CHYP) linked to PEG were tested, along with free

CHYP. As shown, the polymeric constracts (1 and 2) and the free amino acid inhibit rat

fibroblast growth to a similar extent. The differences between the two batches of Poly(PEG-

Lys-cHyp) were not significant. More importantly, the lack of difference between the free and conjugated CHYP activity indicates that the RFL-6 cells readily hydrolyze the CHYP

from the polymer backbone. Similar experiments with other analogs of Pro are summarized

in Table 1 below.

TABLE 1 : Compiled IC₅₀ results for free proline analogs, lysyl dipeptides, and PEG constructs

[0034] The free amino acid CHYP is the least cytotoxic proline analog tested in RFL-6

cultures. But its effects in the lysyl-dipeptide and PEG conjugate forms are comparable to those of the other proline analogs, indicating the ability of the cells to easily cleave the

CHYP-Lys bond. This ability differs from the other analogs; AZC, DHP and THP are considerably more cytotoxic than CHYP as free drags, as well as in their lysyl-dipeptide form, but their cytotoxicity as PEG constracts is reduced by factors of 4x, 8x and 12x, respectively, when compared to the free amino acid.

[0035] Collagen synthesis by anchorage-dependent fibroblasts is required for cell growth. To differentiate between cell growth inhibition and cytotoxicity, the effect of proline analogs

on RFL-6 cells grown on plain or collagen pre-coated wells was examined. Cultures were

incubated in the presence or absence of proline analogs at concentrations found to be

inhibitory (see Table 1). CHYP and DHP inhibited cell growth on plain wells, but not on

collagen-coated wells, demonstrating the fact that pre-coating the wells with collagen abrogated the effects of these proline analogs (Figure 3). THP was too cytotoxic on both

surfaces at the tested concentration to observe any effect.

D. Poly(PEG-Lys-cHyp) Immunoassay

[0036] A polyclonal antiserum was generated in rabbits immunized with Poly(PEG-Lys-

cHyp) coupled to keyhole limpet hemocyanin (KLH). Pre-immune, immune, and boosted

plasma samples were tested for the ability to bind solid-phase Poly(PEG-Lys-cHyp) (1 μg

Poly(PEG-Lys-cHyp)/well adsorbed onto nmulon microtiter plates). Specificity was

determined by the ability of the antibody to bind immobilized conjugate following pre-

incubation with competing haptens. Competing haptens included Poly(PEG-Lys-cHyp) itself, free CHYP, PEG-Lys (the actual copolymer backbone in Poly(PEG-Lys-cHyp)), CHYP-Lys,

as well as structural analogs of hydroxyproline (dehydroproline, or DHP, and azetidine

carboxylic acid, or ACA). From these competition-binding studies it was determined that the

antiserum recognized only the intact Poly(PEG-Lys-cHyp) molecule (50% inhibition at 2-5

μg/ml). None of the other molecules competed effectively (50% inhibition at >50 μg/ml). E. Development of the Delivery Vehicle

[0037] Hyaluronic acid (HA) is a naturally occurring bio-polymer that is a component of various medical products, hi general, HA is a biocompatible material found to retard

movement and proliferation of fibroblasts and related cell types. In vivo studies of hyaluronic acid gels and films that essentially form barriers between tissue planes have been shown to abrogate the formation of surgical adhesions.

[0038] HA beads can be formed that contain anti-fibrotic agents, such as the Poly(PEG-

Lys-cHyp) active agent. The HA beads can be manufactured by a number of standard

techniques, such as spray drying, or the use of non-aqueous solvents (such as alcohols) to

precipitate HA. The HA beads can be subsequently crosslinked using a number of standard chemical methods to enhance bead stability and regulate the bioresorption rate. The final form of the beads maybe a free-flowing, freeze-dried powder.

[0039] A number of formation variables were tested to determine an optimal release characteristic of the Poly(PEG-Lys-cHyp) from the hyaluronic acid beads. Table 2 presents

the various formulations of HA/Poly(PEG-Lys-cHyp) beads and their release kinetics:

TABLE 2: Release kinetics of various polymer formulations

* Weight hyaluronic acid/weight Poly(PEG-Lys-cHyp) during bead formation process.

** Defined as the time at which 50% of the Poly(PEG-Lys-cHyp) is released from the beads f For the first six formulations, the Poly(PEG-Lys-cHyp) was mixed with the hyaluronic acid prior to bead formation. For this last formulation, the beads were made first. The beads were then treated with a solution of

Poly(PEG-Lys-cHyp) (100 mg/ l), then freeze-dried. [0040] HA/Poly(PEG-Lys-cHyp) beads listed in Table 2 were subsequently tested separately in three animals for each variable, observing after day 14. There were no

noticeable adverse reactions from any of the materials. Therefore, the HA/Poly(PEG-Lys- cHyp) beads were investigated in various in vivo models.

F. In Vivo Results

Adhesiolysis Model

[0041] Injuries were made to the right abdominal wall of mature female rabbits (removal

of the periternium and first muscle layer from an area of approx. 2 5 cm), cecum adjacent to

the right abdominal wall (removing serosa), and abrasion of the right uterine horn. The

bladder was protected during surgery in order to minimize unintentional adhesions. The

injury to the side wall was electrocauterized; about 2 to 3 cc of blood was withdraw from the

ear vein and placed on the wound. The animals were allowed to recover; a second

laparotomy was done three weeks later, and the incidence, severity, and extent of each adhesion was scored for each animal (see Table 3 for adhesion grading system). Animals having no adhesions were removed from this study. The adhesions were lyzed, and the

animals were treated and allowed to recover. Three weeks after adhesiolysis and treatment, a final open observation was done, whereby the incidence, severity, and extent of each adhesion

were again scored and compared to the initial observation. For the control group, 88% of all

the adhesions which were lyzed, reformed (Table 4).

TABLE 3: Scale for Scoring Surgical Adhesions

Severit Extent

[0042] i this series of studies, a viscous polymer fluid was used as a carrier gel for the sustained release polymer Poly(PEG-Lys-cHyp). The viscous polymer fluid consisted of PEG

grafted to α-hydroxy acids PLA and PGA, and phase-separated into a semi-solid gel once

hydrated. The gel provided controlled release of Poly(PEG-Lys-cHyp). Four formulations

were developed to test in the adhesiolysis model: A. Gel; B. Gel and 2 mg/ml Poly(PEG-

Lys-cHyp); C. Gel and 8 mg/ml Poly(PEG-Lys-cHyp); and D. Gel and 2 mg/ml free cHyp.

All treatment tubes were coded to "blind" the surgeon.

[0043] Table 4 provides a summary of the data from this experiment. Each treatment group had six or more adhesions which were treated, with a mean incidence per animal of

1.75 for the treated group (e.g., on average, there were approx. two adhesions per animal).

More than 65%_> of the adhesions were Grade 2 or worse at the initial observation

(adhesiolysis and treatment). The post-treatment observations, made three weeks later, are presented on Table 4 (and graphically in FIGS. 4 and 5). The mean incidence per animal of

adhesions after treatment for the combined groups A through D was less than 1, which is a

statistically significant difference (p < 0.05) versus the mean incidence at the initial

observation. The number of adhesions for groups B and C (the sustained release

formulations) were substantially less after treatment, which are statistically significant

differences (p < 0.05), versus the number of adhesions at the initial observation. TABLE 4: Final Observations (3 weeks post adhesiolysis)

[0044] The percentage of returning adhesions were also significantly less for groups B

and C than the control or for groups A or D (see Fig. 9A). There is also a statistically

significant difference between group C (8 mg/ml sustained release polymer) and B (2 mg/ml sustained release polymer), which suggests that the sustained release polymer works in a

dose-dependent manner. Each adhesion was graded for severity and extent; the combination

grade average for each group is presented in Fig. 9B, comparing the initial observation grade with the final observation grade. As with the percent of returning adhesions, the extent and

severity of adhesions was significantly reduced for groups B and C compared to the control or

for groups A or D.

[0045] In a parallel study, the adhesiolysis and treatment model was again used to

compare the current leading product for adhesion prevention, SEPRAFILM™ (Genzyme),

with control (adhesiolysis, no treatment). SEPRAFILM™ is a crosslinked hyaluronic acid

film. Table 8 provides a summary of the data from this experiment. Each treatment group

had six or more adhesions which were treated (Table 5 -A), with a mean incidence of 1.2 for

the treated group and 2.25 for the control group. More than 65% of the adhesions were Grade 2 or worse at the initial observation (adhesiolysis and treatment), which is the same as for the previous experiment.

[0046] The post-treatment observations, made three weeks later, are presented in Table 5- B. The mean incidence per animal of adhesions after treatment for the Seprafilm group was

0.8, which is not statistically different (p < 0.05) versus the mean incidence at the initial observation. The mean incidence of adhesions after treatment for the control group was 1.75,

which is not statistically different (p < 0.05) versus the mean incidence at the initial

observation. The number of returning adhesions for the Seprafilm group was 66.7%, which is

not statistically different (p < 0.05) than the control group, which had a reformation rate in

this experiment of 77.8%. There was one animal in the Seprafilm group which had no

adhesion reformation. In three of the remaining Seprafilm animals, the severity of the

adhesions decreased by one grade.

TABLE 5-A: Adhesion Observation at Adhesiolysis and Treatment

TABLE 5-B: Adhesion Observation Three Weeks After Adhesiolysis and Treatment

[0047] A similar adhesiolysis study was performed using the HA/Poly(PEG-Lys-cHyp) beads as described in Tables 4 and 5. hi this study, formulations HA/MedX (no active), and

HA/Active/LowX, HA Active/MedX (see Table 2) were used. Bead material was placed on each wound to provide a complete and ever coverage (about 100 mg per wound). Each bead

formulation adhered relatively strongly to tissue, and began to hydrate shortly thereafter. The

beads formed a pasty film after about 15 minutes.

[0048] Observations were made three weeks after treatment. As previously observed, there were no observable adverse effects from the material at the site of delivery. Two

animals treated with HA/Active/MedX had residual polymer (most likely HA), which was

encapsulated. There was slight inflammation at the site of the residual polymer.

[0049] Table 6 provides a summary of the data from this experiment. Each treatment group had six or more adhesions which were treated, with a mean incidence of 1.85 per

animal for the treated group and 2.1 for the control group. More than 65% of the initial adhesions were Grade 2 or worse at the initial observation (adhesiolysis and treatment), which

is the same as for the previous experiment. The post-treatment observations, made three

weeks later, confirmed that the incidence of returning adhesions after adhesiolysis in the

control group was high (91.54%). The mean incidence of adhesions and percent of returning adhesions showed that each application group was effective. There was no statistical difference between HA/Active/MedX versus HA/Active/LowX with respect to the mean incidence or percent of returning adhesions (p < 0.05). However, the group HA/MedX (no Poly(PEG-Lys-cHyp) in the HA beads) did not appear to be as effective as the two groups containing Poly(PEG-Lys-cHyp).

TABLE 6: Adhesion Observation Three Weeks After Adhesiolysis and Treatment

Small Bowel Model

[0050] A small bowel adhesion model was developed in rabbits to mimic at least some of

the clinical manifestation of small bowel obstraction in humans. A 4 to 5 cm section of

small bowel was isolated and the surface lightly abraded with gauze. A small bowel loop was formed at the midline of the abraded portion, and the loop of bowel was secured by two interrapted 5/0 prolene sutures, spaced about 0.5 cm apart. A branch of the mesenteric artery

affecting the looped small bowel was tied off with 5/0 prolene suture, and the area was

allowed to dry by exposing the loop to ambient conditions for 2 min. The animals were

allowed to recover for 2 weeks before the extent and severity of the adhesions was

investigated.

[0051] All animals treated (n=17) in this fashion formed adhesions between the small

bowel loops, with 90% of the animals having a grade 3 adhesion (vascularized adhesion needing sharp dissection), and the remaining having grade 2 adhesions (non-vascularized adhesion needing sharp dissection). 30% of the animals showed signs of bowel obstraction.

[0052] Table 7 provides a summary of the data from this experiment. The form of

adhesion was separated into three categories: intra-loop adhesion, suture adhesion, and adhesion between small bowel and other organs. It should be noted that the most severe

adhesions were the intra-loop adhesions, and Grade 3 adhesions in the control group often caused necrosis and obstraction. There were no Grade 3 adhesions for animals treated with

V0426A. All treatment groups showed statiscally significant decreases in incidence of

adhesions compared to the control group. However, the group V0425B (no Poly(PEG-Lys-

cHyp) in the HA beads) was substantially less effective compared to the two groups

containing Poly(PEG-Lys-cHyp). There was no statistical difference between V0425A (50

mM EDC crosslinked) versus V0426A (25 mM EDC crosslinked).

TABLE 7: Summary of Data from Small Bowel Adhesion Studies

Hysterectomy Model

[0053] A rabbit hysterectomy model was developed to mimic some of the clinical

problems of adhesions to surrounding tissue after surgery in humans. This model was created by ligating the left or right pedicle with 5/0 prolene sutures in order to isolate a 4 to 5 cm portion of the uterus. Approximately 4 cm of the uteras was then surgically removed and hemostasis was achieved by electrocarterization. The excised surface was treated and the

wound closed using standard procedures. The site was observed after 14 days.

[0054] Table 8 provides a summary of the data from this experiment. In this study, the

material V0714A contained 30% Poly(PEG-Lys-cHyp), while material V0714B contained

none. All treatment groups showed statiscally significant decreases in incidence of adhesions

compared to the control group. However, the group V0714B (no Poly(PEG-Lys-cHyp) in the

HA beads) was less effective compared to V0714A that contained the active agent Poly(PEG-

Lys-cHyp). Residual polymer (most likely HA) was found in 2 of the 5 animals treated with

V0714A, and 6 out of 6 animals treated with V0714B. There was slight inflammation noted at the site of the residual polymer and an adhesion was formed from the residual polymer to

the bladder in one case.

TABLE 8: Hysterectomy Model with HA Beads

[0055] The same hysterectomy model was used to investigate delivery of the Poly(PEG- Lys-cHyp) agent without the use of a delivery gel. Dosing of Poly(PEG-Lys-cHyp) was done using 7-day Alzet miniosmotic pumps, which provide linear sustained release of the agent over 7 days. 20 mg of the Poly(PEG-Lys-cHyp) in 100 μl was loaded into each pump. After the hysterectomy was performed and prior to closing, three pumps were loaded into the periotoneal space of each animal. Table 9 provides a summary of the data from this experiment. This study shows that the active agent Poly(PEG-Lys-cHyp) alone may be sufficient to inhibit the formation of adhesions.

TABLE 9: Hysterectomy Model with Alzet Minipumps

Claims

What is claimed:

1. A fibrotic tissue inhibiting agent, comprising: a drug/polymer conjugate of formula:

-[P-M]-_n or Q-(P-L-D)_k or (D-L)_m-PP I

L

D wherein,

P is a water-soluble polymer segment,

M is a multifunctional moiety joining water soluble polymer segments P into a co-polymer

backbone and providing attachment for groups -L-D to the backbone,

L is a linker or a chemical bond,

D is a fibrotic tissue-inhibiting compound,

Q is a multivalent coupler,

n is an integer greater than 2,

k is an integer of 2 to 1000,

PP is a polymer with one or more functional groups to attach the -L-D groups,

m is an integer.

2. The agent of claim 1, wherein D is an anti-fibrotic agent.

3. The agent of claim 1, wherein D is an anti-proliferative agent.

4. The agent of claim 1, wherein D is a fibrotic tissue inhibiting proline analogue.

5. The agent of claim 4, wherein D is c/_?-4-hydroxy-L-proline (cHyp), cts-4-hydroxy-D- proline, cw-3-hydroxy-DL-proline, 3, 4-dehydro-L-proline(DHP), cw-4-fluoro-L-proline, cis- 4-chloro-L-proline, cts-4-bromo-L-proline, L-azetidine-2-carboxylic acid (AZA), or L- thiazolidine-4-carboxylic acid (THP).

6. The agent of claim 2, wherein D is pirfenidone, tranilast, halofuginone, pentoxifylline, relaxin, estradiol, interleukin 10, pyridine-2, or 4-dicaboxylic-di(2-methoxyethyl) amide.

7. The agent of claim 3, wherein D is 5-flurouracil, mitomycin-C, or paclitaxel.

8. The agent of claim 1, wherein D is retinoic acid, a retinoic acid analogue, a retinoic acid

antagonist, a plasminogen activator, a vastatin, or a non-steroidal anti-inflammatory

compound.

9. The agent of claim 1, wherein D is released by enzymatic action or metabolic activity on

linker L.

10. The agent of claim 1, wherein P is a water-soluble segment of weight average molecular

weight 400-25,000 Da, derived from compounds having at least two functionalities for

covalent attachment to M, selected from the group consisting of hydroxyl, amino, thiol, alkyl disulfide, aryl disulfide, isothiocyanate, thiocarbonylimidazole, thiocarbonylchloride,

aldehyde, ketone, carboxylic acid, carboxylic acid ester, sulfonic acid, sulfonic acid ester,

sulfonyl chloride, phosphoric acid, alkyl succinimidylcarbonate, aryl succinimidyl- carbonate,

alkyl chlorocarbonate, aryl chlorocarbonate, alkyl succinimidylthiocarbonate, aryl

succinimidylthiocarbonate, alkyl

chlorothiocarbonate, aryl chlorothiocarbonate, halide, and thioester.

11. The agent of claim 1 , wherein P is a polyethylene glycol, polyvinyl alcohol, poly(2-

hydroxyethyl methacrylate), poly(acrylic acid), poly(methacrylic acid), poly(maleic acid), polylysine, or a mixture thereof.

12. The agent of claim 1, wherein P is a poly(carboxylic acid), poly(orthoester), poly(anliydride), pluronic polyol, poly(vinylpyrrolidone), poly(acrylate), polyamide,

polyphosphazine, poly(amino acid),polypeptide, pseudo-poly (amino acid), linear or branched

polymer containing PEG, copolymers of PEG, a dendrimer, or a PEG-dendrimer.

13. The agent of claim 1, wherein PP is a polyethylene glycol, polyvinyl alcohol, poly(2-

hydroxyethyl methacrylate), poly(acrylic acid), poly(methacrylic acid), poly(maleic acid),

polylysine, or a mixture thereof.

14. The agent of claim 1, wherein PP is a poly(carboxylic acid), poly(orthoester),

poly(anhydride), pluronic polyol, poly(vinylpyrrolidone), poly(acrylate), polyamide,

polymer containing PEG, copolymers of PEG, a dendrimer, a PEG-dendrimer, collagen,

hyaluronic acid, fatty acid lipid, polyhydroxyalkanoate, chondrotin sulfate, glycosaminoglycan, chitosan, alginate, starch, dextran, a carbohydrate-based polymer,

cellulose, or cellulose derivative.

15. The agent of claim 1, wherein n is 3 to 100.

16. The agent of claim 1, wherein k is 2 to 100.

17. The agent of claim 1, wherein m is 1 to 100.

18. A method for treatment of tissue adhesions, comprising: administering to a tissue in need

of treatment for surgically-induced adhesions or fibrotic disease, an effective amount of a fibrotic tissue inhibiting agent, comprising: a drag/polymer conjugate of formula:

-[P-M]-_n or Q-(P-L-D)_k or (D-L)_m-PP

L

D

wherein,

P is a water-soluble polymer segment,

M is a moiety joining water soluble polymer segments P into a co-polymer backbone and

providing attachment for groups -L-D to the backbone,

L is a linker or a chemical bond,

D is a fibrotic tissue inhibiting compound, Q is a multivalent coupler,

n is an integer greater than 2, k is an integer of 2 to 1000,

PP is a polymer with one or more functional groups to attach the -L-D groups, and

m is an integer.

19. The method of claim 18, wherein the agent administered topically by inhalation, or by

injection.

20. The method of claim 18, wherein the agent is encapsulated in an eroding polymer or entrapped in a polymer matrix.