WO1997024459A1

WO1997024459A1 - Method for reducing unwanted cellular adhesions

Info

Publication number: WO1997024459A1
Application number: PCT/US1996/020490
Authority: WO
Inventors: Margaret Anne Baker; Betsy Mae Ohlsson-Wilhelm; Alan R. Olzinski; Gregory A. Kopia
Original assignee: Phanos Technologoes, Inc.
Priority date: 1995-12-29
Filing date: 1996-12-27
Publication date: 1997-07-10

Abstract

A conjugate comprising a drug which has an inhibitory effect on cellular adhesion and a lipophilic cell binding agent, when applied to a compromised site, e.g., due to surgical intervention, injury, chemotherapy, disease or inflammation, is effective in reducing unwanted adhesion formation.

Description

METHOD FOR REDUCING UNWANTED CELLULAR ADHE8ION8

BACKGROUND OF THE INVENTION

The present invention relates to a method for placement of therapeutic agents so as to reduce unwanted cellular adhesions. Unwanted cellular adhesions may include, but are not restricted to, adhesions between tumor cells and other tumor cells, adhesions between tumor cells and normal cells, and certain adhesions between normal cells and other normal cells. Tumor cells are found in two distinct modes: as ascites (free floating cells) and as solid tumors (attached/adhered to host tissue/organs as well as to other tumor cells) . Prevention of tumor cell adhesion and growth is a major clinical problem. The control of local tumor recurrence after surgical removal of malignancies is an especially serious concern. The observation that tumor growth occurs at the incision site, even at histopathologically "clean" margins, has led to the conclusion that viable tumor cells have higher affinity for damaged tissue than for normal tissue. This represents one particularly critical aspect of a much broader issue concerning prevention and control of unwanted cellular adhesions. For instance, in cases involving surgical intervention in the abdominal cavity for conditions such as inflammatory bowel disease or endometriosis, there is often the complication of postsurgical adhesions of normal tissue to the resected areas, in a manner similar to that of tumor cell adhesion and colonization or invasion of the wound.

In addition to clinical observations, experimental models of tumor cell adhesion have likewise demonstrated that damaged tissue is an attractive site for tumor growth after systemic injection of viable tumor cells. See, for example, S. Murthy and E. Scanlon, eds. , In urv and Tumor Implantation: Biological Mechanisms and Clinical Implications for Recurrence and Metastasis. R.G.

Landes Co., Austin, TX 1993. It has been reported that specific adhesion molecules and their recognition molecules (receptors) play a role between tumor cell and endothelial cells or extracellular matrix attachment. B. Zetter, Seminars in Cancer Biology, 4_.: 219-229 (1993) . There is a growing list of families of adhesion molecules which are expressed by tumor cells and are stimulated by various environmental conditions or factors, e.g. , cytokines. More recently, there has been a report of evidence showing that display of the receptors or adhesion molecules requires intracellular components known as cytoskeletal elements. F. Pavalko and C. Otey, Proc. Society Exp. Biol. Med., 205: 282-293 (1994); D. Tang, et al., Cancer Res. 54: 1119-1129 (1994). «

The cytoskeleton is composed of microtubules and various filaments of self-assembling polymers of proteins including tubulin, vimentin, and actin. Adhesion molecules allow communication between the cytoplasmic domains and the cytoskeleton triggering a variety of cellular functions including cell-cell interactions, cell motility and receptor-ligand interactions and receptor internalization. See F. Pavalko and C. Otey, supra . Thus, agents which interfere with or disrupt the assembly of microtubules or microfilaments can prevent or inhibit the expression of specific adhesion molecules on cells thereby blocking their recognition, binding, attachment and migration into wounded and healing, or normal tissue. Colchicine is an agent which binds to tubulin and causes depolymerization of microtubules. Treatment of tumor cells with colchicine has been shown to decrease their ability to bind to lymphatic tissue. See, for example, S. Islam et al., Surgery 113: 676-82 (1993) . Treatment of endothelial cells with colchicine likewise blocked tumor cell binding to specific adhesion molecules, as reported by D. Tang, et al., supra . Therefore, microtubule disrupting agents, which are often used in combination regimens for cancer therapy may act not only as cytostatic agents but also as anti-adhesives and possibly as anti-migratory agents thereby decreasing tumor dissemination and metastatic spread. It has been proposed to reduce the occurrence of adhesion formation resulting from chemotherapeutic treatments involving cisplatin and bleomycin by administering these chemotherapeutic agents in combination with a vinca alkaloid substance, such as vindesine. R. Molloy et al., Irish J. Med.

Sci., 159(6) : 175-77 (1990). However, no technique is provided for site specific delivery of the proposed chemotherapeutic combination.

Another approach to controlling unwanted cellular adhesions has been the use of physical barriers, including both mechanical and viscous solutions. See, for example, U.S. Patent 5,250,516. Such barriers tend to prevent adhesion formation by limiting tissue apposition during the critical stages of mesothelial repair. Although use of physical barriers for preventing adhesion formation has been reasonably extensive, general acceptance is constrained because of technical difficulties. In co-pending United States Patent Application Serial Number 884,432, which is commonly owned with the present application, there are described various conjugates comprising a therapeutic agent or drug and a lipophilic cell binding agent, in the form of cyanine dyes substituted with relatively long hydrocarbon "tails". See also, PCT/US92/10076 (WO93/11120, published June 10, 1993). These conjugates are capable of stably binding to the lipid regions of cell membranes, thus enabling site selective delivery of therapeutic agents, either via local in vivo administration, e.g. , by injection, or by means of a carrier, for retention at the disease site.

The above-noted conjugates afford a number of distinct advantages, as compared with compositions and methods currently available for delivery of therapeutic agents to disease sites. Most notably, the above-mentioned conjugates can be delivered and retained at a selected site in the body by stable association with cell structures at that site. Existing modes of delivery either are unable to deliver sufficient dosages to the disease site without adverse systemic side effects, or are unable to allow sufficient retention of the therapeutic agent at the disease site for a time and in an amount sufficient to produce the desired therapeutic effect. Moreover, since the lipid regions comprise the majority of the outer membrane of the cell, it is possible to place larger numbers of lipid binding conjugates, and thus a greater concentration of therapeutic agent, into the plasma membrane. Furthermore, because the above-mentioned conjugates are stably incorporated into membrane lipids due to their hydrophobic tails, they are effectively trapped there and cannot dissociate easily. Consequently, leakage from the cells is minimized, thereby reducing undesired systemic effects. Ongoing research involving the above- mentioned conjugates has led to the discovery of a solution to the problem of reducing unwanted cellular adhesion. When appropriately constituted and utilized, these conjugates can effectively reduce unwanted adhesions, such as: tumor cells to surgical wounds, tumor cells to normal tissue, tumor cells to tumor cells, normal cells to normal cells as in the case of surgical adhesion.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention there is provided a therapeutic agent for reducing undesired cell adhesions at a compromised site. The therapeutic agent comprises a conjugate of anti-adhesive drug and a cell binding compound. The conjugate is selected from the group consisting of conjugates of the formula

wherein R and R, represent hydrocarbon substituents having from 1 to about 30 carbon atoms;

X and Xi may be the same or different and represent 0, S, C(CH₃)₂ or Se;

R₂ is selected from H, CH₃, CH₂CH₃, CH₂CH₂CH₃ or CH(CH₃)₂, m being from 0-3; Za and Zb may be the same or different and represent substituents selected from the group H, alkyl, OH, -0- alkyl, COOH, C0NH₂, S0₃H, S0₂NH₂, SH, S- alkyl, CONH-alkyl, CON-(alkyl)₂, NH-acyl, NH-alkyl, N(alkyl)₂, N0₂, halogen, Si(alkyl)₃ O-Si(alkyl)₃, Sn(alkyl)₃ or -Hg- halogen, the alkyl groups of the Z substituent having from 1 to 4 carbon atoms;

D represents a drug having an inhibitory effect on cell adhesion;

L represents a linking moiety which links said drug to said cell binding compound, with q being 0 or 1; and

A^" represents a pharmaceutically acceptable anion;

or

wherein each R, is the same or different and represents H or CH₃ with n being 1 or 2, and p being 1, 2 or 3; and ^ represents a substituent selected from the group consisting of OH, C1-C3 alkoxy, or acyl, said acyl substituent being unsubstituted or substituted with COOH, COOR, or CONH₂, R,. being a C1-C3 alkyl; and D, L and q are as defined above; and with the proviso that when said cell binding compound is of formula I, said drug is an oligonucleotide having anti-adhesive activity. According to another aspect of this invention, a method for reducing unwanted cellular adhesion is provided. The method involves treating the compromised site with a conjugate as described above, comprising a drug and a cell binding compound, in an amount effective to inhibit attachment to said site of cells susceptible to unwanted adhesion.

In carrying out the method of the invention, the conjugate is conveniently applied to the intended site or region in the form of an irrigation solution.

In addition to reducing cellular adhesions, the therapeutic agent and method of the invention may have a cytostatic or cytotoxic effect on treated cells, or may enhance their sensitivity to x-rays.

Moreover, the affinity of the conjugate for membranes of cell at the site of application effectively "links" the conjugate to that site, so as to minimize any deleterious systemic effect. The therapeutic agents having a tocol or tocopherol structure, e.g., Vitamin E, are believed to function as useful lipophilic cell binding compounds for the delivery of anti-adhesive drugs. Vitamin E is a natural component of plant oils and consumption of quantities 80 times the recommended daily allowance, 800 I.U. (equivalent to 800 mg alpha-tocopherol acetate) , for more than 3 years by individuals did not produce toxicity (Farell, P.M. and Bieri, J.G. "Megavitamin E supplementation in man". Am. J. Clin. Nutr. 18:1381, 1975). The structure of the unconjugated cell binding compound of Formulas II and III provides a free hydroxyl group which can be readily esterified to form conjugates in accordance with this invention. Vitamin E distributes widely throughout the body and forms a natural reservoir especially in fatty tissues, such as liver and body fat, but also circulates with other lipid components of blood. Vitamin E inserts into cellular membranes at a ratio of 1:2100 to polyunsaturated fatty acids. It is known to protect the unsaturated fatty acids from peroxidative damage (Tappel, A.L. and Dillard, C.J., In vivo lipid peroxidation measurement via exhaled pentane and protection by vitamin E. Fed. Proc. 4):174-178, 1981). In addition to providing an effective drug carrier for intracellular anti-adhesion drugs, vitamin E forms a body depot with a long half- life.

A particularly preferred class of anti- adhesive drugs comprises oligodeoxyribonucleotideε having sequences which prevent the synthesis of cellular adhesion molecules or otherwise function to decrease the adhesion of cells to cells.

The method of the present invention enables local treatment of an area such as a tumor or wound, a cavity such as the pelvic, abdominal, thoracic or joint, or the lumen of a blood vessel, by a method which takes advantage of the lipophilic properties of the cell binding compounds used to form the conjugates of formulae I, II and III. When these cell binding compounds are conjugated to drugs having anti-adhesive activity, the cell binding compounds facilitate the delivery of the conjugate to the'compromised site and alter the pharmacokinetics of the anti-adhesion drug.

Additional advantages and features of the present invention are set forth in, and will be apparent to those skilled in the art from the detailed description of the invention presented below considered in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE IA is a graphical illustration showing tumor growth (body weight increase) as a function of time elapsed after tumor transplantation for test animals bearing transplanted tumors that were treated after tumor transplantation with certain preferred drug-cell binding compound conjugates in comparison to the conjugate carrier alone and the unconjugated drug.

FIGURE IB is a graphical illustration showing tumor growth (body weight increase) as a function of time elapsed after tumor transplantation for test animals bearing transplanted tumors that were treated before tumor transplantation, using the same conjugates, carrier and drug referenced in Fig. IA. FIGURE 2A is a graphical illustration showing tumor growth (body weight increase) as a function of time elapsed after tumor transplantation for test animals bearing transplanted tumors that were treated after tumor transplantation with the same conjugates, carrier and drug referenced in Fig. IA. FIGURE 2B is a graphical illustration showing tumor growth (body weight increase) as a function of time elapsed after tumor transplantation for test animals bearing transplanted tumors that were treated before tumor transplantation with the same conjugates, carrier and drug referenced in Fig. IA, as well as the unconjugated cell binding agent.

DETAILED DESCRIPTION OF THE INVENTION

The expression "compromised site", as used herein, refers to a site of a human or animal body at which an environment exists that tends to foster unwanted cell adhesions. Such an environment may exist as a result of surgery, injury, disease, chemotherapy, inflammation or other condition jeopardizing cell viability or function. Compromised sites are beneficially treated with a composition of the invention comprising a conjugate of formulae I, II or III above, in order to reduce adhesion formation. Conjugates of Formula I, above, which are used in the practice of this invention can be prepared according to the synthetic routes described in the above-mentioned U.S. Patent Application Serial No. 884,432, the entire disclosure of which is incorporated by reference in the present specification as if set forth herein in full. The structure of the cell binding compounds in Formulae II and III, both the tocol and tocopherol species, provides a free hydroxyl group that can be readily esterified to anti-adhesive drugs. Vitamin E, for example, may be linked to an anti-adhesive drug according to the procedure described in Will and Brown, Tetrahedron Letters, 3_3:2729-2732, 1992.

The conjugates of the invention reduce the occurrence of undesired cell adhesion due to the action of the drug incorporated therein, which may inhibit undesired cell adhesion in several ways.

Thus, the drug may act to i) prevent the display of specific adhesion molecules on the cell surface; ii) prevent the intracellular signaling in the cells susceptible to undesired adhesion subsequent to the binding of the adhesion molecules which leads to their further attachment to extracellular matrix, basement membrane, or to normal tissue cells; iii) prevent the release of mediators which stimulate the display of specific adhesion molecules on the surfaces of cells susceptible to undesired adhesion, thereby facilitating the adhesion of cells to cells or tissue to tissue in an unnatural or undesired way; or iv) interfere with the binding of the cell adhesion molecules of one cell to those of another cell or to extracellular matrix proteins contained in the tissue by providing a false or competing substrate.

The drug component of the conjugate may be selected from the group of anti-microtubule agents, such as colchicine or vinca alkaloids (e.g. , Vinblastine or Vincristine) ; anti-filament agents, such as cytochalasins B and D or acrylamide or - 11 -

cycloheximide; anti-signaling agents, such as protein kinase C inhibitors, including calphostin, or inhibitors of intracellular calcium mobilization, including l-0-hexadecyl-2-0-acetyl-SN-glycerol, 8- (diethylamino)octyl-3,4,5-trimethoxybenzoate-HCl or EGTA (ethyleneglycol-bis-(beta-aminoethylether)- N,N,N' ,N^,-tetraacetic acid); agents that inhibit release of stimulatory mediators, such mediators including, e.g., interleukin 1, transforming growth factor-beta, or 12-S-hydroxyeicosatetraenoic acid, and antagonists of these mediators, including, e.g., prostacyclin analogue PGI2 (Iloprost®) ; interleukin-1 receptor antagonist; nonsteroidal anti-inflammatory drugs, including ibuprofen and indomethacin; lipoxygenase inhibitors, such as NDGA

(nordinhydroguariaretic acid) or Balcalein (5,6,7- trihydroxyflavone) or other agents effective to interfere with binding of adhesion molecules to their ligands, including the ligands themselves or fragments thereof, such as fibronectin or laminin, or the sequences specific to the binding sites containing the amino acids, arg-gly-glu or tyr-ile-gly-ser-arg, or a combination of such drugs.

According to a preferred embodiment, the conjugates of the invention comprise an oligonucleotide, as the anti-adhesive drug, which functions to selectively inhibit the synthesis of cell adhesion molecules. Oligomers are available which target the cellular synthesis of adhesion molecules or which through specific nucleotide sequences interfere with cell to cell adhesion. The availability of nucleotide sequence information encoding anti-adhesion molecules enables preparation of isolated nucleic acid molecules of the invention by oligonucleotide synthesis. These oligonucleotides are designed to hybridize specifically with sequences on the target polynucleotide. As used herein, a "specifically hybridizing" oligonucleotide is one of sufficient complementarity to a specified region of the target polynucleotide (i.e., the predetermined polynucleotide) to hybridize substantially exclusively with that region under standard hybridization conditions (i.e., conditions normally used for a given polynucleotide amplification reaction) . Fully complementary oligonucleotides are preferred. Conjugation of a lipophilic cell binding compound to an oligomer with anti-adhesive activity should potentiate this activity. An antisense oligonucleotide targeting a c-myc mRNA translation sequence was reported to have anti-adhesive activities (Leonard M. Neckers, of the National Institutes of Health, "The art of antisense" New Orleans, LA, presented September 22, 1995). Oligomers based on the sequence of c-myc have been synthesized and tested for biological activity. Different oligomers targeting the intercellular adhesion molecule 1 (ICAM-1) , have been synthesized and tested by others, Chiang et al., J. Biol. Chem. 266:18162. 1991. Other targeted sequences may include for example, those encoding the translation initiation sites of E-selectin, P- selectin, L-selectin, LFA-1, V-CAM, and PECAM-1. Conjugation of the oligonucleotide to the the lipophilic cell binding compound may be performed utilizing the 3' OH or the 5' phosphate of the DNA strand using chemical methods known to those skilled in the art. Oligomers of this type would be expected to have utility as anti-adhesive drugs when conjugated to the lipophilic cell binding compounds of the present invention.

Experiments have been performed using conjugates of the following formulae and favorable results have been obtained.

The conjugates described above function like pro-drugs, with the drug being linked to the lipophilic cell binding agent through a cleavable linkage, and exerting its therapeutic effect upon release from the conjugate. Cleavage of the linking moiety is believed to occur after binding and is accelerated by a reduction in pH which promotes disruption of chemical bonds within the linking moiety.

These conjugates release an analogue of colchicine, a molecule which binds to tubulin protein in such a way as to prevent the intracellular functioning of microtubules. These functions include but are not limited to the translocation of cell adhesion molecules to the cell membrane and also the intracellular signaling triggered by binding at the outer cell membrane of these adhesion molecules to their respective ligands.

A method for treating arterial atherosclerotic plaques constitutes another embodiment of the present invention. In this condition, circulating macrophage cells are attracted to and become attached to plaques formed on the walls of the artery by cholesterol, fibrin and other biological molecules. Delivery of an anti-adhesive to the arterial lumen via a drug delivery catheter affords an effective method of drug administration. The anti- adhesive activity of the conjugate would be expected to limit the subsequent attachment of platelets and macrophages associated with progression of the condition. The duration of drug presence at the compromised arterial site when linked to the lipophilic cell binding compounds of the present invention has been demonstrated to be significantly longer than that of the drug moiety when administered alone. Particularly good results have been obtained with the therapeutic agent of Formula IV.

As noted above, "unwanted cellular adhesion" refers to adhesions between tumor cells and other tumor cells, adhesions between tumor cells and normal cells, and certain adhesions between normal cells and other normal cells. Thus, cells susceptible to undesired adhesion include any malignant cells or extracellular matrix generated by malignant cells; normal tissue cells, including endothelial cells, endometrial cells, mesothelial cells or epithelial cells that become injured or inflamed, e.g., as a result of surgical intervention; and signaling or mediator releasing cells including the platelets, macrophages, lymphocytes, or neutrophils. The term "unwanted cell adhesion" does not include the adhesion of cells that occurs in the course of forming normal, healthy tissue. The conjugates described herein may be conveniently formulated as an irrigation solution in admixture with a biologically compatible carrier, such as dimethylsulfoxide, polyoxyethylene (20) sorbitan monooleate (e.g., Tween-80) , polyols, e.g., propyleneglycol, aqueous dextrose (5%) solution.

Ringer's solution, saline solution or a combination of such carriers. A particularly preferred liquid carrier is an ethanolic mannitol solution comprising 2% (vol) ethanol in 1.5% (wt) mannitol.

Concentration of the conjugate in the selected liquid carrier should normally be from about 5 μM to about 5 mM. Where appropriate, the action of contaminating microorganisms can be prevented by various anti-bacterial and anti-fungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. It will often be preferable to include with the conjugate isotonic agents, for example, glucose or sodium chloride.

As used herein, the term "biologically compatible carrier" includes any and all vehicles, solvents, dispersion medium, anti-bacterial and anti¬ fungal agents, isotonic agents and the like. The use of such media and agents with therapeutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the conjugates described herein, its use in practicing the methods of the present invention is contemplated.

Certain adjuvants, i.e., agents that enhance the effectiveness of the treatment for reducing undesired cell adhesions, may also be used in conjunction with the above-described conjugates, if necessary or desirable. Suitable adjuvants include anti- inflammatories, e.g., non-steroidal, anti- inflammatory drugs (NSAIDs) , biological response modifiers, e.g., IL-1 antagonists, or a combination of such adjuvants.

The method of the invention may be used to reduce undesired cell adhesions in connection with surgical intervention for the treatment of cancer in various body cavities or organs where the cancer is found to exist, such as the abdominal cavity, the abdominopelvic cavity, the thoracic cavity, including the pleura and pericardium, the dorsal cavities, including the cranial and spinal cavities, joint cavities, i.e., the space between articulating bones in a synovial joint; lumina, such as the interior of the gastrointestinal tract or a blood vessel, or the female reproductive system. In addition to natural cavities, it is within the contemplation of the present invention to treat induced cavities, i.e., those created by surgical intervention, by the methods described herein. The method may also be advantageously applied to reduce post-surgical adhesions of normal tissue to the wound repair site, such as occurs in connection with surgical intervention for conditions such as inflammatory bowl disease or endometriosis.

Treatment of the surgical site prior to attachment of cells susceptible to undesired adhesion can normally be effected by applying the conjugate prior to, during or immediately after the surgical event. In this way, the well-being of normal tissue in the vicinity of the surgical site can best be preserved.

EXAMPLES The following examples are provided to describe the invention in further detail. These examples are intended to illustrate and not to limit the invention. Unless otherwise indicated, all reagents and drugs were acquired from Sigma, St. Louis, MO.

EXAMPLE 1

Pretreatment of Mice Receiving Human Ovarian Carcinoma Cells, i.p. with Thiocolchicine-Conjugated to Lipophilic Cell Binding Agents Pretreatment of mice before introduction of tumor burden was carried out to determine the change in pharmacokinetics of the thiocolchicine analogue, as such, and as conjugated to cell binding agents and having the formulas IV and V, respectively, as set forth above. Whereas 4-formyl-thiocolchicine (product of Zynaxis, Inc., Malvern, PA) had been experimentally determined to have little or only modest effects on tumor growth when introduced after tumor to the peritoneal cavity of mice bearing human ovarian carcinoma cells, the conjugate of formula IV had a more pronounced effect.

In that earlier experiment (A) mice, nu/nu, 5 per group were injected with 5 x 10⁶ A2780 human ovarian carcinoma cell (i.p.) and treated the following day with the compound(s) listed in Table 1, below. Median survival day was the average day of death of the second and third test animal. As can be seen from the data in Table 1, the increase in life span (% ILS) of mice treated with 4-formyl- thiocolchicine ((S)-N-[5,6,7,9-tetrahydro-l,2,3- trimethoxy-4-formyl-10-(methylthio)-9-oxobenzo[a] heptalen-7-yl] acetamide;

was 10%, which was significantly different from the 10% DMSO treated group (p < 0.05). Both groups treated with the conjugate of formula IV had significantly longer survival times than those treated with 4-formyl- thiocolchicine group (p < 0.01). The survival time of mice treated with the conjugate of formula IV was not different than those treated with 500 nmol of Cisplatin.

The compounds listed in Table 1, including 4-F-T, produced no observable adverse effects at any of the concentrations tested. TABLE 1

Dose Day Median nmol

ComDoundiB) Der mouse Treated Survival (D) % ILS

Experiment A:

10% DMSO +1 43 -

Ciβplatin (4.5 mg/kg) 250 +1 45 5

Ciβpla in (9.0 mg/Kg) 500 +1 52 20

4-F-T* 500 +1 48 10

4-F-T + CBA 500 + 500 +1 44 2

Formula II Conjugate 250 +1 51 17 do 500 +1 53 22

" 4-formyl-thiocolchicine ^b cell binding component of formula IV, above

Previous pharmacokinetics studies in other models showed rapid disappearance and/or metabolism of 4-formyl-thiocolchicine. Therefore, a pretreatment protocol was designed to determine the longevity of efficacy. In carrying out the protocol (experiments B and C) , the human ovarian line, A2780 (gift of T.C. Hamilton of Fox Chase Cancer Center, Phila. , PA) , was maintained in tissue culture (RPMI 1640 Bio-Whittaker, Walkersville, MD) , with 10% fetal bovine serum (GIBCO) and 0.26 u/m/bovine insulin (GIBCO). For tumor initiation, 5 x 10⁶ cells were injected i.p. into NIH: Balb/C nu/nu female mice five per group. Two types of treatment schedules were performed, a single i.p. injection on the day before the tumor cells were implanted or a single i.p. injection on the day after tumor cells were implanted.

Syntheses of 4-formyl-thiocolchicine and the conjugates of formulas IV and V were performed at Zynaxis, Inc., Malvern, PA, utilizing the synthetic routes described in the above-mentioned U.S. Patent Application Serial No. 07/884,432. These compounds were shown to be greater than 95% pure by HPLC and NMR and/or mass spectroscopy.

The dosing levels of the compounds were calculated in terms of molar equivalents to assure that the therapeutic concentration was uniform across groups. Compounds were first dissolved in dimethylsulfoxide (DMSO) and administered in 0.5 ml of a maximum of 10% DMSO in water. No toxicity symptoms were noted at the administered dose which was 500 nmol per mouse.

Efficacy was monitored by the change in group mean body weight and scored as increase in median survival time of treated versus the controls which were given carrier only (10% DMSO) . Two identical protocols were run at two different facilities. The protocols were identical except that the source of mice for experiment B was the NCI, Frederick, MD and for experiment C the mice were provided by Jackson Laboratory, Bar Harbor, ME. Experiment C also contained the additional control group of mice treated with cell binding agent, as such, i.e. free of any conjugated therapeutic agent. In experiment C, mice were sacrificed when the tumor burden exceeded 75% of initial body weight for humane reasons.

Kaplan-Meier statistics were calculated using the comparing pairs of groups using the Wilcoxon test.

The results of these two experiments are set forth, in part, in Figures IA, IB, 2A and 2B, wherein the dotted line (• • •) represents a 10% solution of

DMSO in water; the dashed line ( ) represents 4- for ylthiocolchicine; the solid line ( ) represents the conjugate of formula IV, above; and the stippled line (111) represents the conjugate of formula V, above. The effect of pretreatment was to prevent the increase in body weight of the mice associated with the tumor growth, as can be seen from the data in Figures IA and IB, which set forth data resulting from experiment B. In that experiment, the delay in tumor growth for mice pretreated with either of the conjugates of formula IV or V was evident. In particular, Figure IB shows that the effect of pretreatment was to prevent the increase in body weight of the mice associated with the tumor growth as seen in all the groups treated one day after tumor cells had been injected. (Compare Figure IA) .

The results of experiment C were similar to those obtained in experiment B, in that pretreatment with the conjugates of formula IV and V delayed tumor growth. These results are set forth in Figures 2A and 2B. Little or no effect was seen from posttreatment with either conjugate; nor did the unconjugated therapeutic agent or the unconjugated cell binding agent alone have any effect on total tumor burden.

Time to reach 150% of initial body weight was derived from the above described figures and is shown in Tables 2 and 3, below, along with the median survival time or day of sacrifice for each group. All of the groups of mice pretreated with the conjugates of formula IV and V had significantly longer survival times or times of low tumor burden, as compared with mice treated with unconjugated 4-formyl- thiocolchicine, or unconjugated cell binding agent, before tumor injection, or after tumor injection. As can be seen from the data in Tables 2 and 3, posttreatment produced no appreciable effect on the survival time of mice given either conjugated or unconjugated thiocolchicine analogue. By contrast, pretreatment with the conjugates of formula IV and V caused a marked prolongation of life span. TABLE 2

Time to Reach

150% of Day Median

Compound(s1 Initial Weiαht Treated Survival (D) % ILS

10% DMSO 30 +1 38

4-F-T 34 +1 40 4

Formula IV Conjugate 28 +1 42 9

Formula V Conjugate 25 +1 39 3

4-F-T 30 -1 37 -4

Formula IV Conjugate >80 -1 >120* >200

Formula V Conjugate 60 -1 >104* >173

*These groups each had 3/5 animals which survived more than 120 days.

Time to Reach

150% of Day Median Day

ComDound Is ) Initial Weiαht Treated of Sacrifice % ILS

10% DMSO 36 +1 43

4-F-T 32 +1 45 5

Formula IV Conjugate 36 +1 44 2

Formula V Conjugate 43 +1 45 5

4-F-T 35 -1 33 -24

Formula IV Conjugate >80 -1 61 41

Formula V Conjugate >80 -1 52 21

CBA* 28 -1 36 -17 ^m Cell binding agent of formula V, above; represented by dot- dashed line ( ^■) in Figure 2B.

Although the survival times are different in experiments B and C, it should be noted that the mice were sacrificed when they reached 35 g (175% of initial body weight) in experiment C rather than allowing them to succumb to tumor burden. The time to reach 150% body weight, however, indicates that the animals pretreated with the formula V conjugate in experiment C had better tumor control than did the animals in experiment B treated the same way since the time increased from 60 days to >80 days. In addition to body weight change and time to 175% of initial body weight change, animals in experiment C were necropsied and gross pathology was performed. It was observed that the tumor pathogenesis in mice treated either with vehicle control, with 4-formyl-thiocolchicine or with the cell binding component of the conjugate of formula V had widely disseminated tumor covering all surfaces of the peritoneum, the organs and often the lower surface of the diaphragm. Mice treated with the conjugates of formulas IV or V, whether pre or post tumor injection, had more focal lesions (i.e., enhanced tumor margin definition) though large masses had formed by the time of sacrifice. The data resulting from the above-described experiments tend to establish that pretreatment of the peritoneal cavity with the conjugates described above has an effect which cannot be explained as simple decrease in early stage tumor cell number. For the difference between tumor cell number at day 0 and 1 day thereafter will not be greater than a factor of two given the tumor cell doubling time of 22 hours. Thus, the observed effect can be attributable to alteration of the "bed" in terms of the epithelial and endothelial cells and the "environment" of the cavity. By this is meant the cytokine and adhesion molecule balance at the site where the undesired cells may bind. The over 60 different cytokines identified thus far can be variously categorized, depending on whether they are immunomodulatory, inflammatory, chemoattractant or growth regulatory. The three former types can lead to increased adhesion molecule expression and activation and therefore increased cell binding. See, for example, A. Thompson, The Cytokine Handbook. 2nd Edition, Academic Press, 1994 and J. Harlan and D. Liu, Adhesion: Its role in Inflammatory Disease. W. H. Freeman and Co., New York, 1992. The fact that the pathogenesis of the disease was altered in such a way as to produce an operable situation with well defined margins demonstrates the advantage of treating the compromised site prior to attachment of cells susceptible to undesired adhesion. Furthermore, it is believed that treatment of a compromised site soon after attachment of cells susceptible to unwanted adhesions will be similarly effective.

EXAMPLE 2 Oligonucleotide conjugation to Cell Binding Compounds of Formulae I. II and III

The methods for conjugation of oligodeoxynucleotides containing the natural phosphodiester linkages between nucleotides to form conjugates according to formula I have been described in Lin et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 22.:1995. By way of example, an 18-mer phosphodiester antisense sequence targeting the 5' initiation region of the proto-oncogene c-myc was synthesized on a CpG support derivatized with the cell binding compound of formula I. Thereafter, a fluorescein molecule was attached at the 5' end of the oligonucleotide portion for the purpose of intracellular tracking. The resulting purified product was used to follow intracellular uptake. The results demonstrated that after 24 hours of exposure of this product to rabbit aortic smooth muscle cells, the fluoresceinated conjugate of formula I accumulated to a level 130-fold higher in cells than the fluoresceinated oligomer by itself. EXAMPLE 3 Improved Formulations for the delivery of therapeutic agents having anti-adhesive activity

The liquid carrier used in Example 1 for the delivery of anti-adhesive conjugates according to Formula IV consisted of 10% dimethylsulfoxide. Alternative formulations using clinically approved excipients, ethanol and mannitol have been tested for the delivery of the therapeutic agents of the present invention. The following example is provided to demonstrate the relative non-toxicity of the conjugate of formula V in an ethanolic solution of mannitol.

The therapeutic agent of formula V is soluble in 100% ethanol at concentrations in excess of 100 mg/ml. An ethanol stock solution containing the conjugate of formula V can be diluted by addition to an equal volume of glycerol, Pluronic F68 or a larger volume of iso-osmotic (300 mM) mannitol solution at a final concentration of 1 mg/ml to 4 mg/ml. These solutions contain no pelletable material.

The therapeutic agent of formula V was dissolved in 100% ethanol at 150 mg/ml and subsequently diluted 51-fold into 102 mM mannitol to a final concentration of 3 mg/ml. The resulting solution contains 2% v/v ethanol and 100 mM mannitol. A portion of the 3 mg/ml solution was diluted further using the same ethanolic mannitol diluent to give solutions with final concentrations of conjugate of 2, 1, and 0.5 mg/ml. These solutions were administered to groups of mice consisting of 5 males and 5 females weighing an average of 27 g each. All mice received 2.7 ml of solution with or without the conjugate of Formula V according to Table 4. The body weight and survival of the mice was monitored for 14 days. The maximum body weight loss, if any, usually occurred on day 7 after the solutions were administered. None of the mice showed untoward symptoms or behavior and none of the mice died by 14 days after administration of the solution.

TABLE 4 Administration of Conjugate of Formula V i.p. to healthy mice at increasing doses Acute Toxicity Test

*One mouse died immediately following injection for unknown reasons.

The mice were humanely sacrificed and examined internally. The excipient treated groups appeared completely normal as did those receiving 50 mg/kg of conjugate (group 2) . Those receiving 100 mg/kg had some local abnormalities such as liver adhesion to the diaphragm. In the mice receiving 200 and 300 mg/kg of conjugate of Formula V, inter-organ adhesions were more prominent and accompanied by clear fluid accumulation in the cavity. The latter groups had large particles of injected material in the cavity especially around the omentu and fat pads. - 26 -

The liquid carrier used, 2% ethanol in 100 mM mannitol, was non-toxic and an effective excipient for peritoneal delivery of the therapeutic agent of the present invention. However at delivered doses of 100 mg/kg or more, the mechanical properties of the material and subsequent particle formation in the peritoneal cavity, may lead to local irritation and adverse effects.

EXAMPLE IV

Conjugation of Anti-adhesive drugs to lipophilic cell binding compounds generates therapeutic agents with enhanced retention times in mouse ear arteries

Rabbit ear arteries were treated with 4- formyl thiocolchicine (4FT) or 4FT conjugated to the lipophilic cell binding compound¹ according to Formula IV. At various time points up to 14 days following restoration of blood flow through the artery, the agents were extracted and the amount of 4FT quantitated.

Rabbits were anesthetized with ketamine HCl (50 mg/kg) , xylazine (6 mg/kg) and acepromazine (1 mg/kg) i.m. The central ear artery was retrogradely cannulated using a 22 g i.v. catheter. A 10 mm segment of the central ear artery was isolated as follows. Three 2.0 silk ligatures (X) were placed around the artery through the skin 10 mm proximal to the tip of the catheter and tied off. Two silk 2.0 ligatures were placed through the skin around the catheter to secure the catheter to the artery. Intestinal clamps were applied to either side of the artery to minimize collateral circulation. Solutions of 4FT or formula IV at 100 and 300 μM were delivered intraluminally to the central ear artery by means of the 22 g catheter using the following delivery conditions: pressure= 2 atm; volume = 8 ml; time = 200 sec. A 30 g needle was used to puncture the proximal end of the isolated vessel segment to allow for fluid flow at a constant pressure of 2 atm. The same procedure was repeated for the contralateral ear (n= 2/animal) . The treated portion of the vessel was removed from the ear at the following time points: 4FT treatment: t=0, 0.25, 0.5, 1.0, 1.5, 2.0, and 2.5 hr: Formula IV t=0, 1, 2, 3, 7, 10, 14 days following treatment. The arteries were blotted dry and sections weighing 10 - 20 mg were placed in 2.0 ml of 1.0 N HCl with 5 μM colchicine (added as an internal standard) . The tissue was first minced with scissors and then homogenized using a Bio-Homogenizer (BioSpec, Inc.). Formula IV hydrolyzes in the presence of acid to release all of the 4FT. The samples were then incubated for 3 hr at room temperature to allow the complete liberation of 4FT from the conjugate of Formula IV. The sample was clarified by centrifugation and loaded onto a Chem-Sep C-18 column previously conditioned with 2 ml each of water, methanol, and 0.1 N HCl. The column was washed once with 1.5 ml of 0.1 N HCl and then the compounds eluted with 2.25 ml ethyl alcohol. The samples were reduced to 0.2 ml by rotary evaporation. The amount of 4FT was quantitated by HPLC using the ratio of 4FT to colchicine peak area. The samples were run on a phenyl column using a gradient mobile phase consisting of A:0.1% TFA in H20, B: 0.1% TFA in acetyl nitrile, and C: 0.1% TFA in tetrahydrofuran. The gradient was 95% to 40% A, 2 - 50 % B, and 3 to 10% C followed by a wash phase. The peak ratio was compared to a standard curve and the amount of 4FT calculated in ng based on the molecular weight (443 g/mole) .

The results showed that conjugation of the anti-adhesive drug, 4FT to the lipophilic cell binding conjugate enhances the retention time in the artery relative to anti-adhesive drug alone. The initial amount of 4FT was 48 ± 6 ng/mg when given as the unconjugated form (300 μM x 8 ml or 1 mg delivered) . The conjugate was delivered at a three fold lower molar dose (100 μM x 8 ml or 1 mg delivered) and was retained at 237 ± 40 ng/mg tissue. Pharmacokinetic analysis of tissue amounts over time demonstrates that 4FT was cleared rapidly from tissue with a disappearance half-life of 2 minutes to one hour.

When given in the conjugated form, as the therapeutic agent of the present invention, the drug disappeared at a substantially slower rate, 3.9 days. These data suggest that conjugation of anti-adhesion drugs to cell binding compounds in accordance with present invention results in therapeutic'agents that may be effective in the promotion of prolonged retention and release of pharmacologically active agents at parenteral sites after local administration. While certain embodiments of the present invention have been described and/or exemplified above, various other embodiments will be apparent to those skilled in the art from the foregoing disclosure. For example, the conjugates described herein may be attached, e.g. , via adsorption, to a biomaterial, such as barrier materials used for protection of wound repair sites, suture materials and the like, prior to the insertion of that biomaterial into a body cavity, thereby providing enhanced prevention of unwanted cellular adhesion by the biomaterial. The present invention is, therefore, not limited to the particular embodiments described and/or exemplified, but is capable of considerable variation and modification without departure from the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A therapeutic agent for reducing unwanted cell adhesion at a compromised site comprising a conjugate of a drug and a cell binding compound, said conjugate being selected from the group consisting of conjugates of the formula:

wherein R and Rl represent hydrocarbon substituents having from 1 to about 30 carbon atoms; X and XI may be the same or different and represent 0, S, C(CH₃)₂ or Se;

R₂ may be the same or different and is selected from H, CH₃, CH₂CH₃, CH₂CH₂CH₃ or CH(CH₃)₂, m being from 0-3; Z, and Z_b represents substituents independently selected from the group H, alkyl, OH, - 0- alykyl, COOH, C0NH₂, S0₃H, S0₂NH₂, SH, S-alkyl, C0NH- alkyl, C0N-(alkyl)₂, NH-acyl, NH-alkyl, N (alkyl)₂, N0₂, halogen, Si(alkyl)₃, O-Si(alkyl)₃, Sn (alkyl)₃ or -Hg- halogen, the alkyl groups comprising said Z substituent having from 1-4 carbon atoms;

D represents a drug having an inhibitory effect on cell adhesion;

A' represents a pharmaceutically acceptable anion;

or

wherein each R, is the same or different and represent H, or CH₃ with n being 1 or 2 and p being 1,2 or 3;

R_j, represents a substituent selected from the group consisting of OH, C,-C₃ alkoxy, or acyl, said acyl substituent being unsubstituted or substituted with COOH, COOR_j or CONH₂, R,. being a

alkyl and D, L and q are as previously defined; and, with the proviso that when said cell binding compound is of formula I, said drug is an oligonucleotide having anti-adhesive activity.

2. A therapeutic agent as claimed in claim 1 wherein said cell binding compound is a compound of formula I selected from the group consisting of Tocol, /S-Tocopherol, γ-Tocopherol, £-Tocopherol, e- Tocopherol, fj-Tocopherol, f₂-Tocopherol, TJ-Tocopherol, α-Tocopherol, α-Tocopherol acid succinate and α- Tocopherol acetate.

3. A therapeutic agent as claimed in claim 1 wherein said drug is selected from the group consisting of anti-microtubule agents, anti-filament agents, anti-signalling agents, inhibitors of release of stimulatory mediators, antagonists of stimulatory mediators, agents effective to interfere with binding of adhesion molecules to their respective ligands and oligonucleotides having anti-adhesive activity.

4. A therapeutic agent as claimed in claim 1, wherein said drug is an oligonucleotide having anti-adhesive activity.

5. A therapeutic agent as claimed in claim 1 wherein said drug is conjugated to said cell binding agent via a cleavable linking moiety.

6. A pharmaceutical preparation for the treatment of unwanted cell adhesion at a compromised site comprising a therapeutic agent as claimed in claim 1 and a biologically compatible liquid carrier.

7. A pharmaceutical preparation as claimed in claim 6 wherein said biologically compatible liquid carrier comprises a liquid selected from the group consisting of an aqueous solution of ethanol and mannitol, dimethyl sulfoxide, polyoxyethylene sorbitan mono-oleate, a polyol, aqueous dextrose solution, Ringer's solution, saline solution or a mixture of said liquids.

8. A method for reducing unwanted cell adhesion at a compromised site comprising treating said site with a therapeutic agent as claimed in claim 1 in an amount effective to inhibit attachment to said site of cells susceptible to unwanted adhesion.

9. A method for reducing unwanted cell adhesion at a compromised site comprising irrigating said site with a pharmaceutical preparation as claimed in claim 6, the amount of said therapeutic agent in said preparation being effective to inhibit attachment to said site of cells susceptible to unwanted adhesion.

10. A method for reducing unwanted cell adhesion at a compromised site comprising irrigating said site with a pharmaceutical preparation as claimed in claim 7, the amount of said therapeutic agent in said preparation being effective to inhibit attachment to said site of cells susceptible to unwanted adhesion.

11. A method as claimed in claim 8, wherein said site is compromised by surgery.

12. A method as claimed in claim 8, wherein said site is compromised by inflammation.

13. A method as claimed in claim 8, wherein said site is compromised by injury.

14. A method as claimed in claim 8, wherein said site is compromised by disease.

15. A method as claimed in claim 8, wherein said site is compromised by chemotherapy.

16. A method as claimed in claim 8 wherein said cells susceptible to unwanted adhesion are selected from the group of malignant cells, inflammatory cells, diseased cells, chemotherapeutically-treated cells or injured normal cells.

17. A method as claimed in claim 8, wherein said compromised site is the abdominal cavity.

18. A method as claimed in claim 8, wherein said compromised site is the thoracic cavity.

19. A method as claimed in claim 8 wherein said compromised site is the pleural cavity.

20. A method as claimed in claim 8, wherein said compromised site is the pericardial cavity.

21. A method as claimed in claim 8, wherein said compromised site is a joint cavity.

22. A method as claimed in claim 8 wherein said compromised site is a synovial joint cavity.

23. A method as claimed in claim 8 wherein said compromised site is located in the lumen of a blood vessel.

24. A method as claimed in claim 8 wherein said compromised site is located in the gastrointestinal tract.

25. A method as claimed in claim 8 wherein said compromised site is located in the female reproductive system.

26. A method of reducing unwanted cell adhesion at a compromised site in a blood vessel said method comprising treating said site with a pharmaceutical preparation comprising a therapeutic agent as claimed in claim 1, in an amount effective to inhibit attachment to said site of cells susceptible to adhesion, and a biologically compatible liquid carrier comprising a liquid selected from the group consisting of aqueous solution of ethanol and mannitol, dimethyl sulfoxide, polyoxyethylene, sorbitan mono-oleate, a polyol, aqueous dextrose solution. Ringer's solution, saline solution or a mixture of said liquids.

27. A method as claimed in claim 26, wherein said blood vessel is an artery which is compromised by sclerosis.

28. A method as claimed in claim 27, wherein said site is treated with said therapeutic agent in a biologically compatible excipient comprising 2% (v/v) ethanol in 100 mM mannitol.