AU6553994A - Methods of affecting the growth of living tissue in mammals and compounds and compositions therefor - Google Patents

Description

METHODS OF AFFECTING THE GROWTH OF LIVING TISSUE IN MAMMALS AND COMPOUNDS AND COMPOSITIONS THEREFOR

Cross Reference to Related Applications This application is a continuation-in-part of application Serial No. 790,320 (hereinafter "the '320 application"), filed November 12, 1991 (pending).

This application is also a continuation-in-part of application Serial No. 900,592, filed June 18, 1992 (pending), which in turn, is a continuation-in-part of application Serial No. 691,168, filed April 24, 1991 (pending), which is a continuation of application Serial No. 397,559, filed August 23, 1989 (now abandoned) , which in turn, is a continuation-in-part of application Serial No. 434,659, filed November 9, 1989 (now U.S Patent No. 5,019,582), which is a continuation of application Serial No. 295,683, filed January 10, 1989 (now abandoned) , which in turn, is a continuation-in-part of application Serial No. 145,407, filed January 19, 1988 (now abandoned) , all of said applications being hereby incorporated by reference.

Field of Invention

The present invention relates to compounds, compositions and methods for affecting the growth of living tissue. More particularly, this invention relates to saccharide-based compounds and compositions for healing wounded living tissue, including the inhibition of smooth muscle cell growth following injury to vessel walls caused by treatment of atherosclerosis, such as by angioplasty.

Background of the Invention

Atherosclerosis, which is a disorder involving thickening and hardening of the wall portions of the larger arteries of mammals, is a life-threatening affliction that is largely responsible for coronary artery disease, aortic aneurysm and arterial disease of the lower extremities. Atherosclerosis also plays a major role in cerebral vascular disease. Atherosclerosis is responsible for more deaths in the United States than any other disease. See National Center of Heal th Statistics, Vital Statistics Report, Final Mortality Statistics, 1986.

Angioplasty has heretofore been a widely used method for treating atherosclerosis. For example, percutaneous transluminal coronary angioplasty (hereinafter "PTCA") was performed over 200,000 times in the United States alone during 1988. PTCA procedures involve inserting a deflated balloon catheter through the skin and into the vessel or artery containing the stenosis, or blockage. The catheter is then passed through the lumen of the vessel until it reaches the stenoic region, which is characterized by build-up of fatty streaks, fibrous plaques and complicated lesions on the vessel wall. This results in a narrowing of the vessel and blood flow restriction. To overcome the harmful narrowing of the artery caused by the atherosclerotic condition, the balloon is inflated to flatten the plaque against the arterial wall and otherwise expand the arterial lumen.

Although PTCA has produced excellent results and low complication rates, there are difficulties associated with this technique. In particular, the arterial wall being enlarged frequently experiences damage and injury during expansion of the balloon against the arterial wall. While this damage itself is not believed to be particularly harmful to the health or the life of the patient, the healing response triggered by this damage can cause a reoccurrence of the atherosclerotic condition. In this connection, the injury of tissue initiates a series of events that results in tissue repair and healing of the wound. During the first several days following an injury, there is directed migration of neutrophils, macrophages, fibroblasts and smooth muscle cells to the site of the wound. The macrophages and smooth muscle cells which migrate to the wound site are activated, thereby resulting in endogenous growth factor production, synthesis of a provisional extracellular matrix, proliferation of smooth muscle cells and collagen synthesis. From about two weeks to about one year after infliction of the wound, there is remodeling of the wound with active collagen turn-over and cross-linking (Pierce et al., J. Cell Biochem. , Vol. 45, pp. 319-326 (1991)). The manner in which this repair process is regulated is mostly unknown. However, it is known that cell proliferation, migration and protein synthesis can be stimulated by growth factors that act on cells having receptors for these growth factors.

In addition, and as known to those in the art, cells generally involved in proliferation, migration and protein synthesis processes, for example, smooth muscle cells, derive much of their energy from oxidative and glycolytic mechanisms. Glycolysis is the enzymatic splitting of glucose into two molecules of pyruvate, and is the primary sequence in the metabolism of glucose by all cells. There are at least ten steps involved in glycolysis, each of which is catalyzed by a specific enzyme, including hexokinase, phosphoglucose isomerase and phosphofructokinase. These enzymes are generally found in the cytoplasm, dissolved in the cytosol and outside mitochondria. See N.A. Campbell, Biology, 2nd Ed., The Benjamin/Cummings Publishing Co., Inc., pp. 186-187 (1990).

As is also known to those in the art, plasma membranes of cells function, in part, to maintain an ionic composition in the cytosol which is very different from that of the surrounding fluid. In both vertebrates and invertebrates, for example, the concentration of sodium ion is about 10 to 20 to 40 times higher in the blood than within the cell. The concentration of potassium ion is the reverse, generally 20 to 40 times higher inside the cell. The generation and maintenance of such gradients on either side of a semipermeable membrane requires the expenditure of a great deal of energy.

Transport across a membrane may be passive or active.

Passive transport is a type of diffusion in which an ion or molecule crossing a membrane moves down its electrochemical or concentration gradient. No metabolic energy is expended in passive transport.

Active transport uses metabolic energy to move ions or molecules against an electrochemical gradient. For example, low sodium concentration inside the cell is maintained by the sodium potassium ATPase (Na⁺K⁺-ATPase) , which is a specific active transport mechanism located in the membrane that transports sodium from the interior of the cell to the outside.

Ion gradients are utilized in driving many biological processes. For example, the transmembrane concentration gradients of sodium and potassium ions are essential for the conduction of an electrical impulse down the axon of a nerve cell. See, Darnell et al., Molecular Cell Biology, Sci. Am. Books, Inc., 618-19 (1986). Applicants contemplate that such ion gradients are also involved in cell proliferation, migration and protein synthesis processes.

With particular reference to injury of the arterial wall from angioplasty, it has been observed that the smooth muscle cells associated with the stenotic region of the artery initiate cell division. As the smooth muscle cells proliferate and migrate into the intimal layer of the artery, they cause thickening of the arterial wall. Initially, this thickening is due to the increased number of smooth muscle cells. However, further thickening of the arterial wall and narrowing of the lumen is due to increased smooth muscle cell volume and the accumulation of extracellular matrix and connective tissue. This thickening of the cell wall and narrowing of the lumen following treatment of atherosclerosis is referred to herein as restenosis. It has been observed that up to 40% of patients who undergo PTCA are afflicted by restenosis and the recurrent arterial blockage that it causes. Thus, the long-term effectiveness of treatments for atherosclerosis, including angioplasty, has been substantially limited by the reoccurrence of restenosis.

Techniques have heretofore been developed to prevent restenosis or mitigate its negative consequences. In particular, a variety of pharmacological agents have, been evaluated as possible inhibitors of restenosis. One group of pharmacological agents under study are antiplatelet agents. Since platelet aggregation and thrombus formation have been implicated in the development of restenosis, agents that effectively reduce platelet adhesion may be useful in preventing restenosis. An example of this approach is found in U.S. Patent No. 4,929,602, which discloses halogen-methyl ketone-containing peptides for use in the prevention of platelet-dependent arterial thrombosis. Another approach to preventing restenosis has been to use pharmacological agents which inhibit smooth muscle cell proliferation. For example, it has been reported that low molecular weight heparin reduces restenosis after transluminal angioplasty. See Howell et al., "Low Molecular Weight Heparin Reduces Restenosis After Experimental Angioplasty, " -Circulation (Suppl. II), Vol. 80, No. 4, October 1989.

Heparin, a mucopolysaccharide, is a constituent of various tissues, especially liver and lung, and mast cells in several mammalian species. The use of heparin to inhibit restenosis has several disadvantages. For example, heparin is not a homogeneous, well-defined substance. Moreover, heparin is generally manufactured by different processes and is available from different vendors. Heparin may therefore possess differing properties and characteristics, depending on the process used for manufacture and the particular vendor from which it is obtained. Accordingly, the use of heparin involves an undesirable lack of predictability and reproduceability.

Despite the excessive and undesirable cell proliferation, migration and protein synthesis which is believed to be caused by growth factors in connection with restenosis, there nevertheless exists diseases and/or injuries which may be ameliorated by the administration of growth factor(s). For example, in vivo studies have shown that local application of exogenous single growth factors or a combination of growth factors can enhance the healing process following experimental wounding in animals. See Antoniadε et al. , Proc . Natl . Acad. Sci . USA, Vol. 88, pp. 565-569 (1991). The ability of these growth factors to promote wound healing has resulted in efforts to obtain these factors in purified form. It is known that a number of these growth factors, known as heparin binding growth factors (HBGFs) , have a strong affinity for heparin. See Lobb, Eur. J. Clin . Invest . , Vol. 18, pp. 321-328 (1988) and Folkman and Klagsbrun, Science, Vol. 235, pp. 442-447 (1987). These HBGFs have been shown to have mitogenic and non-mitogenic effects on virtually all mesoder - and neuroectoderm-derived cells in vitro . HBGFs are also known to promote the migration, proliferation and differentiation of these cells in vivo . It has been suggested by Lobb {Bur. J. Clin . Invest . , Vol. 18, pp.

321-328 (1988)) that HBGFs could therefore effect the repair of soft tissues. It was further suggested that HBGFs may be used to effect the repair of hard tissue such as bone and cartilage.

Knowledge of the affinity of growth factors for heparin and, as noted above, the difficulty of obtaining heparin in a pure, homogeneous form, has resulted in attempts to obtain a compound which possesses heparin's affinity for growth factors but which can be easily and reproducibly manufactured. As described in the parent applications referenced hereinabove, one group of compounds meeting these requirements are cyclodextrins which are cyclic oligosaccharides consisting of up to at least six glucopyranose units. ₍

Summary of the Invention

In accordance with the teachings of this invention, a composition for affecting the growth of cells of living tissue in mammals is provided. This composition comprises a physiologically acceptable carrier and a compound selected from the group consisting of a metabolic inhibitor of a glycolytic pathway of the cells, an agent which causes an increase in the concentration of intracellular sodium of the cells, and mixtures thereof. In certain preferred embodiments, the compound comprises a saccharide which is preferably selected from the group consisting of 2-deoxyglucose, 2-deoxyglucose derivative, cyclodextrin derivative and a mixture of two or more of these. In more detailed embodiments of this invention, .the 2- deoxyglucose derivative comprises a 2-deoxycyclodextrin derivative. Suitable 2-deoxycyclodextrin derivatives include compounds of the formula

wherein at least two of the R_ and R₂ groups are hydroxy or an anionic substituent selected from the group consisting of sulfate, phosphate, sulfonate and nitrate, and the other of the R-_L and R₂ groups, when present, is a substituent selected from the group consisting of H, alkyl, aryl, ester, ether, thioester, thioether and -COOH; and n is an integer from about 6 to about 12.

Also in accordance with the teachings of this invention, methods are provided for inhibiting the pathological growth of smooth muscle cells in a tissue of a mammal. The methods involve the metabolic inhibition of the glycolytic pathway of smooth muscle cells and/or the increase of the concentration of intracellular sodium of smooth muscle cells.

In preferred form, the present methods involve administering to said mammal one or more saccharides. The saccharides, which are administered in an amount effective to inhibit the pathological growth, are preferably selected from the group consisting of 2-deoxyglucose, 2-deoxyglucose derivative, cyclodextrin derivative and a mixture of two or more of these. In certain embodiments, the methods and compositions of this invention involve compounds which are extremely water- soluble. This high solubility favorably facilitates introduction of the compounds into the body of a mammal and aids in dispersal of the compounds via the bloodstream. The compounds may be administered to mammals, either alone or in combination with growth-inhibiting steroids, to absorb growth factors present in the bloodstream.

In other embodiments, the methods and compositions involve compounds which possess a high affinity for growth factor and which are characterized by very low water solubility. According to one aspect of applicants' discovery, such substantially water-insoluble compounds may be combined with a growth factor prior to administration and may be applied locally to the site of a wound. Due, at least in part, to their low solubility, such compounds remain at the site of application and slowly release the growth factor to optimize the dosage of growth factor at the wound site. Applicants have found also that a compound possessing both a high affinity for growth factor and a low solubility can be used to remain at the site of an injury and to absorb at least some portion of the growth factors released by the injured tissue. This reduces the probability of over-stimulation of the wound healing process, as is observed in restenosis following angioplasty. Also in accordance with the present invention, compounds which have the following formula are provided

wherein R_{l f} R₂ and n are as defined above.

In preferred form, R_α and R₂ are independently hydroxy or sulfonate and n is an integer from about 6 to about 8. More preferably, R_x and R₂ are independently sulfonate and n is an integer of about 7.

Definitions

As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

"Alkyl" means a saturated aliphatic hydrocarbon, either branched- or straight-chained. A "lower alkyl" is preferred, having about 1 to about 6 carbon atoms. Examples of alkyl include methyl, ethyl, n-propyl, isopropyl, butyl, sec- butyl, t-butyl, amyl and hexyl.

"Aryl" means an unsaturated ring system characteristic of benzene. Preferred aryl groups include ring systems of from about 6 to about 10 carbon atoms, and include phenyl, naphthyl, and phenanthryl.

"Ester" refers to a group characteristic of the formula -C(=0)-OR, where R represents alkyl or aryl. In preferred form, R is lower alkyl.

"Thioester" refers to a group characteristic of the formula -C(=0)-SR, where R represents alkyl or aryl. In preferred form, R is lower alkyl.

"Wound healing" refers to the repair or reconstruction of cellular tissue.

"Substantial reduction in restenosis" means a post- treatment restenosis value of no greater than about 50%. According to preferred embodiments, the post-treatment restenosis value is no greater than about 25%.

"Post-treatment restenosis value" refers to the restenosis value measured at about one to about six months after angioplasty. "Restenosis value" refers to the restenosis rate calculated as a loss of greater than or equal to 50% of the initial gain in minimum lumen diameter achieved by angioplasty.

"2-Deoxyglucose derivative" refers to any derivative of 2-deoxyglucose. The 2-deoxyglucose derivatives hereof may comprise, for example, substituted forms of deoxyglucose, including 2-deoxyglucose substituted with one or more substituents, such as ionic and/or non-ionic substituents. 2-Deoxyglucose derivatives may also comprise dimeric, trimeric, tetrameric, oligomeric, and polymeric forms of 2-deoxyglucose, which are collectively referred to hereinafter as "multimers" or "multimeric" . "Oligosaccharide" refers to saccharides having* from about 5 to about 10 sugar units and having molecular weights, when unsubstituted, from about 650 to about 1300.

"Polysaccharide" refers to saccharides comprising greater than about 10 sugar units per molecule. Polysaccharides are understood to be materials having many 2-deoxyglucose units, either alone or in combination with other sugar units.

"Polymer" refers to structures of repeated 2-deoxy¬ glucose derivatives and/or cyclodextrin derivatives and are based on monomers which are linked together to form the polymer. "Low solubility" refers to solubility of much less than about 15 grams per 100 milliliters of water.

"2-Deoxycyclodextrin derivative" refers to compounds which comprise 2-deoxyglucose units forming a ring or toroid shaped molecule and which are analogous to cyclodextrins and cyclodextrin-containing compounds.

"Salt precipitate" means a polyanionic deoxyglucose derivative which has been associated or complexed with a suitable, non-toxic, physiologically acceptable cation to produce a salt which is substantially insoluble at body temperature.

Brief Description of the Drawings

For the purpose of illustrating the invention, there are shown in the drawings forms which are presently preferred; it being understood, however, that this invention is not limited to the precise arrangement and instrumentality shown.

Figure 1 is a schematic representation of the 3-dimensional shape of oc-, β- and γ-2-deoxycyclodextrin. Figure 2 graphically illustrates the effect of 2-deoxyglucose on migration of human smooth muscle cells in tissue culture. Figure 3 graphically illustrates the effect of 2-deoxyglucose on proliferation of human smooth muscle cells in tissue culture.

Figure 4 shows the affinity of β-deoxycyclodextrin tetradecasulfate polymer for basic fibroblast growth factor.

Figure 5 shows polyacrylamide gel electrophoresis of basic fibroblast growth factor and Chrondosarcoma-derived growth factor purified by deoxycyclodextrin copper biaffinity chromatography. Lane 1 shows the protein profile of the protein markers (phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, beta lactoglobulin, and lysozyme) . Lanes 2 and 3 show the 18,000 molecular weight polypeptide bands of basic fibroblast growth factor and Chrondosarcoma derived growth factor, respectively. Figure 6 compares the affinities of heparin and beta- deoxycyclodextrin tetradecasulfate polymer for Chrondosarcoma derived growth factor.

Figures 7 and 8 show the effect of 2-deoxyglucose on the degradation of tissue plasminogen activator by human umbilical vein smooth muscle cells.

Detailed Description of the Invention

Compositions, methods and compounds for affecting the growth of cells of living tissue in mammals are provided by this invention. The compositions comprise, in combination with a physiologically acceptable carrier, a compound which acts as a metabolic inhibitor of a glycolytic pathway of said cells and/or as an agent which causes an increase in the concentration of intracellular sodium of said cells.

The methods, compositions and compounds of this invention are particularly suitable for inhibiting the pathological growth of smooth muscle cells in a tissue of a mammal, including the treatment of restenosis.

This invention is also directed to methods for preparing these compositions and to methods for treating a variety of wounds resulting from accidents or surgical procedures. The wound may be the result of an accident, such as injury or burns. The wounds treatable by the present compositions and methods also include wounds resulting from surgical procedures of any type, from minor intrusive procedures, such as catheterization or angioplasty resulting in wounding of vascular organ surfaces, and to major surgical procedures, such as bypass or organ transplant operations. Included in this concept of wound healing is the repair of injured or fragmented bone or cartilage and the promotion of the establishment of bone grafts or implants.

I. THE COMPOSITIONS Applicants have found that compositions comprising metabolic inhibitors of glycolytic pathways of cells and/or agents which cause an increase in the concentration of intracellular sodium can be useful for affecting the growth of cells of living tissue in mammals. In particular, applicants have discovered that such compositions are useful as wound healing materials, including wounds associated with abnormally proliferating and/or migrating cells, for example, smooth muscle cells, which are associated with restenosis.

While applicants do not intend to be bound to any particular theory or theories, it is believed that the use of metabolic inhibitors of the glycolytic pathway of cells, at any given step of the pathway, and/or the use of agents which cause an increase in the concentration of intracellular sodium, result in the selective deprivation from the cells of energy derived from glucose.

Applicants contemplate that any one of a number of materials or compounds may be utilized to inhibit glycolytic pathways of cells and/or increase the concentration of intracellular sodium of cells, including abnormally proliferating and/or migrating cells, and are all within the scope of the present invention. Such materials include organic compounds, for example, 2-deoxyglucose, variably modified false substrates, and the like, which may be used to inhibit one or more of various enzymes involved in glycolysis, including, for example, hexokinase, phosphoglucose isomerase and phosphofructo- kinase, and to increase intracellular sodium concentration. In addition to organic compounds, various other types of materials that may act by varying mechanisms could be used to inhibit glycolysis and/or increase intracellular sodium.

In this connection, applicants contemplate that down- regulation of one or more of the enzymes mentioned above and which are involved in glycolysis could be accomplished locally to the wound, for example, in the wall of a blood vessel which has been subjected to angioplasty. Such down-regulation could be achieved by using an antisense oligonucleotide strategy to decrease the amount of any of the enzymes which may be proximate to the wound site. For example, the cDNA sequence coding for phosphofructokinase could be obtained and used to make a phosphorothiorate oligonucleotide which is complementary to a portion of the cDNA sequence. The expression of phosphofructokinase may then be decreased locally, for example, in the blood vessel wall.

Applicants contemplate also that, in addition to glucose utilization and sodium transport, other important mechanisms which involve cellular energy could be selectively targeted to inhibit abnormal proliferation or migration of cells, including vascular smooth muscle cells, by the methods and compositions of the present invention.

In certain preferred embodiments, compounds of the present compositions comprise saccharide. The term "saccharide" refers to all known and available sugars and sugar-based compounds, and includes oligosaccharides and polysaccharides. In preferred form, the saccharide is selected from one or more of 2-deoxyglucose, 2-deoxyglucose derivative, and cyclodextrin derivative. Each of these saccharides are discussed more fully hereinafter. A. 2-Deoxyglucose

In certain embodiments of this invention, the saccharide preferably comprises 2-deoxyglucose (hereinafter "DG"). DG, which corresponds to the deoxygenated form of glucose, has the following formula.

All known and contemplated isomers of 2-deoxyglucose are within the scope of this invention, including various anomeric isomers, such as the α and β anomers. DG is commercially available from various vendors, including Aldrich Chemical Co. of Milwaukee, I.

Applicants have discovered that the administration of compositions comprising DG are effective for effecting the growth of cells of living tissue in mammals. In particular, applicants have found that compositions comprising DG are effective, when administered according to the various teachings herein, for inhibiting or preventing the undesired smooth muscle cell development often observed following angioplasty or treatment to remove atherosclerotic plaques which occlude blood vessels.

Applicants believe that, in addition to the metabolic inhibition of glycolysis, DG inhibits abnormal migration and/or proliferation of cells via the mediation of changes in sodium transport. In this connection, if has been observed that when DG is administered to smooth muscle cells, including human smooth muscle cells, there is a corresponding increase of intracellular sodium without changes in high energy phosphates, for example, adenosine triphosphate (ATP) . Upon cessation of the administration of DG, smooth muscle cell intracellular sodium levels return to baseline after about 12-16 hours, while ATP concentrations remain unchanged. While applicants do not intend to be bound to any particular theory or theories, applicants contemplate that, as a metabolic competitive inhibitor of glucose metabolism and/or an agent which causes an increase in the concentration of intracellular sodium, DG is capable of inhibiting vascular smooth muscle cell migration and proliferation without irreversibly damaging the cells, and thus retard the restenosis process after vascular injury, for example, as by angioplasty. DG possesses solubility characteristics which are similar to that of other monomeric sugars, for example, glucose. Thus, DG has a high solubility in distilled water at body temperature. Applicants contemplate that this high solubility enables DG to readily enter the bloodstream of the mammal being treated, and to reach the site of a given wound. Accordingly, DG is particularly suited for oral administration, which is discussed more fully hereinafter.

According to the present methods, mammals, including humans, which have arterial regions subject to angioplasty, are treated by administering to the mammal DG in an amount effective to inhibit arterial smooth muscle cell migration and proliferation. It is contemplated that the degree of restenosis inhibition according to the present methods may vary within the scope hereof, depending upon such factors as the mammal being treated and the extent of arterial injury during the angioplasty. It is generally preferred, however, that DG be administered in an amount effective to cause a substantial reduction in restenosis.

Thus, certain embodiments of the present invention involve methods and compositions for inhibiting restenosis in a patient which comprises administering to the patient an amount of DG effective to inhibit the formation of a restenotic lesion in a patient who has undergone angioplasty.

It is contemplated that the DG may be administered before, during and/or after angioplasty treatment of the stenosed artery. The compositions of the present invention can be administered to a mammalian host in a variety of forms adapted to the chosen route of administration. The various methods of administering the present compositions, including compositions comprising DG, are discussed more fully hereinafter. B. 2-Deoxyglucose Derivative

In alternate embodiments of the present invention, the saccharide preferably comprises 2-deoxyglucose derivative (hereinafter "DG derivative"). As noted above, the DG derivative encompasses a multitude of various forms of DG, including, but not limited to, substituted 2-deoxyglucose and dimeric, trimeric, tetrameric, oligomeric and/or polymeric forms of 2-deoxyglucose. In addition, DG derivative encompasses oligosaccharides and polysaccharides which are based, at least in part, on repeating units of 2-deoxyglucose.

Thus, and in basic form, the DG derivative may comprise DG wherein one or more of the hydroxy groups in the DG moiety are substituted or replaced with other substituents, such DG derivative being represented by the following formula.

In the compound of formula III, at least one or R is an anionic substituent selected from well-known and available substituent groups, including, but not limited to, sulfate, phosphate, sulfonate and nitrate groups. The remainder of the R groups, when present, are non-anionic substituents selected from well- known and available substituent groups, including, but not limited to, hydroxy, hydrogen, alkyl, aryl, ester, ether, thioester, thioether and carboxyl.

The above substituents may be selected so as to provide the substituted DG derivative with varying properties, for example, solubility, hydrophilicity and/or hydrophobicity, and which may be selected as desired based on the particular application. The preparation of the compounds of formula III involves standard synthetic organic substitution reactions, and would be readily apparent to one of ordinary skill in the art. Also in basic form, the DG derivative may comprise a dimer of DG, which corresponds to 2,2'-dideoxymaltose.

Additional DG units may be covalently bonded to the 2,2'- dideoxymaltose dimer to form the trimer, tetramer, and various multimers generally, of DG.

The multimeric forms of DG may comprise sugar units other than DG. For example, the polymeric DG derivative may comprise repeating units of DG as well as one or more of other repeating sugars, for example, glucose. The particular sugars utilized in the multimeric DG derivative and the size (molecular weight) of the multimer may be selected to obtain the desired properties depending on the particular application. In addition, the multimers may be prepared to be branched and/or straight-chained, again depending on the desired properties of the desired product, for example, the solubility of the multimeric derivative. Applicants contemplate also that one or more of the hydroxyl groups of the DG and/or other sugar units contained in the multimer may be substituted with a variety of substituents, as discussed more fully hereinafter. In certain preferred aspects of this invention, the DG derivative comprises anionic substituents. This provides the DG derivative with a high negative charge density and low solubility. It is contemplated that the anionic substituents may be selected from a large group of known and available anionic substituents. However, it is generally preferred that the anionic substituents be selected from the group consisting of sulfate, phosphate, sulfonate, nitrate, carboxylate and combinations of two or more of these. Preferred compositions are based on DG derivatives having six or more sugar units which may be DG units alone or in combination with other sugar units, as described hereinbefore with respect to the multimeric DG derivatives, and which have up to about two substitutents per sugar unit, wherein the substituents preferably comprise sulfate, sulfonate and/or phosphate substituents. In certain aspects of this invention, the present DG derivatives preferably have a low solubility in distilled water at body temperature. This enables the DG derivatives to remain localized in a solid state for a substantial period of time in an aqueous medium, for example, physiological and distilled water. According to certain preferred embodiments, the DG derivatives have substantially no solubility in distilled water at body temperature. Thus, it is preferred that the solubility of the DG derivatives is much less than about 1 gram per 100 ml of distilled water, and even more preferably, less than about 1 milligram per 100 ml. Such insolubility is achieved, for example, by utilizing DG derivative comprising polymer aggregates or dispersions of substantially solid polymer particles. While it is contemplated that various particle sizes and shapes may be utilized, it is preferred that the particles have an average particle size ranging from about 1 millimicron to about 1000 microns in diameter. Expressed in terms of molecular weight, the polymers have, on average, a molecular weight of about 1 billion or greater. The high molecular weight of the preferred polymers is due to the presence of many millions of DG and/or other sugar units within any of the discrete undissolved entities. In addition, particles having the desired insolubility may be produced by forming a salt comprising an anionic DG derivative in combination or associated with a polyvalent cationic constituent.

1. Deoxycyclodextrins and Derivatives Thereof In certain embodiments, the DG derivatives involve 2-deoxycyclodextrins (hereinafter "DCs") and deoxycyclodextrin derivatives (hereinafter "DC derivatives"). In preferred form, the DCs and DC derivatives comprise α-, β- and/or γ- deoxycyclodextrins and derivatives thereof, the structures of which are discussed in detail below.

Cyclodextrins, which are compounds well-known to those skilled in the art, are saccharide compounds containing at least six glucopyranose units forming a ring or toroid shaped molecule, which therefore has no end groups. Although cyclodextrins with up to 12 glucopyranose units are known, only the first three homologs have been studied extensively. These compounds have the following simple, well-defined chemical structure.

The common designations of the lower molecular weight cyclodextrins, namely, α-, β- and γ-cyclodextrins, refer to the chemical structure above wherein n is 6, 7 and 8, respectively. Cyclodextrins and various derivatives thereof are discussed extensively in the chemical literature. See, e.g., "Tetrahedron Report Number 147, Synthesis of Chemically Modified Cyclodextrins, " A.P. Croft and R.A. Bartsch, Tetrahedron 39 (9) :1417-1474 (1983), which is specifically incorporated herein by reference (hereinafter referred to as "Tetrahedron Report No. 147") .

Analogously to the cyclodextrins of the prior art, it is contemplated that the present DCs and DC derivatives comprise saccharide compounds containing at least six sugar units, at least one of which is DG. As with the prior art cyclodextrins, the present deoxycyclodextrins may comprise up to about 12 sugar units. In addition, all or only some of the sugar units contained in the DCs may correspond to DG. Other sugars may be present in the deoxycyclodextrins, including, for example, glucose. In preferred form, the DCs comprise compounds having the following formula

wherein n is 6, 7 and 8, respectively and which are designated herein as α-, β- and γ-deoxycyclodextrin, respectively.

Topographically, and as with the known α-, β- and γ- cyclodextrins, it is contemplated that the present DCs may be represented as a torus, as shown in Figure 1, the upper rim of which is lined with primary hydroxyl groups and the lower rim with secondary hydroxyl groups. Coaxially aligned with the torus is a channel-like cavity of about 5, 6 or 7.5 A.U. diameter for the α-, β- and γ-DCs, respectively. These cavities render the deoxycyclodextrins capable of forming inclusion compounds with hydrophobic guest molecules of suitable diameters.

The compositions of certain alternate and preferred embodiments of the present invention include polyanionic DC derivatives. In general, DC derivative refers to chemically modified DCs formed by reaction of the primary and/or secondary hydroxyl groups attached to carbons 3 and 6 of the DG molecule without disturbing the α (l-→4) hemiacetal linkages. A review of methods for preparing various chemically modified cyclodextrins is given in Tetrahedron Report Number 147. It is contemplated that the procedures disclosed and referenced in Tetrahedron Report No. 147 and the prior art generally for chemically modifying or derivatizing cyclodextrins may be adapted to prepare DC derivatives of the present invention.

The DC derivatives are preferably derivatized deoxycyclodextrin monomers, dimers, trimers, polymers or mixtures thereof. In general, the deoxycyclodextrin derivatives of the present invention are comprised of, or formed from, derivatized DC monomers, each of the DC monomers consisting of at least six sugar units. The DC monomers may comprise all DG units, or at least one DG unit in combination with other. sugar units, and have α (1→4) hemiacetal linkages. The preferred derivatized deoxycyclodextrin monomers of the present invention generally have the following formula:

In formula I above, each of R_x and R₂ are present in the DC derivatives at least six times, n being an integer of at least six. Accordingly, the definitions of each of R-_L and R₂, in a given DC derivative, are independent of each other, unless indicated otherwise. In preferred form, at least two of the R_x and R₂ groups, per monomeric unit, are hydroxy or an anionic substituent and the remainder of the R-_ and R₂ groups, when present, are non-anionic groups selected from well known and available substituent groups. The remaining, non-anionic R_ and R₂ groups may be, for example, hydrogen, alkyl, aryl, ester, ether, thioester, thioether and carboxyl. The remaining non- anionic R_ and R₂ groups may be hydrophilic, hydrophobic or a combination thereof, depending upon the particular requirement of the desired composition. However, it is generally preferred that the remaining non-ionic R_ and R₂ substituents be hydrophobic to minimize the solubility of the compounds.

For deoxycyclodextrin monomers having the structure of formula I above wherein n is from about 6 to about 8, it is preferred that the compound be polyanionic. In other words, it is preferred that the compounds have, on average, at least about 9 anionic substituents per monomer unit, and more preferably, at least about 12 anionic substituents per monomer, and even more preferably, at least about 14 anionic substituents per monomer. In general, it is preferred that the anionic substituents be relatively evenly distributed on the monomer molecule. . Such structures are believed to provide the high negative charge density which is contemplated to be therapeutically beneficial, with the highest charge density molecules being the most therapeutically beneficial. The polyanionic deoxycyclodextrin monomers of the type described above are important components in certain preferred compositions of the present invention. The monomeric units may be present in the composition in the form of, for example, insoluble polymeric or co-polymeric structures, or as insoluble precipitated salts of derivatized deoxycyclodextrin monomer, dimer or trimer. Such salts may be formed by methods which comprise derivatizing the DG or other sugar unit(s) contained within the DC derivative with anionic substituents and then complexing or associating the derivatized sugar with an appropriate polyvalent cation to form an insoluble derivatized sugar salt. In alternate embodiments, the basic monomeric structure identified above is the repeating unit of the novel insoluble polymeric deoxycyclodextrins of the present invention.

In certain embodiments, applicants have found that the saccharide desirably comprises a mixture of two or more of 2- deoxyglucose derivatives, cyclodextrin and/or cyclodextrin derivative, and DG. These various saccharides may be simply combined together when formulating the compositions. Alternatively, applicants have found that it may be desirable to chemically link together, via covalent bond(s) , two or more of 2-deoxyglucose derivatives, cyclodextrin and/or cyclodextrin derivative, and DG. Preferably, the saccharide mixture involves a DG molecule which is covalently bonded to a DG derivative, cyclodextrin and/or cyclodextrin derivative. This linking together of the saccharide compounds involves standard synthetic organic coupling reactions, and would be readily apparent to one of ordinary skill in the art.

The cyclodextrin (hereinafter "CD") derivatives are preferably derivatized CD monomers, dimers, trimers, polymers or mixtures thereof. In general, the CD derivatives of the present invention are comprised of or formed from derivatized cyclodextrin monomeric units consisting of at least six glucopyranose units having α (1 → 4) hemiacetal linkages.- The preferred derivatized cyclodextrin monomers of the present invention generally have the following formula:

wherein at least two of said R groups per monomeric unit are anionic substituents and the remainder of said R groups, when present, are nonanionic groups selected from well known and available substituent groups. The remaining, nonanionic R groups may be, for example, H, alkyl, aryl, ester, ether, thioester, thioether and -COOH. Exemplary alkyl groups include methyl, ethyl, propyl and butyl. The remaining nonanionic R groups may be hydrophilic, hydrophobic or a combination thereof, depending upon the particular requirements of the desired composition. However, it is generally preferred that the remaining nonionic R substituents be hydrophobic in order to minimize the solubility of the compounds.

For CD monomers having the structure of formula V wherein n is from about 6 to about 8, it is preferred that the compound have on average at least about 9 anionic R substituents per monomer unit, more preferably, at least about 12 anionic R substituents per monomer, and even more preferably, at least about 14 anionic R substituents per monomer. In general, it is preferred that the anionic substituents be relatively evenly distributed on the monomer molecule, and accordingly compounds having the structure of formula I wherein n is from about 6 to about 8 preferably have from about 1 to about 3 anionic R substituents per n unit, more preferably, from about 1.3 to about 2.5 anionic R substituents per n unit and even more preferably, from about 1.4 to about 2.2 anionic R substituents per n unit. Such structures are believed to provide the high negative charge density found to be therapeutically beneficial, with the highest charge density molecules providing excellent results.

The polyanionic cyclodextrin monomers of the type described above are important components of certain preferred compositions of the present invention. The monomeric units may be present in the composition as components of, for example, insoluble polymeric or co-polymeric structures, or as insoluble precipitated salts of derivatized cyclodextrin monomer, dimer or trimer. Such salts may be formed by methods which comprise derivatizing the CD with anionic substituent(s) and then complexing or associating the derivatized CD with an appropriate polyvalent cation to form an insoluble derivatized CD salt.

In preferred form, the mixture of saccharides comprises a mixture of DG and sulfated CDs of formula V above. Preferably, the mixture of saccharides comprises DG and β- cyclodextrin tetradecasulfate. In preferred form, this mixture preferably comprises β-cyclodextrin tetradecasulfate, which is covalently linked to one or more 2-deoxyglucose molecules. a. Deoxycyclodextrin Polymers According to important and preferred embodiments, the present compositions comprise derivatized deoxycyclodextrin polymers (hereinafter "the DC polymers"). The DC polymers have a structure corresponding to polymers formed from derivatized deoxycyclodextrin monomers of the type illustrated above. In view of the present disclosure, it will be appreciated that polymeric materials having such structures may be formed by a variety of methods. For example, derivatized deoxycyclodextrin polymers may be produced by polymerizing and/or crosslinking one or more derivatized deoxycyclodextrin monomers, dimers, trimers, etc., with polymerizing agents, for example, epichlorohydrin, diisocyanates, diepoxides and silanes, using procedures known in the art to form cyclodextrin polymers. See Insoluble Cyclodextrin Polymer Beads, Chemical Abstracts 102:94:222444m; Zsadon and Fenyvesi, 1st Int. Symp. on Cyclodextrins, J. Szejtli, ed. , D. Reidel Publishing Co., Boston, pp. 327-336; Fenyvesi et al. , 1979, Ann. Univ. Budapest, Section Chim. 15:13, 22; and Wiedenhof et al., 1969, Die Starke 21:119-123. The polymerizing agents noted above are capable of reacting with the primary and secondary hydroxy groups on carbons 6 and 3 of each of the DG moieties, as well as hydroxy groups on moieties other than DG, for example, glucose, and which may be present in the DC monomers as discussed above. Alternatively, the derivatized deoxycyclodextrin polymers may be produced by first polymerizing and/or crosslinking one or more underivatized deoxycyclodextrin monomers, dimers, trimers, etc.

(for example, DC multimers) , and then derivatizing the resulting polymer with anionic substituents. The derivatized deoxycyclodextrin polymers may also be formed by reacting mixtures of derivatized monomers and underivatized monomers, or by copolymerizing and/or crosslinking derivatized deoxycyclodextrin polymers and underivatized deoxycyclodextrin polymers and DG and DG polymers. For all preparation procedures, it is preferred that the polymerization method employed results in a solid polymer product of sufficient porosity to allow diffusion penetration of molecules between the external solvent and a substantial portion of the internal anionic monomer sites. The solubility of the present deoxycyclodextrin polymers will depend, inter alia, on the molecular weight and size of the polymer. It is contemplated that the present derivatized deoxycyclodextrin polymers are of large molecular weight so as to remain substantially in the solid state. Preferably, the deoxycyclodextrin polymers are solid particulates of generally about 1 to 300 micron size.

The derivatized deoxycyclodextrin polymer of the present invention may be available in a variety of physical forms, and all such forms are within the scope of the present invention. Suitable forms include beads, fibers, resins or films. Many such polymers have the ability to swell in water. The characteristics of the polymeric product, chemical composition, swelling and particle size distribution are controlled, at least in part, by varying the conditions of preparation.

The deoxycyclodextrin polymer derivative preferably comprises a polyanionic derivative of an α-, β- or γ- deoxycyclodextrin polymer. In preferred embodiments,, the anionic substituents are selected from the group consisting of sulfate, sulfonate, phosphate and combinations of two or more thereof. Although it is possible that other anionic groups, such as nitrate, might possess some therapeutic capacity, the sulfate, sulfonate and phosphate derivatives are expected to possess the highest therapeutic potential. In preferred embodiments, at least about 10 molar percent of the anionic substituents, and even more preferably, at least about 50 molar percent, are sulfate groups. Highly preferred are α-, β- and γ- deoxycyclodextrin polymers containing about 10-16 sulfate groups per deoxycyclodextrin monomer, with β-deoxycyclodextrin tetradecasulfate polymer being especially preferred.

As with the DG derivatives above, the DC polymers may be combined and/or covalently linked with other chemical entities in the present compositions. In desired form, the DC polymers are covalently linked to DG units, either in the polymer backbone or as substituents to the monomeric units of the DC polymers.

2. Insoluble Salt Precipitates The present compositions may include derivatized, insoluble DG salt precipitates, and preferably, derivatized insoluble oligomeric DG salt precipitates. Suitable polyvalent cations which may be used to produce an insoluble salt precipitate of the present invention include Mg, Al, Ca, Ce, and Ba. The cations herein listed are presented generally in order of decreasing solubility, although this order may be different for DG derivatives of different types and degrees of anionic substitution. While all such derivatized insoluble DG salt precipitates are believed to be operable within the scope of the present invention, the derivatized oligomeric DG salt derivatives are preferred. Such oligomeric DG salts will typically have unsubstituted molecular weights ranging from about 650 to about 1300. In certain preferred embodiments, the DG salt precipitates may be obtained by reacting the desired DG derivative with agents that will produce the desired anionically substituted product and subsequently exchanging the cations which were introduced by the synthesis for cations of the desired polyvalent type. This latter step will result in precipitation of the insoluble DG salt precipitate derivative.

In preferred form, the DG salt precipitate comprises derivatived DC salts, including polyanionic DC salts. Preferred DC salts include the Al, Ca and Ba salts of α-, β- and γ-deoxycyclodextrin sulfate. β-Deoxycyclodextrin sulfate salts are particularly preferred. As with the DG derivatives generally, various degrees of sulfation per sugar unit can be employed. It is generally preferred, however, that the derivatized 2-deoxycyclodextrin salts have an average of at least about 1.3 sulfate groups per sugar unit, and even more preferably, about two sulfate groups per sugar unit. Especially preferred is β-deoxycyclodextrin tetradecasulfate, which has an average of about two sulfate groups per glucose unit. It is believed that sulfonate-or phosphate- containing DG derivatives, combined with polyvalent cations such as Mg, Al, Ca, Ce or Ba, may result in compositions of low solubility which can be combined with growth factors to facilitate therapeutic delivery of these growth factors to the site of a wound. According to the present invention, salts of DG derivatives may be used to deliver growth factor proteins to tissues or bone in need of repair, by prior complexing with growth factors, and delivering the complex physically to the site of repair. The frequent and/or high dosage use of aluminum salts is well known to have certain health risks associated with it. Aluminum uptake is known or suspected to be associated with a number of diseases. See, for example, the extensive discussions in the books ALUMINUM AND HEALTH; A CRITICAL REVIEW (Hillel and Gitelman, Ed.), Mark Decker, Publisher, 1989 and ALUMINUM IN RENAL FAILURE, Mark E. de Broi and Jack W. Coburn, Klewer, Publisher, 1990. Given the possible toxicity of aluminum, the non- aluminum salt forms of the DG salt derivatives are preferable over the aluminum salts forms in some and perhaps all therapeutic applications. Several specific embodiments of the compositions of the present invention are contemplated to be useful for oral administration in the healing of stomach ulcers. In particular, the non-aluminum salt-containing forms and the polymeric solid forms of highly sulfated deoxycyclodextrin are believed to be especially advantageous because of the absence of aluminum and its side effects.

3. Preparation of DG Derivatives As noted above, the DG derivatives may be prepared using ordinary and customary synthetic organic techniques, including techniques which are particularly applicable to carbohydrate compounds. In addition, applicants contemplate that various published techniques can readily be modified, without undue experimentation, to provide the various DG derivatives of the present invention. In this connection, Zemek and coworkers

(Transglycosylic Reaction of Deoxysugars: Biosynthesis of

Deoxyanalogs of Starch, Eur. J. Biochem. , 89:291 (1978)) have demonstrated that with the use of plant α(l-4)-glucan phosphorylase, 2-deoxy-D-glucose can be incorporated readily into starch. Various forms of plant starch are convertable to β-cyclodextrins. See J. Szejtli et al., Arzneim. Forsch . /Drug Res . , 30:808 (1980). Gerloczy, A. et al. have utilized plant starch isolated from tobacco leaves to generate β-cyclodextrin (Gerloczy, A. et al., Arzim. Forsch. /Drug Res . , 35:1042 (1985)). Applicants contemplate that feeding 2-deoxyglucose to tobacco plants would result in incorporation of DG into tobacco plant starch.

Zemek et al. have also utilized yeast glycogen synthetaεe (UDPG-glycogen glucosyltransferase, EC 2.4.I.II) to incorporate 2-deoxy-D-glucose into glycogen. See Zemek et al. , Transglycosylic Reactions of Nucleotides of 2-Deoxysugars II. 2-Deoxyglucose Incorporation into Glycogen, Biochemica et Biophysics Acta, 252:432 (1971). These studies demonstrate the lack of necessity of the hydroxy at the C-2 position of the DG moiety for various glucosyltransferases. In addition, Zemek et al. have demonstrated that various hydrolases, for example, β- amylase, are capable of cleaving bonds between disaccharides of 2-deoxyglucose. This indicates that the release of 2-deoxy-D- glucose would occur also in vivo .

Applicants contemplate also that DG or multimers thereof may be converted to deoxycyclodextrins by using cyclodextrin-glucanosyltransferase. See Vetter et al. , Directed Enzymatic Synthesis of Linear and Branched Glucooligo- saccharides, Using Cyclodextrin-Glucanosyltransferase, Carbohydrate Research, 223:61 (1992). In addition, various bacteria or yeast may be capable of using 2-deoxyglucose as a substrate and could thus be used to incorporate DG into various complex carbohydrates. Similarly, a very simple series of experiments could be performed, without undue experimentation, whereby screening for naturally occurring mutant strains could be achieved. To accomplish this, bacteria or yeast or other similar simple organisms may be grown in glucose-deficient minimal media which has been supplemented with 2-deoxyglucose. Variants that had developed mutations capable of utilizing DG as an energy substrate would then be identified. Such organisms could be used, not only to prepare complex carbohydrates and/or cycloamyloses, for example, deoxycyclodextrins, but could be studied for the type of mutation which occurred and in which enzyme to enable its utilization. Further cloning of this enzyme could then facilitate mass production of the DG- containing carbohydrate. II. THE FORM OF THE COMPOSITIONS

In view of the disclosure contained herein, those skilled in the art will appreciate that the present wound healing compositions are capable of having a beneficial effect in a variety of applications. It is therefore contemplated that the compositions of this invention may take numerous and varied forms, depending upon the particular circumstance of each application. For example, compositions containing highly water- soluble metabolic inhibitors of glycolytic pathways and/or agents which cause increases in the concentration of intracellular sodium (collectively and individually referred to hereinafter as "active material(s) " or "active compound(s) ") may be administered to a mammalian host in a variety of forms and which are adapted to the chosen route of administration, including, for example, oral, parenterally, and/or via local delivery. The active material may be incorporated into a solid pill or may be in the form of a liquid dispersion or suspension. In general, therefore, the compositions of this invention may comprise one or more of the active materials and a suitable, non-toxic, physiologically acceptable carrier therefor. As the term is used herein, carrier refers broadly to materials which facilitate administration or use of the present compositions for wound healing. A variety of non-toxic physiologically acceptable carriers may be used in forming these compositions, and it is generally preferred that these compositions be of physiologic salinity.

As noted above, the present compositions may be adapted to the chosen route of administration to a mammalian host, including parenteral and oral administration. Parenteral administration includes administration by the following routes: intravenous, intramuscular, subcutaneous, intraoccular, intrasynovial, transepthelially, including transdermal, opthalmic, sublingual and buccal; topically, including opthalmic, dermal, occular, rectal and nasal adminstration via insufflation and aerosol and rectal systemic.

The present compositions may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shelled gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active material may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixers, suspensions, syrups, wafers and the like. Such compositions and preparations should contain at least 0.1% of active material. The percentage of the compositions and preparations may, of course, vary. The amount of active material in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as gum, tragacanth, acacia, cornstarch or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as cornstarch, potato starch, alginic acid and the like; and a lubricant, such as magnesium stearate. The vehicle for oral administration may also comprise a sweetening agent, such as sucrose, lactose or saccharine, and/or a flavoring agent, such as peppermint, oil of wintergreen or cherry flavoring. When the dosage uniform is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain active material, sucrose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active material may be incorporated into sustained release preparations and formulations.

The active material may also be administered parenterally or intraperiotoneally. Solutions of the active material as a neutral compound or as a pharmacologically acceptable salt, as noted hereinbefore, can be prepared in water which is optionally mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may also contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It may be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or disperison medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimersal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions of agent delaying absorption may be included, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active material in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for . the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques which yield a powder of the active ingredient, plus any additional desired ingredient from previously sterile-filtered solutions thereof.

The therapeutic compounds of this invention may be administered to a mammal alone or in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice. The physician will determine the dosage of the present therapeutic agents which will be most suitable for prophylaxis or treatment and will vary with the form of administration and the particular compound chosen, and also, it will vary with the particular patient under treatment.* The physician will generally wish to initiate treatment with small dosages and increase the dosage by small increments until the optimum effect under the circumstances is reached. The therapeutic dosage will generally be from 0.1 to 20 mg or from about 0.01 mg to about 50 mg/kg of body weight per day and higher although it may be administered in several different dosage units from once to several times a day. Higher dosages may be required for oral administration.

It may be desirable to administer a suitable antibiotic as prophylaxis during treatment in accordance with the present invention. Such antibiotics can be mixed with the active material and administered as a mixture. Alternatively, antibiotics can be administered alone and/or contemperaneously with the present compositions either by the same or a different route of administration.

For some applications involving wound healing in the broadest sense, it is desirable to have available a physically applicable or implantable predetermined solid form of material containing the therapeutically active material of the invention. Accordingly, it is contemplated that the compositions of this invention may be incorporated in solid forms such as rods, needles, or sheets. They may thus be introduced at or near the sites of tissue damage or sites of implantation, or applied externally as wound dressings, etc. In such embodiments, the compositions and compounds of the present invention are preferably combined with a solid carrier which itself is bio-acceptable, or the compositions comprise suitably shaped polymer 'or co-polymer of the present active material. For many applications, it is preferred that the compositions of the present invention are prepared in the form of an aqueous dispersion, suspension or paste, including pleuronic gels, which can be directly applied to the site of a wound. To prepare these compositions, an active material, for example, polymeric DG derivative, such as polyanionic 2-deoxycyclodextrin polymer, can be used as synthesized in solid form after suitable purification, dilution and addition of other components, if desirable, including a fluid carrier, such as saline water. This will be the case when the active material has . been synthesized such as to produce a particle form of precipitate, dispersion or suspension. After synthesis, the solid derivative may also be dried, milled, or modified to a desired particle size or solid form. The particle size can be optimized for the intended therapeutic use of the composition. In some preferred embodiments, the solid particles range in size from about 1 micron to about 600 microns, with from about 200-600 microns being even more preferred. Particles ranging from about 1 to about 30 microns offer the best dispersion of growth factor and fast reactivity. For a given weight quantity of particles delivered to the biological environment, a smaller particle size assures exposure of greater particle surface area allowing greater diffusion of proteinic active ingredients into or out of the administered solid. Particles ranging from about 30 to about 100 microns offer fair dispersion of growth factors, medium reactivity and a longer period of delivery of growth factor. Particles possessing a size in excess of 100 microns will have low reactivity, but provide the longest delivery time for growth factors. In certain preferred embodiments, these large particles (>100 micron) will be used to absorb, rather than deliver, growth factors in vivo .

In preferred embodiments, the carrier is an aqueous medium and the compositions are prepared in the form of an aqueous suspension of solid particulate active material. The amount of the active material preferably ranges from about 1 to 30% by weight of the composition, and even more preferably, from about 5 to about 15% by weight.

A. Biologically Active Protein

In certain embodiments, the compositions and compounds include and/or are combined with biologically active proteins. According to preferred embodiments, the biologically active protein exhibits a specific affinity for heparin, and, more specifically, is heparin-binding growth factor, i.e., a class of growth factors, many of which are mitogenic for endothelial cells. An example of such a growth factor is basic fibroblast growth factor. Generally, it will be the heparin- binding growth factor proteins, commonly referred to as HBGF's, which may be combined with the compounds of the present invention. Some of these are listed in Table I.

To determine whether a protein is suitable for the therapeutic compositions of the present invention, one can determine whether it has a specific affinity for heparin. An HBGF protein is one that remains substantially bound to heparin (e.g., using a derivatized column), even in the presence of an aqueous medium having a salt concentration of substantially greater than about 0.6 molar strength of NaCl. Generally, the term substantially bound refers to at least about 80% of such bound protein remaining attached under such conditions.

TABLE I PROTEIN FACTORS

Symbol Name Reference IL-1 (Interleukin-1) Henderson & Pettipher,. 1988 Biochem. Pharmacol. 37:4171; End et al., 1988, BBRC 136:1007 Hopkins et al., 1988, Clin. Exp Immunol. 72:422

IL-2 (Interleukin-2) Weil-Hillman et al., 1988, J Biol. Response Mod. 7:424; Geml et al., 1988, Cancer Res. 48:586

IFNα (Interferon α) Pitha et al., 1988, J. Immunol 141:3611; Mangini et al., 1988 Blood 72:1553 IFNγ (Interferon γ) Blanchard &^■ Djeu. 1988, J Immunol. 141:4067; Cleveland e al., 1988, J. Immunol. 141:3823

TNFα (Tumor necrosis Plate et al., 1988, Ann. NY Acad factor oc) Sci. 532:149; Hopkins & Meager 1988, Clin. Exp. Immunol. 73:88 Granger et al., 1988, J. Biol Response Med. 7:488

EGF ( Epidermal Carpenter and Cohen, 1979, A growth factor) Rev. Biochem. 48:193-216 FGF (Fibroblast Folkman and Klagsbrun, 1987 growth factor, Science 235:442-447 ac idi c and basic)

IGF-1 ( Insulin-like Blundell and Humbel, 1980, Natur growth factor- 287:781-787; Schoenle et al. 1) 1982, Nature 296:252-255

IGF-2 ( Insulin-like Blundell and Humbel growth factor- 2) PDGF ( Platelet- Ross et al., 1986, Cell 46:155 derived growth 169; Richardson et al. , 1988 factor) Cell 53:309-319

TGF-α Trans forming Derynck, 1988, Cell 54:593-595 growth factor- ex)

TGF-β (Transforming Cheifetz et al, 1987, Cel growth factor- 48:409-416 β⁾ It is known that the complexing capabilities of heparin toward growth factor proteins are paralleled by its complexing capabilities for certain cationic dye structures, such as azure-A, methylene blue and others. Other glycosaminoglycan saccharides are known not to function similarly. Thus, such dyes have been used for many years in histology as specific stains for the presence of heparin-like polysaccharides. Metachromasia, which corresponds to the spectral shift resulting from heparin binding on the dye, has been used to identify active heparin-like compounds having the capability of modulating angiogenesis. Such dye complexing of the active protein also is similarly resistant to salt concentration as is the complexing to heparin.

In relation to this invention, it is contemplated that such dye complexing, serving as a model for proteinic growth factor complexing, can usefully serve as an indicator for the desired activity of the compositions of the invention. Thus, the proteinic growth factor complexing ability of the precipitates, polymers, or co-polymers of the compositions of the present invention may be determined using dye complexing assays.

In the course of practicing heparin binding separation or chromatography for the separation of proteinic factors, it has been customary and accepted that desorption of the complexed growth factor requires the added step and involvement of contacting with a very strong salt solution. It is contemplated that release of protein complexed to the saccharide herein specified does not require the added step of contacting with high concentration electrolyte. While such operation would be needed for an immediate large scale desorption process, as may be desired for a separation technology, the relatively very low external concentration of desorbed factor may be maintained by an equilibrium process involving the complexed phase on the solid and the low biologically required solute phase in the physiological surrounding liquid. There is thus a recognition that applicants' compositions may be used as delivery agents for biomedical purposes. Generally, to prepare the growth factor- containing compositions, one or more of the present active materials, and preferably, DG derivatives, including DCs and/or DC derivatives, is contacted with a solution containing a growth factor or combination of growth factors. The active material(s) are thereafter separated from the contact fluid, resulting in an enrichment of the growth factor on the active material(s), and a corresponding removal of the growth factor from the fluid. The contacting solution may contain a single, preseparated, preconcentrated growth factor purified from tissue or bodily fluids or growth factor obtained from recombinant DNA methods. Alternatively, the contact solution may comprise viable tissue or organ materials (hereinafter organic sources) which contain a variety of growth factors. When combined with tissue or organ material containing growth factors, the compounds of the present invention may act as extractants of these growth factors. When organic sources are used as the source for growth factors, it is preferred that the organic source used for the contacting solution have a volume greater than about 10 to about 100 times the volume of the tissue to be treated by the combined derivative and growth factor(s) .

After contacting the partially or wholly complexed active material, the solid phase can be easily separated from the fluid phase that was the source of protein to be complexed. It is preferable that the source of growth factor contains the protein as a dissolved component in the absence of solids other than the active materials to be complexed. However, some solids in the growth factor source solution may not necessarily be undesirable or disturbing contaminants. Separation of solids, such as tissue or organ fragments from the saccharides, may be accomplished by sedimentation, suitable filtering, centrifugation or other mechanical or other methods. III. METHODS FOR THERAPEUTIC REGULATIONOF WOUND HEALING

One aspect of the present invention relates to methods for the therapeutic regulation, and preferably in vivo regulation, of wound healing, and particularly to in vivo regulation of the concentration and diffusion of protein factors. Such methods generally comprise therapeutic biodelivery of the present compositions and compounds to the wound site. The low solubility, i.e., the solid immobilized state which is associated with certain of the present materials, allows various of the compositions and compounds to be administered directly to the site of a wound and for the active ingredients to remain at the site of application for an extended period of time. In other embodiments, and in particular, methods and compositions involving lower multimers, the present compositions and compounds are extremely water-soluble. These materials may be orally administered and will travel through the bloodstream to the wound site.

Vascular cell proliferation and abnormal accumulation of extracellular matrix in the vessel wall are common pathological features observed in arteriosclerosis, hypertension and diabetes. Such conditions are also observed following vascular injuries, such as angioplasty. Intimal hyperplasia is thought to be mediated in part by a variety of growth factors, such as platelet derived growth factor (PDGF) , which act through receptors to stimulate vascular smooth muscle cell proliferation and migration from the media into the intima. Thus, applicants have discovered methods for regulating migration and proliferation of the smooth muscle cells, thereby affecting the degree of intimal thickening noted after vascular injury. Applicants have found that the present active materials can inhibit human vascular smooth muscle cell proliferation and migration in vi tro when stimulated with fetal calf serum, which contains potent growth factor activity. It is seen, therefore, that the presence or absence of growth factors at the site or vicinity of a wound has an impact upon the healing process. Applicants have found that certain of the present compositions and compounds can be used to beneficially regulate and control biologically active proteins, such as growth factor, at the site of a wound. For example, when the present compounds and compositions are combined with growth factors prior to biodelivery as described herein, the compositions and compounds slowly release this growth factor into the immediate vicinity of the wound, thereby accelerating the wound healing process. It is contemplated that all growth factors known to accelerate or facilitate wound healing are usable in the present compositions and methods. Growth factors suitable for this acceleration of wound healing include those listed in Table I, as well as brain endothelial cell growth factor and retina-derived growth factor. As described above, heparin binding growth factors can be used to effect the repair of both soft and hard tissue. The potential uses for interferons, interleukins, and tissue growth factors are well known in the art.

The invention also relates to methods for the therapeutic administration of active materials with a protein factor, wherein the active material is combined with or comprises a portion of a biocompatible porous solid. The phrase, "biocompatible porous solid", as used herein, means a solid which may be applied or administered to a mammal without provoking a substantial inflammatory response or other substantial adverse effect. Such biocompatible porous solids include membranes, such as collagen-based polymeric membranes, amniotic membranes, and omentum membranes (reviewed in Cobb, 1988, Eur. J. Clin. Investig. 18:321-326). The active materials may be immobilized on such membranes in a preferred embodiment by contacting the derivatized saccharide with electrostatic binding partners on the membrane. Biocompatible porous solids may also include polymers of ethylene vinyl acetate, methylcellulose, silicone rubber, polyurethane rubber, polyvinyl chloride, polymethylacrylate, polyhydroxyethylacrylate, polyethylene terephtha1ate , polypropylene, polytetrafluoroethylene, polyethylene, polyfluoroethylene, propylene, cellulose acetate, cellulose and polyvinyl alcohol (reviewed in Hoffman, Synthetic Polymeric Biomaterials in Polymeric Materials and Artificial Organs, ACS Symposium Series #256, (G. Gebelein, ed.) 1988). In preferred embodiments involving polymeric active materials, the starting materials are co-polymerized with monomers of the biocompatible polymer material of the final product composition, so as to create a porous co-polymer. This co-polymer is subsequently reacted chemically to provide the active material with the preferred anionic substituents. For example, 2-deoxycyclodextrins can be coupled with reactive groups, such as amine, amide, carboxylate end groups, etc. , contained in the biocompatible polymer and then subsequently derivatized with ionic substituents. More preferably, the DG derivative, such as a 2-deoxycyclodextrin, is introduced as a co-reagent in a monomer formulation to be polymerized to a solid polymer or co-polymer, and the product is contacted subsequently with suitable agents to derivatize the DG derivative to add anionic substituents to the degree taught by this invention. Particularly advantageous for such process and products are those methods that will produce a polymer or co- polymer example of a flat polymer product of polyamide polymer, manufactured by 3M Corporation, and used as a bio-compatible patch or dressing on wounds. This biocompatible patch or dressing is designed to physically protect a wound from invasion of pathogens, and yet to have sufficient porosity to allow passage of moisture, air, etc. Applicants' invention contemplates, for example, the coupling of active polyanionic active materials with a carrier comprising such polymer, or, the coupling of the active anionic DG and a proteinic factor together with a polymeric carrier. Such combinations are designed expressly for applications of deliberate promotion or inhibition of cellular growth processes. The HBGFs bind to the immobilized, derivatized DG-basedmolecules, either incorporated into or already present in biomembranes. Biological membranes such as omentum and amnion are well known in the art as wound dressings. Collagen based synthetic biomembranes are being used in the treatment of burns. The presence of derivatized active materials of the present invention in natural membranes, such as amnion, and the ability of these compounds to bind collagen which is used as a base for synthetic membranes will allow such biomembranes, when combined with the compositions of the present invention, to be used as novel delivery vehicles for HBGFs. A. Restenosis

As noted above, arteriosclerosis is a disorder involving thickening and hardening of the wall portions of the larger arteries of mammals, and is largely responsible for coronary artery disease, aortic aneurisms and arterial diseases of the lower extremities. Arteriosclerosis also plays a major role in cerebral vascular disease.

Although applicants do not wish to be bound by any theory or theories for the basis of restenosis, it is believed that restenosis is due in part to the presence of growth factors produced by injured endothelium which activates excessive proliferation of the smooth muscle cells which are exposed after endothelial injury. Accordingly, applicants have found that DG and the present DG derivatives, when substantially free of growth factors prior to biodelivery, are extremely effective for preventing or at least substantially reducing intimal thickening following balloon angioplasty. By virtue of their affinity for growth factors, such compositions can provide an in vivo absorption or reduction of the local concentration and/or diffusion of such growth factors. That is, such wound site growth factors, whether they are produced by the cells at the wound site or are otherwise in the bloodstream, can be taken up by the present compounds, thereby reducing the restenoic effect of such materials on the wounded tissue. According to the present methods, mammals, including humans, which have arterial regions subject to angioplasty, are treated by administering to the mammal a compound of the present invention in an amount effective to inhibit arterial smooth muscle cell proliferation. It is contemplated that the degree of restenosis inhibition may vary within the scope hereof, depending upon such factors as the patient being treated and the extent of arterial injury during angioplasty. It is generally preferred, however, that the DG or DG derivative be administered in an amount effective to cause a substantial reduction in restenosis.

Thus, the present invention contemplates a method of inhibiting restenosis in a patient which comprises administering to the patient an active material, in an amount effective to inhibit formation of a restenotic lesion in a patient who has undergone angioplasty. It is contemplated that the compound may be administered before, during and/or after angioplasty treatment of the stenosed artery. It is generally preferred that the administration comprise administering the compound locally at the wound site. In preferred embodiments, local administration comprises infusing the saccharide derivative directly into the injured tissue. In the case of restenosis, such step preferably comprises infusing the compound directly into the arterial wall at the site of the angioplasty.

Applicants contemplate that particularly beneficial antirestenoic results are obtained for embodiments in which the step of administering the compounds also comprises the step of dilating the vessel lumen to effect angioplasty. For example, applicants contemplate that a preferred administration step comprises infusing an aqueous suspension or dispersion of compound directly into the arterial wall at the site of balloon angioplasty. This is preferably accomplished using a modified infusion balloon catheter having a plurality of holes in the wall of the balloon portion of the catheter. These holes are configured and sized to allow the balloon to be both inflated and to leak the inflation solution through the wall of the balloon. According to preferred embodiments, the balloon is inflated under relatively low pressure conditions, such as 2 - 3 atmospheres. Examples of porous balloon catheters which may be used to apply the compositions of the present invention are made by U.S.C.I.-Bard and Schneider. Balloons of this type are referred to as Wolinsky balloons or "sweating balloons." It is anticipated that a variety of infusion angioplasty balloon catheters may be used for application of the compositions of the present invention and that one skilled in the art would be readily able to determine which types of balloon infusion catheters would be appropriate. Other techniques which involve the local administration of the compounds of the present invention utilize bioabsorbable intravascular stents and pleuronic gels. The compounds of the present invention, for example, the 2-deoxycyclodextrin polymer derivatives, may be incorporated into a bioabsorable stent or gel which is placed at or near the site of tissue damage.

It will be appreciated by those skilled in the art that the particular characteristics and properties of the suspension containing the present compounds may vary widely depending upon numerous factors not necessarily related to the present invention. However, the administration step preferably comprises infusing an aqueous solution, suspension or dispersion of particles of active material, for example, a suspension of sulfated β-2-deoxycyclodextrin polymer particles, ranging in size from about 1 to 600 microns, directly into the arterial wall at the site of balloon angioplasty. Applicants believe that such particles, and particularly the polymeric particles which are instilled into the arterial wall, will remain present at the site of application for several days, in sufficient quantity to result in an inhibition of restenosis.

The aqueous suspension comprises an aqueous carrier of physiological salinity and a compound of the present invention. The active compound is preferably present in an amount ranging from about 1 to about 30% by weight, and even more preferably, from about 5 to about 15% by weight of the composition. In preferred embodiments, the present compounds are applied at about the time of angioplasty.

In some instances, it may be desirable to prevent restenosis but allow angiogenesis. To meet these requirements, it is preferred to use a dispersion of an Al, Mg, Ca, and/or Ba salt of the present active materials.

B. Inhibition of Intimal Thickening of Vein Grafts Venous segments are frequently harvested at the time of surgery and used as bypass grafts to treat vascular occlusive disorders. Specifically, they have been used in the coronary, renal, femoral and popliteal arterial circulations, by way of example. One major limitation of this form of therapy is that intimal thickening occurs which compromises the luminal cross-sectional area and results in reduced flow. This frequently, but not exclusively, occurs at the anastomosis. Applicants contemplate that the placement of the compounds of the present invention, and preferably, compounds in polymeric particulate form, in the perivascular space at the time of surgery, will substantially limit the ingrowth of smooth muscle cells into the inti a and will improve the long term success of these grafts. ' Applicants contemplate also that the bypass grafts themselves may be treated with the active materials prior to anastomotic implantation. Preferably, the graft is pre¬ treated with a solution comprising soluble active materials for example, DG. This pre-treatment may involve wetting and/or soaking the graft with a solution of active material before, during and/or after effecting the anasmototic implantation.

C. Angiogenesis

Angiogenesis is the formation of new blood vessels. Angiogenic stimuli cause the elongation and proliferation of endothelial cells and the generation of new blood vessels. A number of the HBGFs are known to promote angiogenesis. The new blood vessels produced by angiogenesis result in neovascularization of tissue.

There are a variety of diseases associated with deficient blood supply to tissue and organs. A deficiency of this kind, known as ischaemia, may be due to the functional constriction or actual obstruction of a blood vessel. These diseases can be grouped into cardiac, cerebral and peripheral ischemic diseases. Cardiac ischaemia may result in chronic angina or acute myocardial infarction. Cerebral ischaemia may result in a stroke. Peripheral ischaemia may result in a number of diseases, including arterial embolism and gangrene. In severe cases of peripheral ischaemia, necrosis of the tissues supplied by the occluded blood vessels necessitates amputation. To overcome ischaemia, an alternative blood supply to the affected tissue must be established.

According to preferred embodiments, angiogenesis is promoted by first contacting the active materials of the present invention with growth factor(s), and then administering the composition locally to the location of the ischemic tissue, by hypodermic injection for example, to promote angiogenesis and the formation of collateral blood vessels. As the term is used herein, collateral blood vessels are blood vessels which are absent under normal physiological conditions, but which develop in response to appropriate stimuli, such as the presence of HBGFs. It is contemplated that administration of compositions which include the present compounds and growth factor will result in the formation of collateral blood vessels • and revascularization of ischemic tissue.

In preferred embodiments, angiogenesis is promoted by methods in which the present compounds comprise a highly anionic DG and/or DG derivative or a salt form of same. It is preferred that the present compounds be combined with basic fibroblast growth factor at a basic fibroblast growth factor weight ratio of from about 10:1 to 100:1.

D. Tissue and Organ Grafts or Transplants

As described above, HBGFs are known to stimulate neovascularization and endothelial cell growth. In transplan¬ tation, the graft represents a wound, and success of the grafting procedure depends critically on the rapidity of establishing an adequate blood supply to the grafted or transplanted tissue. Thus, we envision the application of the compositions of the present invention, combined with growth factor(s) , at the site of the graft to promote the establishment of an adequate blood supply to the grafted or transplanted tissue. The growth factor-containing compositions may be coated on the surfaces to be joined, sprayed on the surfaces, or applied in the form of an aqueous suspension with or without viscosity enhancers, such as glycerol. In addition, the organ or tissue to be grafted or transplanted may be presoaked in a treating solution containing the compositions of the present invention, prior to transplantation. The compositions of the present invention may also be injected into the transplant site or surface of both items to be joined.

In a preferred method for preparing the compositions used in treating grafted or transplanted tissue and organs, the compounds of the present invention are precontacted with growth factor-containing organic sources (e.g., tissue or organ debris, ground matter, or liquid extract) so as to extract the growth factors present in these sources. In highly preferred methods, the organic source used for contact is about 10 to about 100 times greater in volume than the transplanted or grafted tissue to be treated by the composition. A more direct and often more economic method will involve contacting the compounds of the present invention with growth factor substances created by recombinant biochemical and biotechnological procedures. In this manner, specific growth factor proteins are more readily chosen for a contemplated therapeutic application.

Abnormal cellular proliferation and/or migration processes are also associated with tissue and organ grafts or transplants. For example, chronic transplant atherosclerosis generally occurs after organ transplants, including heart transplants. As in restenosis, chronic transplant atherosclerosis involves intimal thickening and is the major reason for organ transplant failure, including heart transplants. Applicants contemplate that the present compositions may be administered before, during and/or after organ/tissue transplants to inhibit the proliferation and migration processes associated with chronic transplant atherosclerosis. Preferably, the compositions are administered orally, and thus involve water soluble active materials, for example, DG.

E. Bone Grafting and Transplantation The response of bone to injuries, such as fractures, infection and interruption of blood supply, is relatively limited. In order for the damaged bone tissue to heal, dead bone must be resorbed and new bone must be formed, a process carried out in association with new blood vessels growing into the involved area. HBGFs can induce neovascularization and the proliferation of bone forming cells. It is therefore contemplated to use the present compounds in combination with growth factor for the purposes of aiding the healing of bone fractures, the joining of implanted and host bone, and the mineralization of bone (where such is intended) .

In preferred embodiments, the present compounds are combined with growth factors and powdered bone substance and/or finely dispersed demineralized bone matter to form a paste. Suitable methods for preparation of such a paste are presented in Repair of Major Cranio-Orbi tal Defects wi th an Elastomer Coated Mesh and Autogenous Bone Paste, Mutaz B. Habal et al., 61:3, Plastic and Reconstructive Surgery, 394, 396 (1978) . The bone tissue used to produce the paste may be obtained from iliac crest or calvarium. It is preferred to use autogenous bone for implant purposes and to use partially demineralized bone over fully demineralized bone powder. Demineralized bone powder obtained from allogenic and xenogeneic sources may be used in preparing the bone powder. To make a soft paste, absorbable cellulose cotton or similar material may be used. Although applicants do not wish to be bound by any theory or theories, it is thought that the bone paste produced by these methods functions as an induction matrix from which new bone will form after being invaded with a network of blood vessels. The paste is applied to the surfaces of bone to be joined in implant procedures or used to fill fractures of contour bone to be repaired.

F. Skin Ulcer Healing

One debilitating disorder affecting millions of people including, but not limited to the aged, paraplegics, trauma victims, and diabetics, are cutaneous nonhealing skin ulcers or decubiti. In many cases, inadequate blood supply to the damaged tissue prevents the delivery of adequate nutrients for healing. It is anticipated that the application of polymeric beads of the present compounds, preabsorbed with combinations of compounds, such as epidermal growth factor and basic fibroblast growth factor, to the ulcer directly, will lead to increased angiogenesis, improved blood supply, increased keratinocyte ingrowth, and faster ulcer closure and healing.

G. Dermatological Applications The control of blood vessel growth is an important aspect of normal and of pathological states encountered in dermatology. In particular, the abnormal growth of cellular materials and vessels accompanies several pathological states, psoriasis being one prominent example. In many cases, excesses of growth stimulating protein factors are involved. Abnormalities of this type are often associated with imbalances in proteinic growth factors. For example, in the case of patients with cutaneous mastocytosis, extracts from involved skin had 15-fold higher levels of chymotryptic activity than extracts of uninvolved skin or from control samples of patients without such deficiency. (See Human Skin Chymotryptic Protease, N. M. Schechter, J. E. Fraki, J. C Geesin, G. S. Lazarus, J. Biol. Chem., 258, 2973-2978, 1983. The Chymase Involved Is a Heparin Binding Factor (See S. Saya a, R. V. Iozzo, G. S. Lazarus, N. M. Schechter, Human Skin Chymotrypsin-like Proteinase Chymase, J. Biol. Chem. 262, 6808-6815, 1987. It appears that the chymotrypsin like proteases can degrade the epidermal junction and can result in epidermal-dermal separation (See Sayama et al. above) .

Another example of a growth promoting factor involved in dermal abnormalcies is epidermal plasminogen activator, which is elevated in a variety of dermal pathologies (See Epidermal Plasminogen Activator is Abnormal in Cutaneous Lesions ' , P. J. Jensen et al., J. Invest. Dermat. 90-777-782, 1988) . Certain embodiments of this invention, namely highly sulfated solid dispersions or other physical variants of highly sulfated polymeric compounds, are particularly amenable to dermal therapy in those cases where excess growth of cellular components is involved. In such cases, the agents of the present invention can be introduced at or near the tissue involved. This may be accomplished by cutaneous or sub¬ cutaneous injection of fine particle dispersion of the agent, or the implantation of solid polymer shapes suitably shaped for effective contact, or the agent may be comprised in material such as patches, or other suitable forms of externally applied materials containing agents of the invention.

It will be understood that depending on the pathology and disease condition, the application of the agents of this invention without pre-contacting with proteinic growth factor is contemplated. This will be the case in conditions as exemplified above, where it is intended to reduce any growth promoting factor or factors. In other cases of dermal damage or disease, and in certain phases of treatment, it may be desirable to use the combined proteinic factors. This would be the case in connection with healing processes where angiogenesis, that is the establishment of new and added blood supplies are desired.

EXAMPLES

The following examples are provided to illustrate this invention. However, they are not to be construed as necessarily limiting the scope of the invention, which scope is determined by the appended claims. All amounts and proportions shown are by weight unless explicitly stated to be otherwise. EXAMPLE 1

This example illustrates the inhibition of human smooth muscle cell migration in tissue cultures. Fetal calf serum was added to DMEM/Ham's F-12 mixture, commercially available from Gibco, Grand Island, New York. 2- Deoxyglucose was dissolved in either Media 199 (Gibco) or DMEM/Ham's F-12 mixture. The cells were cultured using standard techniques and media containing 2-deoxyglucose was placed in the chamber slides and microtiter plates, respectively. Inhibition and proliferation of the human smooth muscle cells was then measured and is graphically illustrated in Figures 2 and 3, respectively.

This example demonstrates that, as the concentration of 2-deoxyglucose in human smooth muscle cell cultures is increased, the rate of migration and proliferation decreases substantially.

EXAMPLE 2

PREPARATION OF GROWTH FACTORS Human recombinant basic fibroblast growth factor

(bFGF) was provided by Takeda Chemical Industries, Ltd. It was purified from E. coli as previously described (Kurokawa et al.,

1987, FEBS. Letters 213:189-194 and Iwane et al. , 1987, Biochem.

Biophys. Res. Commun. 146:470-477). Rat chondrosarcoma-derived growth factor (ChDGF) was isolated from the transplantable tumor as previously described (Shing et al., 1984, Science 223:1296-1298). About one hundred ml of the crude extract prepared by collagenase digestion of the tumor was diluted (1:1) with about 0.6 M NaCl in about 10 mM

Tris, pH 7 and loaded directed onto a heparin-Sepharose® column

(1.5 x 9 cm) pre-equilibrated with the same buffer. The column was rinsed with about 100 ml of about 0.6 M NaCl in about.10 mM

Tris, pH 7. ChDGF was subsequently eluted with about 18 ml of about 2 M NaCl in about 10 mM Tris, pH7.

EXAMPLE 3

BETA-DEOXYCYCLODEXTRIN AFFINITY CHROMATOGRAPHY OF FGF The insoluble sulfated beta-2-deoxycyclodextrin polymer (about 0.5 ml bed volume) is incubated with about 0.5 ml of about 0.1 M NaCl, about 10 mM Tris, about pH 7 containing about 1,000 units of human recombinant bFGF at about 4°C for about 1 hour with mixing. The polymer is then rinsed stepwise with about 2 ml each of about 0.1, 0.6, and 2 M NaCl in about 10 mM Tris, pH 7. All fractions eluted from the polymer are assayed for growth factor activity. EXAMPLE 4 GROWTH FACTOR ASSAY Growth factor activity is assessed by measuring the incorporation of [³H]thy idine into the DNA of quiescent, confluent monolayers of BALB/c mouse 3T3 cells in 96-well plates. One unit of activity is defined as the amount of growth factor required to stimulate half-maximal DNA synthesis in 3T3 cells (about 10,000 cells/0.25 ml of growth medium/well) . For determination of specific activities, protein concentrations of the crude extract and the active fraction eluted from heparin- Sepharose column are determined by the method of Lowry et al. (1952, J. Biol. Chem. 193:265-275). Protein concentrations of the pure growth factor are estimated by comparing the intensities of silver-stained polypeptide bands of SDS- polyacrylamide gel to those of the molecular weight markers.

EXAMPLE 5

AFFINITY OF FIBROBLAST GROWTH FACTOR FOR BETA-DEOXYCYCLODEXTRIN TETRADECASULFATE POLYMER

Human recombinant bFGF (about 1000 units) is incubated with sulfated beta-cyclodextrin polymer. The polymer is eluted stepwise with about 0.1 M, 0.6 M, and 2 M NaCl. The contemplated results are shown in Figure 4. While most of the growth factor activity is bound to the polymer at about 0.6 M NaCl, about 230 units of the activity is recovered when eluted with about 2 M NaCl. These results indicate that basic fibroblast growth factor has a very strong affinity for β-deoxycyclodextrin tetradecasulfate and is at least comparable to that of FGF for heparin. The activity peak is analyzed by SDS polyacrylamide gel electrophoresis followed by a silver stain. Lane 2 in Figure 5 shows the contemplated polypeptide band of basic fibroblast growth factor. The affinities of heparin and β-deoxycyclodextrin tetradecasulfate for chondrosarcoma derived growth factor is also tested. Chondrosarcoma extracts which contained about 500 units of growth factor activity are incubated individually with heparin-Sepharose® and β-deoxycyclodextrin tetradecasulfate polymer. The beads are eluted stepwise with about 0.1 M, 0.6 M, and about 2 M NaCl. The contemplated results are shown in Figure 6. Approximately 32% and 68% of the total activity is recovered at 2 M NaCl with heparin Sepharose® and β- deoxycyclodextrin tetradecasulfate polymer, respectively. EXAMPLE 6

EFFECT OF 2-DEOXYGLUCOSE ON t-PA DEGRADATION IN HUMAN UMBILICAL VEIN SMOOTH MUSCLE CELLS

The effects of DG on the degradation of t-PA by human umbilical vein smooth muscle cells were examined. t-PA degradation in smooth muscle cells and various other cell types is mediated by a receptor known as the LDL receptor related protein (LRP) , which is also known as the α-2 macroglobulin receptor. See Bu, G. et al., Proc. Natl . Acad. Sci . USA, Vol. 89, pp. 7427-7431 (1992); Orth, K. et al. , Proc . Natl . Acad. Sci . USA, Vol. 89, pp. 7422-7426 (1992); and Grobmyer, S.R. et al., J. Biol . Chem. , (1993) (in press). LRP has been shown to mediate the internalization of various ligands, including urokinase (u-PA) , activated α-2 macroglobulin, chylomicron remnants, and apo-E enriched β-VLDL, each of which has unique effects on cells. Additionally, LRP has been hypothesized to play a role in cell migration and invasion by its ability to clear inactivated plasminogen activator complexes from the cell surface. See Herz et al., Cell , Vol. 71, pp. 411-421 (1992). LRP belongs to the family of receptors that includes the LDL receptor and which has been hypothesized to function via a similar mechanism.

Experiments were conducted wherein human umbilical vein smooth muscle cells were plated onto a 96 well microtiter plate at a density of about 25,000 cells per well for approximately 16 hours. Standard smooth muscle culture media (23 millimolar in glucose) was removed and replaced with standard media to which was added different concentrations of DG. After 48 hours, the standard media was removed and replaced by RPMI which is a commonly used tissue culture media and which contains 1-125 radiolabelled t-PA (0.2 μg/ml) in the presence and absence of a 50-fold molar excess of unlabelled t-PA. After two hours at 37°C, the RPMI media was removed and the amount of t-PA which degraded was measured as trichloroacetic acid soluble counts. As shown in Figure 7, the amount of t-PA which specifically degraded, and which is defined as the amount of radiolabelled t-PA degraded in the absence of excess unlabelled t-PA, minus the amount of t-PA degraded in the presence of excess unlabelled t-PA, was reduced 70% by 10 millimolar DG. As shown in Figure 8, the reduction by DG of the amount of t-PA specifically degraded is dose responsive and a maximal inhibition is obtained at 5 millimolar. To affirm that the differences observed in t-PA degradation were due primarily to the anti-proliferative effects of DG on smooth muscle cells, the cells were stained with naphthol blue-black stain which stains cell protein and is indicative of differences in cell number. Protein staining in DG (10 mM) treated cells was about 27% less, whereas t-PA degradation in the same cells was reduced 67%. This indicates that the decreased t-PA degradation was not due primarily to a decrease in the number of cells.

Applicants contemplate that DG inhibits the ability of LRP to internalize and/or induce production or expression of ligands which can interfere with the interaction of t-PA with LRP. Applicants conclude that inhibition of recycling receptor mediated internalization of t-PA by DG is important in that (1) clearance of plasminogen activators from the cell surface may play a role in cell migration or invasion, and when this clearance process is inhibited, cell migration may be reduced; (2) numerous growth factors and cytokines are known to be internalized by LRP when they are complexed to α-2 macroglobulin, and reduction of LRP function via DG may be a way of inhibiting the proliferative and/or migratory effects of growth factors or cytokines taken into the cell in this manner; (3) inhibition of uptake of lipids by similarly functioning receptors, for example, the LDL receptor, by DG may lead to prevention of pathological foam-cell formation, smooth muscle cells and macrophages that are lipid filled, which are cells characteristic of lesions of native atherosclerosis; and (4) since LRP internalizes t-PA and u-PA which normally function to break down fibrin clots, inhibition of LRP via DG may prevent local internalization of t-PA and u-PA and thus exert a local anti-thrombotic effect.

Claims (48)

CLAIMSWhat is claimed is:

1. A composition for affecting the growth of cells of living tissue in mammals comprising, in combination with a physiologically acceptable carrier, a compound selected from the group consisting of a metabolic inhibitor of a glycolytic pathway of said cells, an agent which causes an increase in the concentration of intracellular sodium of said cells, and mixtures thereof.

2. The composition of claim 1 wherein said compound comprises saccharide.

3. The composition of claim 2 wherein said saccharide is selected from the group consisting of 2- deoxyglucose, 2-deoxyglucose derivative, cyclodextrin derivative, and a mixture of two or more of these.

4. The composition of claim 3 wherein said saccharide comprises 2-deoxyglucose.

5. The composition of claim 4 further comprising cyclodextrin derivative.

6. The composition of claim 5 wherein said 2- deoxyglucose and cyclodextrin derivative are chemically linked together via a covalent bond.

7. The composition of claim 6 wherein said cyclodextrin derivative comprises sulfated cyclodextrin.

8. The composition of claim 7 wherein said sulfated cyclodextrin comprises β-cyclodextrin tetradecasulfate.

9. The composition of claim 3 wherein said saccharide comprises 2-deoxyglucose derivative.

10. The composition of claim 9 wherein said derivative comprises 2-deoxycyclodextrin derivative.

11. The composition of claim 10 wherein said derivative comprises a compound of the formula:

wherein at least two of said R-_ and R₂ groups, per monomeric unit, are hydroxy or an anionic substituent selected from the group consisting of sulfate, phosphate, sulfonate and nitrate, and the other of said R_x and R₂ groups, when present, is a substituent selected from the group consisting of H, alkyl, aryl, ester, ether, thioester, thioe^'ther and -COOH; and n is an integer from about 6 to about 12.

12. The composition of claim 10 wherein said 2- deoxycyclodextrin derivative is comprised of one or more 2- deoxycyclodextrin monomers having on average at least about 10 substituents per monomer, said substituents being hydroxy or anionic substituents selected from the group consisting of sulfate, phosphate, sulfonate and nitrate.

13. The composition of claim 12 wherein said monomer has on average from about 10 to about 16 substituents per monomer, said substituents being hydroxy or anionic substituents selected from the group consisting of sulfate, phosphate, sulfonate and nitrate.

14. The composition of claim 13 wherein R₁ and R₂ are independently hydroxy or sulfonate, and n is an integer from about 6 to about 8.

15. The composition of claim 14 wherein _λ and R₂ are independently sulfonate and n is about 7.

16. The composition of claim 9 further comprising 2-deoxyglucose.

17. The composition of claim 10 wherein said 2- deoxycyclodextrin derivative comprises 2-deoxycyclodextrin polymer.

18. The composition of claim 17 wherein said polymer is solid particulate dispersed or suspended in said carrier.

19. The composition of claim 11 wherein said derivative comprises α-, β- or γ-deoxycyclodextrin.

20. The composition of claim 11 wherein said derivative comprises a salt of polyanionic α-, β- or γ-2- deoxycyclodextrin.

21. The composition of Claim 20 wherein the cationic constituents of said salt are selected from the group consisting of Mg, Al, Ca, Ce, Ba and combinations of two or more of these.

22. The composition of claim 9 wherein said derivative comprises a compound of the formula

wherein at least one of said R groups is an anionic substituent selected from the group consisting of sulfate, phosphate, sulfonate and nitrate, and the other of said R groups, when present, is a substituent selected from the group consisting of H, alkyl, aryl, hydroxy, ester, ether, thioester, thioether and -COOH.

23. The composition of claim 9 wherein said derivative comprises multimeric 2-deoxyglucose.

24. The composition of claim 23 wherein said derivative comprises oligomeric 2-deoxyglucose.

25. The composition of claim 1 which is substantially soluble in water at body temperature.

26. The composition of claim 1 which is substantially insoluble in water at body temperature.

27. The composition of claim 1 wherein at least a portion thereof is solid particulate dispersed or suspended in said carrier.

28. The composition of claim 1 further comprising growth factor.

29. A method for inhibiting the pathological growt of smooth muscle cells in a tissue of a mammal comprising th metabolic inhibition of a glycolytic pathway of said cells o the increase in the concentration of intracellular sodium o said cells.

30. The method of claim 29 comprising administerin to said mammal, in an amount effective to inhibit said pathwa or increase said intracellular sodium concentration of sai cells and thereby inhibit said pathological growth, saccharide.

31. The method of claim 30 comprising administerin to said mammal a saccharide selected from the group consistin of 2-deoxyglucose, 2-deoxyglucose derivative, cyclodextri derivative, and mixtures thereof.

32. The method of claim 31 comprising inhibitin restenosis.

33. The method of claim 29 comprising administerin to said mammal 2-deoxyglucose.

34. The method of claim 33 further comprisin administering to said mammal cyclodextrin derivative.

35. The method of claim 34 comprising administerin to said mammal 2-deoxyglucose and cyclodextrin derivative whic are chemically linked together via a covalent bond.

36. The method of claim 31 comprising administerin to said mammal 2-deoxyglucose derivative.

37. The method of claim 36 comprising administering to said mammal a compound of the formula:

wherein at least two of said R_x and R₂ groups, per monomeric unit, are hydroxy or an anionic substituent selected from the group consisting of sulfate, phosphate, sulfonate and nitrate, and the other of said R_ and R₂ groups, when present, is a substituent selected from the group consisting of H, alkyl, aryl, ester, ether, thioester, thioether and -COOH; and n is an integer from about 6 to about 12.

38. The method of claim 31 comprising administering to said mammal said saccharide with a non-toxic pharmaceutically acceptable carrier of physiological salinity.

39. The method of claim 36 comprising administering to said mammal a sulfated derivative of β-2-deoxycyclodextrin polymer.

40. The method of claim 30 further comprising administering to said mammal growth factor.

41. The method of claim 30 comprising orally administering said saccharide to said mammal.

42. The method of claim 30 comprising parenterally administering said saccharide to said mammal. - 61 -

43. The method of claim 30 comprising infusing sai saccharide directly into the tissue of said mammal.

44. The method of claim 43 comprising infusing a aqueous suspension or dispersion of said saccharide directl into the tissue using an infusion balloon catheter having plurality of holes in the wall of the balloon portion thereof.

45. A compound having the formula

wherein at least two of said R_x and R₂ groups, per monomeri unit, are hydroxy or an anionic substituent selected from th group consisting of sulfate, phosphate, sulfonate and nitrate, and the other of said R_ and R₂ groups, when present, is substituent selected from the group consisting of H, alkyl, aryl, ester, ether, thioester, thioether and -COOH; and n is a integer from about 6 to about 12.

46. The compound of claim 45 wherein R_x and R₂ ar independently hydroxy or sulfonate, and n is an integer fro about 6 to about^* 8.

47. The compound of claim 46 wherein R_x and R₂ ar independently sulfonate and n is about 7.

48. The compound of claim 46 wherein R_ and R₂ ar independently hydroxy.