EP0697874A1 - Procedes pour influer sur la croissance des tissus vivants chez les mammiferes et composes et compositions correspondantes - Google Patents

Procedes pour influer sur la croissance des tissus vivants chez les mammiferes et composes et compositions correspondantes

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Publication number
EP0697874A1
EP0697874A1 EP94913338A EP94913338A EP0697874A1 EP 0697874 A1 EP0697874 A1 EP 0697874A1 EP 94913338 A EP94913338 A EP 94913338A EP 94913338 A EP94913338 A EP 94913338A EP 0697874 A1 EP0697874 A1 EP 0697874A1
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Prior art keywords
composition
derivative
deoxyglucose
compositions
cells
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German (de)
English (en)
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Elliot Barnathan
Mary Osbakken
Shunichiro Okada
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University of Pennsylvania Penn
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University of Pennsylvania Penn
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • 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.
  • 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.
  • 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.
  • the balloon is inflated to flatten the plaque against the arterial wall and otherwise expand the arterial lumen.
  • PTCA has produced excellent results and low complication rates, there are difficulties associated with this technique.
  • 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.
  • 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.
  • 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).
  • 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.
  • concentration of sodium ion is about 10 to 20 to 40 times higher in the blood than within the cell.
  • concentration of potassium ion is the reverse, generally 20 to 40 times higher inside the cell.
  • 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.
  • Na + K + -ATPase sodium potassium ATPase
  • Ion gradients are utilized in driving many biological processes.
  • 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.
  • Applicants contemplate that such ion gradients are also involved in cell proliferation, migration and protein synthesis processes.
  • Heparin a mucopolysaccharide
  • heparin 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.
  • heparin is not a homogeneous, well-defined substance.
  • 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.
  • HBGFs heparin binding growth factors
  • 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.
  • cyclodextrins which are cyclic oligosaccharides consisting of up to at least six glucopyranose units.
  • a composition for affecting the growth of cells of living tissue in mammals 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.
  • 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.
  • the 2- deoxyglucose derivative comprises a 2-deoxycyclodextrin derivative.
  • Suitable 2-deoxycyclodextrin derivatives include compounds of the formula
  • R_ and R 2 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 R 2 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.
  • 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.
  • 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.
  • 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.
  • the methods and compositions involve compounds which possess a high affinity for growth factor and which are characterized by very low water solubility.
  • 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.
  • 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.
  • compounds which have the following formula are provided
  • R l f R 2 and n are as defined above.
  • R ⁇ and R 2 are independently hydroxy or sulfonate and n is an integer from about 6 to about 8. More preferably, R x and R 2 are independently sulfonate and n is an integer of about 7.
  • 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.
  • “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.
  • Figure 1 is a schematic representation of the 3-dimensional shape of oc-, ⁇ - and ⁇ -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 ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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.
  • various other types of materials that may act by varying mechanisms could be used to inhibit glycolysis and/or increase intracellular sodium.
  • compounds of the present compositions comprise saccharide.
  • saccharide refers to all known and available sugars and sugar-based compounds, and includes oligosaccharides and polysaccharides.
  • 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.
  • the saccharide preferably comprises 2-deoxyglucose (hereinafter "DG").
  • DG which corresponds to the deoxygenated form of glucose, has the following formula.
  • compositions comprising DG are effective for effecting the growth of cells of living tissue in mammals.
  • 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.
  • DG inhibits abnormal migration and/or proliferation of cells via the mediation of changes in sodium transport.
  • smooth muscle cells including human smooth muscle cells
  • ATP adenosine triphosphate
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the saccharide preferably comprises 2-deoxyglucose derivative (hereinafter "DG derivative").
  • 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.
  • DG derivative encompasses oligosaccharides and polysaccharides which are based, at least in part, on repeating units of 2-deoxyglucose.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the DG derivative comprises anionic substituents.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the DG derivatives involve 2-deoxycyclodextrins (hereinafter “DCs”) and deoxycyclodextrin derivatives (hereinafter “DC derivatives”).
  • DCs and DC derivatives comprise ⁇ -, ⁇ - and/or ⁇ - 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.
  • cyclodextrins namely, ⁇ -, ⁇ - and ⁇ -cyclodextrins
  • n 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”) .
  • the present DCs and DC derivatives comprise saccharide compounds containing at least six sugar units, at least one of which is DG.
  • the present deoxycyclodextrins may comprise up to about 12 sugar units.
  • 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.
  • the DCs comprise compounds having the following formula
  • n 6, 7 and 8, respectively and which are designated herein as ⁇ -, ⁇ - and ⁇ -deoxycyclodextrin, respectively.
  • 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 ⁇ -, ⁇ - and ⁇ -DCs, respectively. These cavities render the deoxycyclodextrins capable of forming inclusion compounds with hydrophobic guest molecules of suitable diameters.
  • compositions of certain alternate and preferred embodiments of the present invention include polyanionic DC derivatives.
  • 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 ⁇ (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.
  • 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 ⁇ (1 ⁇ 4) hemiacetal linkages.
  • the preferred derivatized deoxycyclodextrin monomers of the present invention generally have the following formula:
  • each of R x and R 2 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 R 2 , in a given DC derivative, are independent of each other, unless indicated otherwise.
  • at least two of the R x and R 2 groups, per monomeric unit, are hydroxy or an anionic substituent and the remainder of the R-_ and R 2 groups, when present, are non-anionic groups selected from well known and available substituent groups.
  • the remaining, non-anionic R_ and R 2 groups may be, for example, hydrogen, alkyl, aryl, ester, ether, thioester, thioether and carboxyl.
  • the remaining non- anionic R_ and R 2 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 R 2 substituents be hydrophobic to minimize the solubility of the compounds.
  • the compound be polyanionic.
  • 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.
  • 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.
  • the basic monomeric structure identified above is the repeating unit of the novel insoluble polymeric deoxycyclodextrins of the present invention.
  • 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.
  • 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.
  • 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 ⁇ (1 ⁇ 4) hemiacetal linkages.
  • the preferred derivatized cyclodextrin monomers of the present invention generally have the following formula:
  • 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.
  • 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.
  • 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.
  • the mixture of saccharides comprises a mixture of DG and sulfated CDs of formula V above.
  • the mixture of saccharides comprises DG and ⁇ - cyclodextrin tetradecasulfate.
  • this mixture preferably comprises ⁇ -cyclodextrin tetradecasulfate, which is covalently linked to one or more 2-deoxyglucose molecules.
  • 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.
  • polymeric materials having such structures may be formed by a variety of methods.
  • 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.
  • 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.
  • the derivatized deoxycyclodextrin polymers may be produced by first polymerizing and/or crosslinking one or more underivatized deoxycyclodextrin monomers, dimers, trimers, etc.
  • 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.
  • 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 ⁇ -, ⁇ - or ⁇ - deoxycyclodextrin polymer.
  • the anionic substituents are selected from the group consisting of sulfate, sulfonate, phosphate and combinations of two or more thereof.
  • anionic groups such as nitrate, might possess some therapeutic capacity, the sulfate, sulfonate and phosphate derivatives are expected to possess the highest therapeutic potential.
  • at least about 10 molar percent of the anionic substituents, and even more preferably, at least about 50 molar percent are sulfate groups.
  • ⁇ -, ⁇ - and ⁇ - deoxycyclodextrin polymers containing about 10-16 sulfate groups per deoxycyclodextrin monomer, with ⁇ -deoxycyclodextrin tetradecasulfate polymer being especially preferred.
  • the DC polymers may be combined and/or covalently linked with other chemical entities in the present compositions.
  • 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.
  • 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.
  • 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.
  • the DG salt precipitate comprises derivatived DC salts, including polyanionic DC salts.
  • Preferred DC salts include the Al, Ca and Ba salts of ⁇ -, ⁇ - and ⁇ -deoxycyclodextrin sulfate. ⁇ -Deoxycyclodextrin sulfate salts are particularly preferred.
  • 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.
  • ⁇ -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.
  • 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.
  • the non- aluminum salt forms of the DG salt derivatives are preferable over the aluminum salts forms in some and perhaps all therapeutic applications.
  • the compositions of the present invention are contemplated to be useful for oral administration in the healing of stomach ulcers.
  • 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.
  • DG derivatives may be prepared using ordinary and customary synthetic organic techniques, including techniques which are particularly applicable to carbohydrate compounds.
  • Zemek and coworkers are particularly applicable to carbohydrate compounds.
  • Zemek et al. have also utilized yeast glycogen syntheta ⁇ e (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, ⁇ - 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 .
  • yeast glycogen syntheta ⁇ e UD
  • DG or multimers thereof may be converted to deoxycyclodextrins by using cyclodextrin-glucanosyltransferase.
  • cyclodextrin-glucanosyltransferase See Vetter et al. , Directed Enzymatic Synthesis of Linear and Branched Glucooligo- saccharides, Using Cyclodextrin-Glucanosyltransferase, Carbohydrate Research, 223:61 (1992).
  • 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.
  • a very simple series of experiments could be performed, without undue experimentation, whereby screening for naturally occurring mutant strains could be achieved.
  • 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.
  • compositions of this invention may take numerous and varied forms, depending upon the particular circumstance of each application.
  • compositions containing highly water- soluble metabolic inhibitors of glycolytic pathways and/or agents which cause increases in the concentration of intracellular sodium 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.
  • compositions of this invention may comprise one or more of the active materials and a suitable, non-toxic, physiologically acceptable carrier therefor.
  • 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.
  • 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.
  • 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.
  • 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.
  • a liquid carrier When the dosage uniform is a capsule, it may contain, in addition to materials of the above type, a liquid carrier.
  • any material may be present as coatings or to otherwise modify the physical form of the dosage unit.
  • 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.
  • 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.
  • 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.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimersal, and the like.
  • 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.
  • 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.
  • 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.
  • antibiotics can be mixed with the active material and administered as a mixture.
  • antibiotics can be administered alone and/or contemperaneously with the present compositions either by the same or a different route of administration.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • the compositions and compounds include and/or are combined with biologically active proteins.
  • 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.
  • 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.
  • HBGF's heparin- binding growth factor proteins
  • 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.
  • substantially bound refers to at least about 80% of such bound protein remaining attached under such conditions.
  • IL-2 Interleukin-2
  • EGF Epidermal Carpenter and Cohen, 1979, A growth factor
  • 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
  • TGF- ⁇ Transforming Cheifetz et al, 1987, Cel growth factor- 48:409-416 ⁇
  • azure-A methylene blue
  • Other glycosaminoglycan saccharides are known not to function similarly.
  • 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.
  • such dye complexing serving as a model for proteinic growth factor complexing
  • 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.
  • one or more of the present active materials 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.
  • the contact solution may comprise viable tissue or organ materials (hereinafter organic sources) which contain a variety of growth factors.
  • the compounds of the present invention may act as extractants of these growth factors.
  • 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) .
  • the solid phase 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.
  • 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.
  • 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.
  • PDGF platelet derived growth factor
  • 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.
  • compositions and compounds can be used to beneficially regulate and control biologically active proteins, such as growth factor, at the site of a wound.
  • biologically active proteins such as growth factor
  • the compositions and compounds slowly release this growth factor into the immediate vicinity of the wound, thereby accelerating the wound healing process.
  • 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.
  • 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.
  • biocompatible porous solid means a solid which may be applied or administered to a mammal without provoking a substantial inflammatory response or other substantial adverse effect.
  • 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).
  • 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.
  • 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.
  • the DG derivative such as a 2-deoxycyclodextrin
  • the product is contacted subsequently with suitable agents to derivatize the DG derivative to add anionic substituents to the degree taught by this invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • such wound site growth factors can be taken up by the present compounds, thereby reducing the restenoic effect of such materials on the wounded tissue.
  • 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.
  • 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.
  • 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.
  • 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.
  • local administration comprises infusing the saccharide derivative directly into the injured tissue.
  • such step preferably comprises infusing the compound directly into the arterial wall at the site of the angioplasty.
  • a preferred administration step comprises infusing an aqueous suspension or dispersion of compound directly into the arterial wall at the site of balloon angioplasty.
  • 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.
  • the balloon is inflated under relatively low pressure conditions, such as 2 - 3 atmospheres.
  • 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.
  • the administration step preferably comprises infusing an aqueous solution, suspension or dispersion of particles of active material, for example, a suspension of sulfated ⁇ -2-deoxycyclodextrin polymer particles, ranging in size from about 1 to 600 microns, directly into the arterial wall at the site of balloon angioplasty.
  • particles of active material for example, a suspension of sulfated ⁇ -2-deoxycyclodextrin polymer particles, ranging in size from about 1 to 600 microns
  • 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.
  • the present compounds are applied at about the time of angioplasty.
  • 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.
  • intimal thickening occurs which compromises the luminal cross-sectional area and results in reduced flow. This frequently, but not exclusively, occurs at the anastomosis.
  • 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.
  • the bypass grafts themselves may be treated with the active materials prior to anastomotic implantation.
  • 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.
  • 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.
  • a deficiency of this kind 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.
  • an alternative blood supply to the affected tissue must be established.
  • 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.
  • growth factor(s) for example, to promote angiogenesis and the formation of collateral blood vessels.
  • 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.
  • 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.
  • HBGFs are known to stimulate neovascularization and endothelial cell growth.
  • 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.
  • 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.
  • 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.
  • 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.
  • growth factor-containing organic sources e.g., tissue or organ debris, ground matter, or liquid extract
  • 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.
  • compositions may be administered before, during and/or after organ/tissue transplants to inhibit the proliferation and migration processes associated with chronic transplant atherosclerosis.
  • the compositions are administered orally, and thus involve water soluble active materials, for example, DG.
  • 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) .
  • 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.
  • absorbable cellulose cotton or similar material may be used.
  • 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.
  • epidermal plasminogen activator 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.
  • 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.
  • 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.
  • Rat chondrosarcoma-derived growth factor 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
  • ChDGF was subsequently eluted with about 18 ml of about 2 M NaCl in about 10 mM Tris, pH7.
  • 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 [ 3 H]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) .
  • 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.
  • 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.
  • 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 ⁇ -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 ⁇ -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 ⁇ - deoxycyclodextrin tetradecasulfate polymer, respectively.
  • LRP LDL receptor related protein
  • LRP has been shown to mediate the internalization of various ligands, including urokinase (u-PA) , activated ⁇ -2 macroglobulin, chylomicron remnants, and apo-E enriched ⁇ -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.
  • the amount of t-PA which specifically degraded was reduced 70% by 10 millimolar DG.
  • the reduction by DG of the amount of t-PA specifically degraded is dose responsive and a maximal inhibition is obtained at 5 millimolar.
  • 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.
  • 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.
  • 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 ⁇ -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

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Abstract

L'invention décrit des procédés et des compositions pour influer sur la croissance des cellules de tissus vivants chez les mammifères. Les compositions comprennent un inhibiteur métabolique de la voie glycolytique des cellules et/ou un agent qui provoque une augmentation de la concentration de sodium intracellulaire, et un vecteur physiologiquement acceptable. Les compositions sont administrées au patient soit seules, soit en combinaison avec un facteur de croissance. Selon la voie d'administration désirée, les compositions sont formulées pour avoir soit une solubilité élevée dans l'eau, soit une solubilité faible dans l'eau. Les compositions hautement solubles dans l'eau peuvent être administrées oralement, alors que les compositions faiblement solubles dans l'eau peuvent être appliquées directement à l'emplacement d'une blessure. Les compositions sont administrées pour apporter le facteur de croissance à une blessure ou pour absorber le facteur de croissance présent dans le corps pour empêcher la stimulation excessive de la réaction de la blessure. Les compositions sont particulièrement utiles comme agents de cicatrisation des blessures, notamment les blessures associées à des cellules proliférant et/ou migrant anormalement, par exemple les cellules de muscles lisses qui sont associées à la resténose.
EP94913338A 1993-03-31 1994-03-31 Procedes pour influer sur la croissance des tissus vivants chez les mammiferes et composes et compositions correspondantes Withdrawn EP0697874A1 (fr)

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WO2003086283A2 (fr) * 2002-04-09 2003-10-23 Greenville Hospital System Activite de modulation de metastase d'oligosaccharides hautement sulfates
EP1581544A1 (fr) * 2002-12-31 2005-10-05 Council of Scientific and Industrial Research Processus de synthese de deoxy-d-glucose
EP1735326A4 (fr) * 2004-01-29 2011-04-20 Pinnacle Pharmaceuticals Derives de beta-cyclodextrine et leur utilisation contre la toxine letale d'anthrax
CA2571055C (fr) * 2004-06-17 2011-11-29 Wisconsin Alumni Research Foundation Composes et procedes pour traiter des crises et des troubles paroxystiques
WO2012026614A1 (fr) * 2010-08-24 2012-03-01 国立大学法人宮崎大学 Composition supprimant une activité de métalloprotéinase de matrice
TW201617084A (zh) * 2014-09-25 2016-05-16 山口龍二 包含β－環糊精之抗腫瘤劑
CN113057960B (zh) * 2021-04-15 2022-06-21 浙江理工大学 β-环糊精类衍生化合物在制备促进伤口愈合药物或制剂中的应用

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