HK1027970A - Method of treating chronic progressive vascular scarring diseases - Google Patents

Description

Method for treating chronic progressive vascular scarring diseases

The present invention relates to methods and pharmaceutical compositions for treating chronic progressive vascular scarring diseases.

Chronic Progressive Vascular Scarring Disease (CPVSD) is a complication of several common diseases, including diabetes, hypertension, various hyperlipidemias, etc., which afflict developed countries. The current therapeutic approaches to CPVSD are directed to the underlying reason. Unfortunately, most of them are not cured or their control in the general population is difficult to achieve. In addition, CPVSD is often not only easy to produce but also develops rapidly, and until now no medical attention has been given to its root cause. Attempts are therefore being made to treat the second complication, with CPVSD being the most severe because it leads to renal failure, stroke, heart disease and blindness.

CPVSD is generally characterized by changes in vascular smooth muscle cells. One of the major changes is the increase in numbers and the change in the type of connective tissue they bind. This can lead to scarring and significant changes in function. In a blood vessel, this results in a loss of elasticity, resulting in the inability of the blood vessel to expand and contract and the blood vessel having a thickened vessel wall and a narrowed lumen. The net result is reduced blood flow or complete occlusion. Examples of vascular scarring diseases characterized by these pathophysiological processes include chronic progressive glomerular diseases such as diabetes-induced glomerulosclerosis (scarring); progressive renal failure after transplantation; occlusion of a branch providing blood access to a patient treated for end-stage renal failure by hemodialysis; other chronic small vessel diseases (as in some hypertensive patients); patients who have undergone coronary artery bypass surgery have a recurrence of stenosis and diabetic retinopathy.

The therapeutic goal of any therapy for CPVSD must be to reduce the extra extracellular matrix (scar) that has formed to restore normal vascular patency and function, or at least prevent or actually slow down progression. However, there is currently no method of directly interfering with the metabolic abnormalities of smooth muscle tissue or modulating connective tissue synthesis, although they are important in chronic progressive disease. The progression of these diseases has now been viewed as inevitable and irreversible.

Therefore, it is particularly important to develop a treatment for CPVSD, preferably one that involves the oral administration of a low toxicity drug that is effective in treating and reversing CPVSD by causing the degeneration and degradation of established lesions.

Pentosan Polysulfonate (PPS) is a highly sulfonated semisynthetic polysaccharide with a molecular weight of about 1,500 to 6,000 daltons, depending on the method of separation. PPS is in the same general class of heparin and heparinoids, but PPS differs from heparin in many ways in chemical structure, method of derivation, and physicochemical properties. Wherein heparin is generally isolated from the muscle, liver and intestine of mammalian tissues such as cattle and pigs, and PPS is a semi-synthetic compound whose polysaccharide backbone xylan is extracted from the bark of beech or other plant sources, and then treated with a sulfonating agent such as chlorosulfonic acid or trichlorosulfonyl and an acid. After sulfonation, the PPS is typically treated with sodium hydroxide to obtain the sodium salt.

The following formula corresponds to heparin and pentosan polysulfonate,

heparin pentosan polysulfonate heparin is a sulfonated polymer of repeating disaccharide monomers, (D) -glucosamine and (D) -glucuronic acid (both 6-carbon hexose) with an amino functional group on the glucosamine; PPS is a sulfonated linear polymer of repeating single monomer (D) -xylose, in which the 5-carbon xylose is in its pyranose ring form. When the polar light of the heparin rotation plane is in the right-hand direction, the PPS rotation light is in the left-hand direction.

In terms of biochemical properties, PPS is able to prolong partial thromboplastin and has been used to prevent deep vein thrombosis, but it has only fifteen molecules one of heparin anticoagulation potency (see mainly, Wardle, J. Med. Res., 20: 361-370, 1992). PPS is also disclosed for the treatment of urinary tract infections and interstitial cystitis (US 5,180,715), and in combination with a vasodilating steroid for the treatment of angiogenesis and leakage of capillaries, cells or membranes (US 4,820,693).

PPS has been demonstrated by several investigators to inhibit smooth muscle cell proliferation and to reduce hyperlipidemia, suggesting that PPS can be used to prevent the formation of limited atherosclerotic plaques, inhibit proliferation of mesangial cells and prevent collagen formation and glomerulosclerosis (Paul et al, thrombosis research, 46: 793-801, 1987; Wardle, ibid.). However, none have ever noted the scarring aspect of CPSVD such as atherosclerosis (as opposed to inhibiting cell proliferation) or demonstrated its suitability for arresting and/or reversing vascular scarring, i.e., PPS was not thought to be in this regard. Moreover, there is no prior art support for the suggestion that PPS may be used for scar disease by any substantial scientific experimental efficacy data generated in whole animals, but instead tests are based on in vitro animal tissue, which often fails to predict in vivo efficacy.

Although publications are currently available which describe the use of PPS in inhibiting fibrosis and scar formation (see, e.g., Roufa et al, US 5,605,938), these teachings address inhibiting the invasion of skin fibroblasts into the skin and adjacent tissue regions, but without scar disease of smooth muscle cells, which are completely different in etiology and pathology.

It is an object of the present invention to provide a method of treating CPVSD which not only halts the progression of the disease but also substantially reverses the progression and causes the existing scar or lesion to degenerate. It is another object of the present invention to provide such a treatment, i.e., a commonly available drug that is non-toxic and does not cause serious side effects, and is very effective in treating CPVSD, when administered by conventional methods.

In order to make these and other objects clear hereinafter, the present invention is briefly directed to a method of treating a mammalian patient suffering from CPVSD which is capable of halting the progression of the disease and of eliminating or reducing scarring or fibrous damage that has formed in the affected organ or vascular system, the method comprising administering to the patient a pharmaceutical composition comprising an amount of pentosan polysulfate or a pharmaceutically acceptable salt thereof effective to treat vascular scarring disease. The mode of administration is preferably oral administration of PPS, e.g., in tablet, capsule or liquid form.

Brief description of the drawings

FIG. 1 shows α obtained by competitive PCR on one tenth of glomeruli from normal 5-week-old mice₁IV glueThe amount of the original mRNA (as described in example 1 below), describes:

a) in its top, the reaction route after PCR amplification and the corresponding 3, 8-diamino-5-ethyl-6-phenylphenanthridinium bromide stained gel; and

b) in the lower part, a plot is made of the ratio of the mutated collagen cDNA in each glomerulus to the amount of mutated cDNA in each of the 9 tubes input containing all PCR reagents.

FIG. 2 depicts:

a) in the upper part, PAS-stained sections of renal cancer specimens from two nephrectomies (a-normal glomerular histology; b — significant hardening);

b) in the middle (C-D), immunofluorescence microscopy, antibody versus collagen IV in the same kidney; and

c) in the lower part (E), one graph reflects the hardening coefficient in the same kidney; determination of alpha by competitive PCR quantification in 50 microdissected glomerular pools₂IV collagen CDNA (values: 145. + -.22 vs 1046. + -. 74X 10)^-4Attomole/number of glomeruli).

FIG. 3 is a graph reflecting the hardening coefficient in the kidney of 5 patients without glomerulosclerosis versus 5 patients with sclerosis, using the relative cell number of glomeruli and alpha₂IV collagen cDNA content.

FIG. 4 is a graph reflecting the α -values of patients with membranous glomerulonephritis (MN) and diabetic nephropathy (DM) and with nephrectomy for glomerulosclerosis (NX GS) and nephrectomy without glomerulosclerosis₂/α₃IV collagen mRNA ratio.

FIG. 5 is a graph showing the effect of PPS sodium on DNA synthesis in normal mesangial cells, as measured by titration thymidine insertion (24-hour culture) every 10 th³Titration per minute of individual cells is plotted against the concentration of PPS sodium (. mu.g/ml).

FIG. 6 is a graph showing the effect of PPS sodium on cell growth in cells of normal mesangial membranes, which is plotted by the number of cells cultured for three days against the concentration of added PPS sodium (. mu.g/ml).

FIG. 7 is a graph showing a comparison of the effect of PPS sodium and heparin (with an untreated control group) on cell growth of normal mesangial cells after three and five days of incubation.

FIG. 8 is a graph showing the proliferation of normal mesangial cells over time in cells cultured with plasma and PPS sodium compared to control cells cultured with plasma alone.

FIG. 9 is a table of MRNA values for normal mesangial cell layers exposed to PPS sodium (10. mu.g/ml) at various time periods and reverse transcribed₁IV and alpha₁Increased, decreased or no change in the levels of collagen mRNA, collagenase (metalloprotease) 72kDa and 92kDa mRNA, growth factor TGF-beta mRNA and cellular protein beta-actin mRNA.

FIG. 10 is a graph showing a₁Graph of collagen/GAPDH ratio, determined by competitive PCR, detailing the administration of 10-12 weeks of glomeruli to GH gene transfer mice with PPS sodium drinking water and the glomeruli of control GH mice receiving untreated water, respectively.

FIG. 11 depicts painless dead Watanabe rabbits and other subcutaneous PPS sodium (Elmiron) in untreated controls^) Photographs of abdominal aorta longitudinal sections of Watanabe rabbits in the treatment group.

FIG. 12 is a graph reflecting the intimal longitudinal cut area of the various aortic branches of Watanabe rabbits receiving a high cholesterol diet only and the intimal area of comparable longitudinal sections obtained from other groups of Watanabe rabbits receiving drinking water containing a high cholesterol diet and PPS sodium.

FIG. 13 is a comparative example of the ratio of intrabronchial to mesenteric crossing zones in the aorta of Watanabe rabbits receiving only high cholesterol in response to treatment and measured in longitudinal sections from Watanabe rabbits receiving drinking water containing high cholesterol diet and PPS sodium from other groups.

The present invention relates to a method of treating a mammalian patient suffering from Chronic Progressive Vascular Scarring Disease (CPVSD) which is capable of halting or objectively slowing the progression of the disease in the affected vascular system, particularly in arteries such as the aorta, and which is capable of causing the elimination and/or reduction of already formed scarring lesions. The method comprises administering to the patient a pharmaceutical composition comprising Pentosan Polysulfate (PPS) or a pharmaceutically acceptable salt thereof in an amount effective to treat the vascular scarring.

Diseases to be treated according to the novel methods include, but are not limited to, chronic progressive glomerular disease including scar-type diabetes induced glomerulosclerosis; arterial scarring caused by arteriosclerosis, including atherosclerosis; progressive renal failure caused by interstitial scarring after kidney transplantation; a branch occlusion providing blood access to a patient with end stage renal disease treated with hemodialysis; other chronic scar small vessel diseases (as in some hypertensive patients); patients who have undergone coronary artery bypass surgery have recurrent scarring stenosis and diabetic retinopathy.

Because of the prevalence and risk of these diseases, it is particularly important to employ new methods of treating chronic atherosclerotic scar pathology to reverse or prevent the progression of the disease and to resolve existing vascular scars and injuries. For example, administration of PPS in accordance with the present invention can halt and reverse arteriosclerosis of the major blood vessels, such that the already-formed scar contained in the arterial wall of an atherosclerotic plaque is eliminated and/or reduced and the longitudinal intimal region is substantially increased to allow greater blood flow through the lumen of the blood vessels.

The term "an amount effective to treat vascular scarring" as used herein refers to an amount of PPS or a salt thereof incorporated into a pharmaceutical composition that is effective to halt and reverse the progressive symptoms of CPVSD when administered one or more times per day during the administration period. In human patients, a total daily dosage of about 5 to 30mg/kg of patient body weight, or about 350 to about 2,000mg per day, and preferably about 500 to about 1,500mg per adult, of PPS or a salt thereof may be administered in divided doses that are equally divided once to four times a day to effectively achieve therapeutic goals for the treatment and reversal of CPVSD. In smaller mammals, the dosage range may be adjusted downward according to the nature of the body weight, breed and condition.

A preferred embodiment of the novel method of treatment is to administer to a patient a pharmaceutical composition comprising an effective amount of PPS and at least one pharmaceutically acceptable inert ingredient. The composition may be in any standard pharmaceutical dosage form, but oral dosage forms are preferred.

Oral dosage forms include conventional tablets, coated tablets, capsules or pellets, sustained release tablets, capsules or pellets, troches, liquids, elixirs or any other oral dosage form known in the pharmaceutical art.

As pharmaceutically acceptable inert ingredients, fillers, binders, solvents, etc. that do not interfere with the CPVSD activity of PPS in the treatment of CPVSD are included. Fillers such as clays or siliceous types may also adjust the size of the dosage form, if desired.

Other ingredients such as excipients and carriers may also be necessary to impart the desired physical characteristics to the dosage form. Such physical properties are, for example, release rate, characteristics and size. Examples of excipients and carriers for oral dosage forms are waxes such as beeswax, castor wax, sugar wax and carnauba wax, cellulose compounds such as methyl cellulose, ethyl cellulose, carboxymethyl cellulose, cellulose acetate phthalate, hydroxypropyl cellulose and hydroxypropyl methyl cellulose, polyvinyl chloride, polyvinyl pyrrolidone, stearyl alcohol, monoglycerides of stearic acid, methacrylate compounds such as polymethacrylate, methyl methacrylate and ethylene glycol dimethacrylate, polyethylene glycol and hydrophilic gums.

In the compositions of the present invention, the PPS active ingredient is desirably present in an amount between about 50 and about 300mg per unit dose. The precise dosage to be administered to each patient will be a function of the condition being treated and the physiological characteristics of the patient, such as age and weight.

The pharmaceutically active ingredient can be PPS or a pharmaceutically acceptable salt thereof, such as a sodium salt. A preferred oral dosage form for use in the method of the invention is Elmiron^Gelatin capsules (Baker Norton pharmaceuticals, Miami, Florida) containing 100mg of PPS sodium and microcrystalline cellulose and magnesium stearate as excipients.

Although oral administration is preferred, the method of treatment of the present invention also includes administration of PPS or a salt thereof by parenteral, transdermal, transmucosal routes, or by other conventional routes of administration known in the medical and pharmaceutical arts. Similarly, the compositions of the present invention may include PPS in combination with suitable inert solvents, excipients, and additives in pharmaceutically acceptable parenteral, transdermal, transmucosal, or other conventional carriers and dosage forms. Many examples of such pharmaceutically acceptable carriers can be found in Remington pharmaceutical sciences (17 th edition (1985)) and other standard texts. Regardless of the route of administration or pharmaceutical dosage form employed, the dosage of the PPS active ingredient ranges from about 5 to about 30mg/kg of patient body weight or from about 350 to about 2,000mg, and preferably from about 500 to about 1,500mg, although dosages at the lower end of this range should be appropriate for parenteral administration.

The pharmaceutical compositions used in the methods of the present invention may include other active ingredients other than PPS or a salt thereof, such as other drugs useful in the treatment of CPVSD.

The novel method enables convenient, safe and effective treatment of patients suffering from various forms of CPVSD, which in many cases can be life-threatening or organ-threatening, by not only halting chronic progressive vascular scarring disease that has long been considered to be an incurable but also substantially preventing and/or reversing already-formed scarring lesions to restore normal vascular potential and function, using drugs that have proven to be of low toxicity and low incidence.

The following examples include (a) descriptions of tests that have been published in the medical literature that demonstrate certain competitive PCR (polymerase chain reaction) techniques used in the quantification of scar-type collagen mRNA and related factors in the glomeruli; and confirms that the corresponding glomerular cell number is independent of the amount of scar-type collagen produced; (b) experiments conducted under the supervision of the inventors to demonstrate the efficacy of PPS in vivo and in vitro in downregulating scar collagen and cell growth factors and upregulating collagenase activity to degrade existing scar collagen deposits; and (c) testing under the supervision of the inventors to demonstrate the efficacy of PPS in reversing atherosclerosis in vivo, including substantially reducing the amount and distribution of atherosclerotic plaques in affected blood vessels. These tests are not intended to set the material, technique or dosage ranges that must be used to practice the invention, or to limit the invention in any way.

Example 1

Quantification of collagen

Such as Penten et al, journal of physiology in the united states, 32: f951-957(1992) for administering alpha in the glomeruli of mice by the following method₁IV and alpha₂Collagen quantification of IV: the amount of cDNA representing mRNA in one tenth of the glomeruli of normal 5-week-old mice and a standard amount of alpha were added to each of several tubes₁IV and alpha₂IV, wherein the tubes contain all PCR reagents from the GeneAmp DNA amplification kit (Perkinelmer Cetus, Norwalk, Connecocut). To this mixture before amplification, serial dilutions of mutated cDNA containing a new restriction enzyme cleavage site or deletion were added (step shown in FIG. 1, top band). The concentration of the mutant was determined in a previous experiment set on the bracket at the same point (y 1).

After PCR amplification, the entire reaction matrix was loaded directly into a H5 Horizon gel apparatus (Life Technologies) at 4% Nusieve: seakem (3: 1) (FMCBioproducts, Rockland, ME) was applied to the agarose gel and electrophoresed. The electrophoretic bands of DNA were visualized by staining with ethidium bromide and UV light clearing. Photographs were taken with 55 polar front/back films (polar, Cambridge, MA) (see middle band in fig. 1). Competitive PCR analysis was performed by scanning the gel negatively by single-reflection densitometry (Shimadzu; scientific instrument, Columbia, MD).

Densitometry values and mutant bands of the assay were calculated and the ratio of each reaction tube was plotted as a function of the amount of mutant template added (FIG. 1, lower band). For alpha₂IV collagen mutants, the intensity of the measured densitometry bands was corrected by factor 562/479 before plotting the mutant/test band ratio. For alpha₂The mutant bands of IV were said to have their density values added before dividing by the original (test) band values. Straight lines were drawn by linear regression analysis. The amount of cDNA in the test sample, i.e.the amount at which the mutant/test strip density ratio equals 1, is calculated. Competitive PCR analysis was performed in duplicate or triplicate.

Example 2

Changes in sclerosing glomeruli

Such as Peten et al, journal of experimental medicine, 176: 1571-1576(1992) unilateral nephrectomy samples of renal cancer were taken from human patients. The patient has no history of diabetes, hypertension or other systemic diseases associated with glomerular disease. Cortical tissue samples distal to the apparent tumor were embedded in methacrylic resin or paraffin and placed in Carnoy's fixative and sections stained with periodic acid-Schiff (PAS). Glomerulosclerosis, defined by mesangial matrix swelling, was assessed separately by histological examination of PAS-stained material (FIG. 2, top band) and immunofluorescence microscopy of cryo-sections after exposure of collagen type IV (PHM-12, Silens, Westbury, NY) to antibodies (FIG. 2, middle band).

Determination of alpha by performing competitive PCR assay as described in example 1₂IV (scar form) amount of extracellular collagen. The corresponding concentration of collagen type in the previously found normal or hardened glomeruli was determined as shown in the lower band of figure 2.

The corresponding cell numbers in the glomeruli of 5 patients without glomerulosclerosis were compared with those of 5 patients with sclerosis. As reflected in FIG. 3, the difference in glomerular-associated numbers between groups was not significant (p > 0.8), but on α₂The concentration of collagen IV cDNA was statistically significantly different (0.01 < p < 0.025).

Example 3

mRNA ratio of collagen in glomeruli of normal and diseased kidneys

Administration of alpha to glomeruli Using the methods described in examples 1 and 2₂/α₃The relative ratio of IV collagen mRNA was quantified from diagnostic biopsies of human patients with mucosal glomerulonephritis (MN) and diabetic nephropathy (DM) and human patients with glomerulosclerosis Nephrectomy (NXGS) and nephrectomy without glomerulosclerosis (NX NI). As reflected in fig. 4, α₂/α₃The ratio of IV collagen mRNA was significantly higher in DM and in NSGS than in NX NI. (xp ═ 0.0002, x p ═ 0.02).

Example 4

In vitro assay with PPS assay design:

normal mesangial cells (8) were cultured at 2-2.5X 10⁴Density of individual cells/well plated in 24-well plates (Nunc, PGC Scientific corp., Gaithersburg, MD) in basal medium supplemented with 20% fetal bovine serum (Gibco, Grand Island, NY). At 24 hours, the medium was removed, the cells were washed twice with PBS and incubated in serum-free medium containing 0.1% bovine serum albumin (RIA grade, Sicjma) for 24-72 hours. The medium was replaced with fresh basal medium supplemented with 20% fetal bovine serum with or without 5-100. mu.g/ml PPS or compared to standard heparin (100. mu.g/ml). Cells in both wells were trypsinized and plated with an Elzone on days +3 and +5^Cell countingThe count was taken by a counter (Particle Data inc., Elmhurst, IL). In parallel wells, by adding 1. mu. Ci/well of [ 2 ], [³H]Thymidine ([ methyl-³H]Thymidine); 2.0 Ci/mM; DuPont NEN, Boston, MA) to determine thymidine incorporation. Numbers were determined on day 1 or day 3. As a result:

on day 1 (24 h), 50. mu.g/ml was the maximum dose-response (FIG. 5), while on day 3, 25. mu.g/ml was the maximum inhibitory response (FIG. 6).

Controls were run between no addition (control) and heparin (100. mu.g/ml) and PPS (100. mu.g/ml) and showed approximately twice the efficacy of the original heparin at the basal moles of PPS (FIG. 7). The reaction is very reproducible (very narrow error).

Summary the figure (fig. 8) compares the efficacy of PPS added to serum against control cells exposed to serum alone. Test B

Normal mesangial cell layers were exposed to PPS (100. mu.g/ml) at different times, and the reverse transcribed mRNA content of the selection molecules was determined on day 1 and compared to the mRNA content on days 3 and 5 (see FIG. 9), collagen mRNA of type IV was unchanged, collagen mRNA of type I was actually decreased, TGF-. beta.mRNA was decreased by 50% and 92kDa enzyme activity was increased by more than 50%. The control was β -actin, which was unchanged and consistently did not proliferate in the treated cells.

Example 5

Experimental trial design with GH transgenic mice:

bovine growth hormone cDNA from 12 6-week-old GH transgenic mice, which did not cross-react with murine GH sequences, was identified by PCR analysis of detergent extracts from tail biopsies using special primers. PPS sodium (Elmiron) was added orally to the drinking water of 6 GH mice^Baker Norton pharmaceutical) for 10-12 weeks and 6 congener GH transgenic mice were allowed to drink tap water at the same time period. The amount of PPS sodium in drinking water is largeAbout 100mg per kg animal body weight. Glomerular isolation and in situ reverse transcription:

glomeruli were isolated by microscopic sectioning in the presence of RNase inhibitor. The left kidney was perfused with saline followed by a collagen solution containing a soluble ribonuclease inhibitor. The lower polarity was removed and the enzyme was rapidly frozen on dry ice prior to collagenase perfusion. After collagenase enzymatic hydrolysis, 40-60 glomeruli were isolated for Reverse Transcription (RT) in the presence of vanadyl ribonucleoside complexes at 4 ℃. RT was performed in situ as described above except that the glomeruli were freeze-thawed once with dry ice acetone and sonicated for 5 minutes at 2 ℃ in the presence of 2% deuterons and 4 units/. mu.l human placental ribonuclease inhibitor (Boehringer Mannheim, Indianapolis, NJ) prior to addition of the RT component. Samples were frozen during sonication using a microsound cell disruptor (Kontes, Vineland, NJ). Standard and competitive PCR analysis:

synthesis of mouse alpha on PCR-Mate (Applied Biosystems, Foster City, Calif.)₁IV and alpha₁Collagen I primers, alpha smooth muscle cell actin, beta-actin, laminin B1, tenascin, 92kDa metalloprotease, and 7kDa metalloprotease mRNAs, as well as DNA of the bovine growth hormone genome. The identity of each amplification product was confirmed by size and by restriction enzyme analysis. Primer specificity for mRNA was determined by omitting reverse transcriptase. PCR was performed using the GeneAmp DNA amplification kit (Perkin Elmer Cetus, Norwalk, CT). cDNA from the 40-60 glomerular/mouse pool was initially analyzed by standard PCR using the linear log portion of the PCR amplification. This is a rapid, non-quantitative determination of mRNA content. Thereafter, α was determined using competitive PCR₁IV collagen (and calculate alpha)₁IV collagen to GAPDH enzyme ratio to normalize data between animals), PDGF-B, alpha smooth muscle cell actin, beta-actin, and laminin B1 cdnas, cDNA mutants of each molecule were constructed by using small internal deletions or new restriction enzyme sites. By means of a container with Quantity One^The PDI densitometer of the image analysis software performs the analysis of the PCR products. Will be competitivePCR analysis was performed in duplicate or triplicate. As a result:

as shown in fig. 10, the average ratio of type IV collagen/GAPDH was less than half in the mice group treated with oral PPS sodium compared to the untreated (control) mice group. This difference indicates that significantly less type of collagen is present in the glomeruli of the treated animals compared to untreated animals, a fact which is confirmed by histological examination and immunofluorescence microscopy.

Example 6

Watabe Rabbit test

Watanbe rabbit¹As an animal model of natural endogenous hypercholesterolemia. This trait is fully expressed in the homozygous state, partially expressed in the heterozygous state and due to a single gene deletion. The homozygous Watanabe rabbits have a plasma cholesterol concentration 8 to 14 times higher than that of normal Japanese white rabbits.

1. This class of rabbits is known as Watarabe inherited hyperlipidemic rabbit (WHHL).

The Watanabe rabbits have a very high incidence of atherosclerotic plaques especially in the aorta. The rabbits were fed a high cholesterol diet to increase the rate of progression and severity of atherosclerosis.

The following two experiments were performed to demonstrate the anti-atherosclerotic activity of PPS against Watanabe rabbits. Test A: subcutaneous evaluation of PPS

12 Watanabe rabbits were divided into two groups of six (groups A and B) and fed a high cholesterol diet (0.5% cholesterol). Animals in group A were treated daily subcutaneously with normal saline, while animals in group B were treated daily with 10mg/kg PPS sodium (Elmiron)^) And (4) performing subcutaneous treatment.

Four PPS treated animals (group B) died, one on day 22 and three between days 80 and 86, before the end of the trial. On day 89, animals from group a and the two remaining animals from group B were sacrificed and necropsied and their tissues evaluated, particularly in sections from different major branches of the aorta. As a result:

as shown in table I below, animals of treatment group B were found to have many smaller plaque deposits and a higher ratio of smooth muscle layer to plaque (6.8 fold higher) in all examined aortic longitudinal sections relative to group a control rabbits. These findings are visually displayed in the picture shown in fig. 11. Longitudinal sections of abdominal aorta of control animals showed a higher incidence of atherosclerotic plaques in almost all transected areas. Longitudinal sections of abdominal aorta of animals treated with PPS sodium showed almost no trace of plaque, although the treated group was fed with the same high cholesterol diet as the control group.

TABLE 1

Watanabe rabbit

Morphometric determination of aortic lesions

		Control (cm)2)	Smooth muscle/plaque	PPS(cm2)	Smooth muscle/plaque	Multiple of plaque size reduction (X)
							Ascending aorta	Smooth muscle layer	0.328	0.61	0.243	4.12	6.8X
	Plaque	0.54		0.59
							Aortic arch	Smooth muscle layer
	Plaque
							Aorta of chest	Smooth muscle layer	0.244	0.47	0.334	1.184	2.5X
	Plaque
							Abdominal aorta	Smooth muscle layer	0.265	0.74	0.303	7.58	10.2X
	Plaque	0.358		0.04

Test B: oral evaluation of PPS

20 Watanabe rabbits were divided into four groups A to D of 5 rabbits each. All rabbits were fed the same high cholesterol diet (0.5% cholesterol). The animals of groups A and B were given tap water, while the animals of groups C and D contained 0.5mg/ml PPS sodium (Elmiron)^) The tap water is drunk. The total daily dose of PPS consumed by each animal in the treatment group was approximately 30mg/kg, based on observations of water consumed by the animals prior to the trial.

Two of the treated rabbits were removed on days 4 and 11, respectively, because of obvious abscesses not associated with PPS.

Animals in groups a and C were sacrificed on day 50 of the trial and necropsies and their aortas were examined. A significant difference was visually observed between the intima of group a (control) animals and the intima of group C (treated) animals, with the latter presenting less development of atherosclerotic plaques.

Rabbits from groups B and D were sacrificed and necropsied on day 64 of the experiment. These groups of aortas were examined from the suprahistologic and intimal transection areas and transected transection areas of intima and media layers in different aortic branches were determined.

FIG. 12 is a graph showing the mean area of the intima measured in longitudinal sections from the aortic branches of rabbits in the control group (group B) and the treated group (group D), respectively. Figure 12 shows that there were significantly fewer aortic branches in the intimal region examined in treated animals compared to untreated animals, indicating that there was actually less atherosclerotic lesion and plaque deposition in the vascular tissue of the treated group.

FIG. 13 shows the mean values of the intima-to-media region ratios in the same aortic longitudinal sections taken from groups B and D of rabbits as described in FIG. 12. This ratio reflects the relative amount of scar tissue and plaque deposited on the vein wall (such deposition increases the transected area of the intima). The ratio was lower in each aortic branch in treated rabbits (group D) compared to untreated animals (group B).

The above data generated by scientifically proven experimental procedures confirm the efficacy of PPS in reducing the synthesis of additional extracellular matrix collagen and specific cell growth factors while increasing collagen degrading enzyme activity. These effects suggest that PPS is highly effective in the clinical treatment of reversal of CPVSD, particularly in arteriosclerosis and atherosclerosis.

Thus, the various objects of the present invention are accomplished by the methods and compositions provided, and are well suited for use in practical applications.

Since various possible embodiments constitute the invention and since various changes may be made in the above embodiments, it is to be understood that all matter herein set forth is intended to be interpreted in detail as illustrative and not in a limiting sense.

Claims (16)

1. A method of treating a mammalian patient suffering from Chronic Progressive Vascular Scarring Disease (CPVSD) which causes narrowing and reduction of distensibility of the vascular lumen in the affected vascular system, which method is capable of halting the progression of the disease and of causing elimination or reduction of already-formed scarring lesions, which method comprises administering to the patient a pharmaceutical composition comprising Pentosan Polysulfate (PPS) or a pharmaceutically acceptable salt thereof in an amount effective to treat the vascular scarring disease.

2. The method of claim 1, wherein the affected vascular system is an arterial vessel.

3. The method according to claim 2, wherein the artery is the aorta or a major branch thereof.

4. A method according to claim 2 wherein the disease is a form of arteriosclerosis characterized by scarring and wherein the arteriosclerotic scarring process is reversible by the method.

5. The method of claim 4, wherein the atherosclerotic form is atherosclerosis and the scar comprises an arterial blood vessel wall affected by atherosclerotic plaque.

6. The method according to claim 1, wherein the pharmaceutical composition is administered to the patient in an amount sufficient to provide a total daily dose of about 5 to 30mg/kg body weight of the patient or about 350 to 2,000mg of PPS or a pharmaceutically acceptable salt thereof.

7. The method according to claim 6, wherein the daily dose is about 500 to about 1,500 mg.

8. The method of claim 6, wherein the daily dose is administered in one to four divided doses.

9. The method according to claim 1, wherein the pharmaceutical composition is an oral dosage form.

10. The method according to claim 9, wherein the dosage form is selected from the group consisting of conventional or sustained release tablets, coated tablets, capsules, pellets, troches, liquids, and elixirs.

11. The method according to claim 9, wherein the dosage form comprises at least one pharmaceutically acceptable inert ingredient.

12. The method according to claim 11, wherein the inert ingredient is a filler, binder, solvent, excipient or carrier.

13. A method according to claim 9, wherein the dosage form comprises about 50 to about 300mg PPS or a pharmaceutically acceptable salt thereof per serving.

14. The method according to claim 1, wherein the pharmaceutically acceptable salt is a sodium salt.

15. The method according to claim 14, wherein the composition is a gelatin capsule containing PPS sodium, microcrystalline cellulose and magnesium stearate.

16. The method according to claim 1, wherein the patient is a human.