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WO1999036074A1 - Use of 8-cl-camp in prevention of restenosis of arterial walls - Google Patents

Use of 8-cl-camp in prevention of restenosis of arterial walls

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Publication number
WO1999036074A1
WO1999036074A1 PCT/US1999/000143 US9900143W WO1999036074A1 WO 1999036074 A1 WO1999036074 A1 WO 1999036074A1 US 9900143 W US9900143 W US 9900143W WO 1999036074 A1 WO1999036074 A1 WO 1999036074A1
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Prior art keywords
camp
cl
stenosis
blood
restenosis
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PCT/US1999/000143
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French (fr)
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Ciro Indolfi
Giampaolo Tortora
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Icn Pharmaceuticals, Inc.
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid

Abstract

Restenosis caused by damage to a blood vessel is lessened by administration of 8-CI-cAMP or a compound that metabolizes to 8-CI-cAMP. The methods are contemplated to be particulary advantageous in instances where the damage was caused by balloon or other angioplasty.

Description

USE OF 8-CL-CAMP IN PREVENTION OF RESTENOSIS OF ARTERIAL WALLS

This application claims the benefit of U.S. provisional application number 60/071085 incorporated herein by reference in its entirety.

Field of The Invention

The field of the invention is blood vessel stenosis.

Background

Arteriosclerosis, also known as hardening of the arteries, and a related disease artherosclerosis, are characterized by a loss of elasticity in blood. Arteriosclerosis is a long-term, progressive process, and often regarded as being asymptomatic until the disease is in an advanced state. Arteriosclerosis, artherosclerosis, and other related conditions can lead to fatal heart attacks, kidney disease, stroke, and even gangrene, which may require amputation. Most of the treatments for arteriosclerosis and artherosclerosis focus on restoring the path of blood flow to the heart, and thus, the treatments deal directly with a stenosis, that is characteristic of both diseases.

Stenosis can be defined as constriction or narrowing. A coronary artery that is constricted or narrowed is referred to as stenosed. A stenosed artery can be formed by a buildup over time of fat, cholesterol, and other substances. The term "plaque" is commonly used to refer to this buildup. Stenosis can also occur with aging of a patient, since blood vessels tend to lose elasticity over time. Other causative factors of stenosis are dietary insult, high blood pressure, diabetes and cigarette smoking.

Restenosis can be defined as "re-narrowing" of arteries following a procedure designed to alleviate stenosis in those arteries. In fact, many such procedures currently used to alleviate stenosis in arteries are regarded as themselves causing vascular injury. Conventional theory states that vascular injury induces formation of neointima and the proliferation of smooth muscle cells (SMCs). Once SMCs begin to proliferate, the affected arteries may re-narrow over a short period of time, and thus undergo the process of restenosis. However, the theory of smooth muscle cell proliferation ultimately causing restenosis has been debated recently.

Common procedures that are thought to induce restenosis include: 1) cardiac catheterization, including percutaneous transluminal coronary angioplasty and atherectomy; 2) insertion of a stent; 3) transmyocardial revascularization; and 4) coronary artery bypass grafting.

Cardiac catheterization comprises a large class of procedures, including percutaneous transluminal coronary angioplasty (PTCA) and atherectomy. The general procedure is to guide a thin plastic tube through an artery or vein in the arm or leg, and thence into the heart and the coronary arteries supplying the heart. This procedure may be used to measure blood pressure and the amount of oxygen in the blood. The catheter may also be modified by addition of a balloon. Once the catheter is inserted into an appropriate artery or vein, the balloon is expanded to the point where the present stenosis is somewhat reduced and the vessel is made more malleable. This procedure is termed PTCA, and is commonly referred to as angioplasty.

Atherectomy is another subset of the class of cardiac catheterization procedures, and is also designed to eliminate stenosis in a patient. The general procedure is that a high-speed rotating device is placed on the end of the catheter that is inserted in the artery or vein. The attached device is designed to grind the "plaque" that is causing the stenosis in the patient into minute particles. An example of a device of this nature is the

Rotoblator™. The Rotoblator™ rotates at a rapid speed, such as 200,000 rpm, and grinds away the stenosing fatty material.

Cardiac catheterization procedures have often been criticized by doctors and health professionals on the grounds that up to 50% of the patients treated by these methods experience restenosis or a "re-narrowing" of the treated arteries within six months of the original procedure. Such patients often require additional PTCA, and possibly a coronary artery bypass.

Insertion of a stent traditionally follows an angioplasty procedure and can detrimentally affect the patient with regards to restenosis. Stent insertion is a process whereby a tube made of metal or plastic is inserted into a vessel or passage to keep the lumen open, and prevent closure from stricture or external compression. Stents are commonly used to keep blood vessels open in the coronary arteries.

Stents used to initially improve blood flow to the heart muscle may remain in the artery permanently to hold it open. However, following a procedure designed to relieve stenosis, it has been found that the insertion of a stent can actually decrease the blood flow velocity in the arteries over time, which is detrimental to the recovery process of the patient. Also, following a stent insertion, the patient must take one or more blood thinning agents, such as aspirin, Ticlopidine and/or Coumadin. Aspirin may be used indefinitely, and the other drugs, which can cause very serious side effects, may be used for four to six weeks.

Within four to six weeks of the stent procedure, patients are almost always advised to take antibiotics after subsequent minor surgical procedures such as dental cleaning. Also, patients cannot participate in a magnetic resonance imaging (MRI) scan within six to eight weeks after the stent procedure without first obtaining a cardiologist's approval. American Heart Association: Stent Procedure, 1998.

Transmyocardial revascularization is a relatively new procedure that uses a laser to cut a series of chambers into the heart muscle of a sick heart. This procedure is designed to increase blood flow to the heart. The surgeon makes incisions on the left side of the chest and the laser is inserted into the chest cavity. The surgeon uses the laser to shoot holes through the heart's left ventricle in between heart beats. Once the procedure is finalized inside the heart, the surgeon can seal up the holes on the outer perimeter of the heart, while leaving the holes inside the heart open. However, the holes inside the heart may be subject to stenosis or restenosis resulting from injuries to the heart.

Coronary artery bypass grafting is a technique in which a doctor takes blood vessels from other places in the body of the patient, and uses these vessels to create a new, unblocked channel that bypasses a blocked area. Although this technique is commonly used to relieve patients of current plaque problems in the arteries, unless the patient radically changes his lifestyle and diet, a new site of stenosis will generally occur in the newly grafted artery. Therefore, this procedure is usually regarded as only a temporary fix to a persistent problem.

About 40% of the patients that have procedures performed to alleviate stenosis undergo significant restenosis within months of the initial procedure. A relatively large additional percentage of patients suffer significant restenosis within several years of the initial procedure. It is thought that several factors contribute to the development of restenosis, including an unstable angina, large residual stenosis, diabetes mellitus, and male gender.

It has also been suggested that that the proliferation of smooth muscle cells (SMCs) plays a major role in the restenotic process after arterial stenting through the stimulation of neointima formation, and may contribute to restenosis after balloon angioplasty.

Circulation, 94, pp. 1247-1254, (1996); J. Am. Coll. Cardiol, 21, pp. 1166-1174, (1993);

J. Am. Coll. Cardiol. 26, pp. 720-724, (1995); Physiol. Rev., 70, pp. 1177-1209, (1990);

Lab. Invest, 49, pp. 208-215, (1983). On the other hand, many of the restenosis prevention strategies that are based on inhibition of smooth muscle cell proliferation, which successfully limited restenosis in animal models, have failed in human models. It is therefore still debated as to whether smooth muscle proliferation is really responsible for restenosis. Ann. Cardiol Angeol. (Paris). 44(7), pp. 349-53, 1995.

There are several techniques currently under review to prevent or otherwise lessen the impact of restenosis, including intravenous radiation, vascular gene transfer, and drug treatment. Intravenous radiation of gamma or beta radiation, otherwise known as intracoronary radiation therapy, has been studied with regard to relieving a patient of possible restenosis. Gamma radiation has been shown to be effective in stent restenosis in humans. Herz, 23(6), pp. 356-61 (1998). However, it has been noted that because of the energy of therapeutic gamma sources and the shielding requirements, it would be preferable to deliver ionizing radiation using a beta emitter, and some of the most popular beta emitters include 90Sr/Y, and 32P. Herz, 23(6), pp. 366-72 (1998). Beta radiation has been found to be initially successful in animals, however, there are disadvantages. The most prominent disadvantage with this method is that small quantities of radiation must be used in order to safely treat a patient. In other words, patients often cannot be safely exposed to the higher doses of radiation necessary for treatment of restenosis.

Vascular gene transfer is a new procedure that has been offered as an approach to treating restenosis in a patent. However, the safety of the procedure remains in question. This process is used to transfer genes locally to the vascular wall in hopes of treating artherosclerosis-related diseases at cellular and molecular levels. Curr. Opin. Lipidol. , 9(5), pp. 465-9, (1998). It appears that blood vessels would be easy targets for gene therapy because of the recent invention of novel catheter-based treatment methods. However, this method has not been significantly tested to date, and the safety of the procedure is a concern of researchers in this area.

Pharmaceutical treatment of arteries through internal devices such as catheters has also been attempted to minimize the onset of restenosis. US 5674192 issued to Sahatjian (October 1997); US 5833651 issued to Donovan et al. (November 1998); US 5180366 issued to Woods (January 1993); US 5453442 issued to Bryant et al. (September 1995); US 5407955 issued to Bryant et al. (April 1995); US 5384332 issued to Fontana (January 1995); US 5308862 issued to Ohlstein (May 1994); US 5306724 issued to Goldberg (April 1994); US 5464827 issued to Soil (November 1995); US 5482925 issued to Hutsell (January 1996); US 5498775 issued to Novak et al. (March 1996); US 5525602 issued to Patterson et al. (June 1996); US 5552401 issued to Cullinan et al. (September 1996); US 5567828 issued to Dodge (October 1996); US 5843976 issued to Bryant et al. (December 1998); US 5688796 issued to Cullinan et al. (November 1997); US 5691353 issued to Bryant et al. (November 1997); US 5728724 issued to Bryant et al. (March 1998); US 5770609 issued to Grainger et al. (June 1998); US 5773420 issued to Nguyen et al. (June 1998); US 5643896 issued to Cullinan et al. (July 1997); US 5643939 issued to Ohlstein (July 1997); US 5631247 issued to Dodge (May 1997); and US 5733925 issued to Kunz et al. (March 1998). However, many of the drugs that have been tested, including aspirin and dipyridamole have not only been unsuccessful in decreasing the incidence of restenosis, but have actually increased the incidence of restenosis. Ann. Intern, Med., 116(9), pp. 731- 6 (1992). Other drugs that have been beneficial in preventing restenosis are actually toxic to the patient in the required doses. And despite an overwhelming and intensive investigation into drug therapy for preventing restenosis after cardiac catheterization procedures, the literature reveals that no pharmaceutical therapy has yet been found to be useful. Drugs Aging, 13(4), pp. 291-301, (1998).

A few researchers have been working with cancer cell growth inhibitors, specifically site selective derivatives of cAMP. See, for example, US 5792752 issued to Cho-Chung (August 1998), and US 5843916 issued to Cho-Chung et al. (December 1998). However, to date cell growth inhibitors have only been investigated with respect to cancer, leukemia and tumor formation, and have not been mentioned in connection with any other cell growth inhibition, including inhibition of smooth muscle cell proliferation or in connection with stenosis, restenosis, or arteriosclerosis.

Therefore, there is still a need for treatments that will delay or otherwise lessen the onset of restenosis in a patient who has recently participated in stenosis-reduction therapy.

Summary of the Invention

The present invention is directed to a method of lessening the onset of restenosis in a mammal that was previously treated for pathological stenosis in a blood vessel, by administering 8-Cl-cAMP or a compound that metabolizes to 8-Cl-cAMP.

The method is contemplated to be particularly advantageous in instances where the previous treatment included balloon angioplasty, or other treatment that tends to distress smooth muscle tissue in the treated vessels.

Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description, along with the accompanying drawings in which like numerals represent like components.

Brief Description of The Drawings

Fig. 1 is a diagram of an experimental protocol.

Fig. 2 shows the effects of 8-Cl-cAMP on Heart Rate (HR) and Systolic Blood Pressure (SBP). Fig. 3 shows neointima/media radio (top panel) and neointimal cross-section (bottom panel) of common carotid arteries from rats subjected to only balloon injury (CONTROL) and 8-Cl-cAMP-treated rats after balloon injury (8-Cl-cAMP). All data are shown as mean + SEM; *p 0.01 vs. CONTROL.

Detailed Description

The present researchers have discovered the surprising fact that 8-Cl-cAMP can be used to lessen, (i.e., to decrease and/or eliminate), the occurrence of restenosis in blood vessels that have previously undergone stenosis reduction. Methods utilizing this discovery can be straightforward, and appear to have wide applicability, regardless of the nature or condition of the recipient, and the type of stenosis reduction previously administered.

Methods contemplated herein are expected to be applicable to all mammals, including primates, rodents, felines, and canines. The methods are especially contemplated to be applicable to humans. Moreover, without relying on the validity of any particular theory, characteristics that normally affect other types of treatment are thought to be irrelevant to successful treatment herein. For example, the age and sex of the animal or human is thought to be largely irrelevant, as are fat, cholesterol, fiber, and protein intake. Success is also expected to be more or less independent of the patient's general health and genetic predisposition, including predisposition to diabetes or high blood pressure.

Since 8-Cl-cAMP is a pharmaceutical carried by the blood stream, essentially all blood vessels are suitable for treatment. For example, even though major coronary arteries may have been directly treated with, and consequently damaged by, balloon or other angioplasty, treatment with 8-Cl-cAMP is contemplated to be effective in both the previously damaged vessels and secondarily damaged vessels. Such secondarily damaged vessels may include upstream or downstream vessels, and may include both arteries and veins. The methods contemplated herein are thus applicable to vessels having been previously treated for arteriosclerosis, or more generally for artherosclerosis. A corollary is that treatment with 8-Cl-cAMP is contemplated to be suitable for vessels in any area of the body, including coronary arteries,

It is contemplated that treatment with 8-Cl-cAMP will in most cases be given to lessen restenosis secondary to angioplasty, and especially balloon angioplasty, cardiac catheterization (including percutaneous transluminal coronary angioplasty and atherectomy), stent insertion; transmyocardial revascularization; and coronary artery bypass grafting. On the other hand, the nature of the stenosis reduction is thought to be largely irrelevant. Thus, 8-Cl-cAMP may also be given to lessen restenosis secondary to any other type of stenosis treatment, including other physical treatments, or even chemical treatments.

The vessels being treated with 8-Cl-cAMP need not have had "pathological" stenosis, i.e., stenosis accompanied by significant structural and functional changes. Instead, the "stenosis" contemplated herein is any undesirable narrowing, whether characterized by accretion of plaque, or merely build up of fat and cholesterol. Still further, the etiology of the stenosis is considered to be irrelevant. For example, treatment with 8- Cl-c AMP is contemplated to be effective in lessening restenosis whether the stenosis was caused by any combination of aging, dietary insult, high blood pressure, diabetes, and cigarette smoking.

The basic treatment regimen is relatively simple 8-Cl-cAMP is administered to a patient following injury to blood vessels secondary to stenosis reduction. Administration is typically via intravenous route, with a typical dosage being 0J to 6.0 per kg body weight per day.

Of course, it is also contemplated that administration of 8-Cl-cAMP can be supplemented or replaced by any compound that can metabolize to 8-Cl-cAMP. Examples are compounds in which a sugar molecule is linked to an 8-Cl-cAMP molecule, as in a glycoside or glinkoside.

Of course, all other applicable routes of administration are also contemplated, including intramuscular injection, subcutaneous injection, and so forth. To this end appropriate pharmaceutical formulations would comprise the necessary excipients, stabilizers, emulsifiers, binding agents, thickening agents, salts, preservatives, and the like.

Oral administration, for example, is contemplated, as long as the active component is coated, microencapsulated, or otherwise protected from destructive acids, proteases and other components of the digestive tract. For oral administration, the active compound(s) may be administered with an inert diluent or with an assimilable edible carrier, or the compounds may be incorporated directly with the food of the diet. Orally administered compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspension syrups, wafers, and the like.

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 corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and sweetening agents, such as sucrose, lactose or saccharin; a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may also be present as coatings, or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or range flavor. Such additional materials should be substantially non-toxic in the amounts employed. Furthermore, the compounds may be incorporated into sustained-release preparations and formulations.

Formulations for parenteral administration may include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile, injectable solutions or dispersions. The solutions or dispersions may also contain buffers, diluents, and other suitable additives, and may be designed to promote the cellular uptake of the compounds in the composition, e.g., the compounds may be encapsulated in suitable liposomes. Preferably the solutions and dispersions for parenteral administration are sterile and sufficiently fluid for proper administration, sufficiently stable from manufacture and use, and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and 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 maintenance of the required particle size in the case of dispersion and by the use of surfactants.

Sterile injectable solutions are prepared by incorporating the active compound(s) in the required amount in the appropriate solvent with one or more of the various other ingredients described above, followed by sterilization. Dispersions may generally be 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 listed above. In the case of sterile powders used to prepare sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solutions.

Pharmaceutical formulations for topical administration may be especially useful with certain bioactive compound(s) for localized treatment. Formulations for topical treatment include ointments, sprays, gels, suspensions, lotions, creams, and the like. Formulations for topical administration may include, in addition to the subject compounds that metabolize to 8-Cl-cAMP, known carrier materials such as isopropanol, glycerol, paraffin, stearyl alcohol, polyethylene glycol, etc. The pharmaceutically acceptable carrier may also include a known chemical absorption promoter. Examples of absorption promoters are e.g., dimethylacetamide (US Patent No. 3472931), trichloro-ethanol or trifluoroethanol (US Patent No. 3891757), certain alcohols and mixtures thereof. British Patent No. 1,001,949 and British Patent No. 1,464,975.

Solutions of the active compound(s) may be stored and/or administered as free base or pharmacologically acceptable salts, and may advantageously be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. These compositions and preparations may advantageously contain a preservative to prevent the growth of microorganisms, for example, parabens. chlorobutanol, phenol, sorbic acid, and the like. In many cases, it will be preferable to include isotonic agents such as sodium chloride. Prolonged drug release from injectable compositions can be brought about by the use of agents which delay absorption, such as aluminum monostearate and gelatin.

The compositions and preparations described preferably contain at least 5% of active compound. The percentage of the compositions and preparations may, of course, be varied, and may contain between about 1% and 25% of the weight of the amount administered. The amount of active compound(s) in such therapeutically useful compositions and preparations is such that a suitable dosage will be obtained.

Dosages are contemplated to be administered all at once, divided into a number of smaller doses which are then administered at varying intervals of time, or administered continuously, as through an IV drip or a transcutaneous patch. At present, the most preferred administration of the compounds of the present invention comprises parenteral administration of between about 0.05 to 3.0 mg of compound per kg of body weight of the patient, two times per day for approximately 5 days.

The specific treatment regimen given to any individual patient will, of course, depend upon the experience of the clinician in weighing the disease involved, the health and responsiveness of the patient, side effects, and many other factors as is well known among such clinicians. For example, greater of lesser dosage levels, and treatment regimens covering greater or lesser periods of time would be dependent upon the judgment of the attending clinician, and may include periods of rest during which treatment with the compound is temporarily halted. Treatment may also be combined with other treatments as appropriate. Progression/remission of the disease being treated may be determined by monitoring blood flow through the vessel in question using methods and other means known in the art, and the existence and extent of side effects may be determined by following functioning of the liver and/or kidneys, and by following the blood circulation as for example through the use of EKG, which again are well known in the art.

In addition to the expressly therapeutic uses discussed above, compounds of this class may also be used in the study of absorption, distribution, cellular uptake, and efficacy. Accordingly, it is contemplated that pathological stenosis in any blood vessel having smooth muscle tissue (whether in vivo or ex vivo), can be affected by reducing the pathological stenosis in a manner that distresses the smooth muscle tissue, and administering to the blood vessel an amount of 8-Cl-cAMP or a compound that metabolizes to 8-Cl-cAMP in a dosage that inhibits restenosis in the blood vessel. In such cases, the fluid flowing in the blood vessel may advantageously include of 8-Cl-cAMP or a compound that metabolizes to 8-Cl-cAMP in a concentration of OJμM to 5.0μM. All of the myriad methods of reducing stenosis discussed herein are contemplated for distressing the smooth muscle tissue, as well as other methods known in the art.

As used herein , "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the therapeutic active ingredients, its use in the therapeutic compositions and preparations is contemplated. Supplementary active ingredients can also be incorporated into the compositions and preparations.

The abbreviation "8-Cl-cAMP" is used herein to mean 8-chloro-cAMP 3', 5'-cyclic monophosphate. 8-Cl-cAMP can be prepared as described by K. Muneyama et al., J. Carbohyd. Nucleosides Nucleotides, 1, p. 55, (1974), via an 8-bromo intermediate compound or by utilizing the processes described in US 5763419 issued to Robins (June 1998). 8-Cl-cAMP can also be synthesized as a sodium salt from ICN (Costa Mesa, CA, USA).

It is also contemplated that 8-Cl-cAMP can be supplemented or replaced by site selective derivatives of cAMP to target particular organs. See US 5792752 issued to Cho- Chung (August 1998), and US 5843916 issued to Cho-Chung et al. (December 1998). Examples

General Considerations

The animals used in this study were handled according to the animal welfare regulation of the University of Federico II of Naples and the protocol was approved by the animal use committee of that institution. All animals received human care in accordance with the animal use principles of the American Society of Physiology. All rats were maintained under identical conditions of temperature (21 + 1 °C), humidity (60 + 5 %) and light/dark cycle, and had free access to normal rat chow.

Animal Preparation

35 Wistar rats weighing 350-400 g (at 14 weeks of age) purchased from Charles

River (Calco, Italy) were included in the present study. Rats were anaesthetized with an intramuscular injection of 100 mg/kg ketamine (Sigma Chimica, Milan, Italy) and 5 mg/kg xylazine (Sigma Chimica, Milan, Italy). Angioplasty of the carotid artery was performed using a balloon embolectomy catheter (2F Fogerty balloon catheter, Edwards Laboratory, Santa Ana, CA) as described and validated in the laboratory. Nature Med., 1, pp.541-45 (1995); Circulation, 92, pp. 1230-35 (1995); Bas. Res Cardiol, in press, (1997) . The balloon catheter was introduced through the right external carotid artery into the aorta and the balloon was inflated at 1.5 atmospheres using a calibrated commercially available inflation device (Indeflator Plus 20, Advanced Cardiovascular System, Inc., Temecula, CA).

The vessel was damaged by passing an inflated balloon three times through the lumen. Previous studies performed in the laboratory demonstrated that the time necessary to pass the inflated balloon catheter back and forth into the carotid artery is 18 seconds. Circulation, 92, pp. 1230-35 (1995). Therefore, in order to keep constant the time of the injury (that might influence the smooth muscle cells proliferation per se) the time of balloon inflation was maintained at 18 seconds. Drug Dosage and Administration

8-Cl-cAMP was synthesized as sodium salt by ICN (Costa Mesa, CA, USA). 8-C1- cAMP (0.012 mg/gr) was administered intraperitoneally in two times (N=9) at the time of the balloon injury and 3 days later (Figure 1). In an additional 8 rats that were used as a control group, a saline solution was administered intraperitoneally.

Toxicity

To study the 8-Cl-cAMP toxicity, laboratory studies and renal histology were performed at baseline and 2 weeks after drug administration (N=6).

Hemodvnamic Measurements

Arterial pressure and heart rate were measured indirectly by a tail-cuff plethysmographic technique (Harvard Apparatus, model 50-0002, South Natick, MA). Drug Res., 18, pp. 1285-87. The effect of 8-Cl-cAMP on blood pressure and heart rate was assessed at baseline and after 1, 3, and 7 hours after the first dose and 3 and 7 hours after the second 8-Cl-cAMP administration 3 days later. (Figure 2).

Morphology

Two weeks later the animals were anaesthetized with an intramuscular injection of 100 mg/kg ketamine and 5 mg/kg of xylazine and the carotid arteries were fixed by perfusion at 120 mm Hg with 100 ml of phosphate-buffered saline (PBS; pH 7.2), followed by 80 ml of prepared PBS containing 4% paraformaldehyde via a large cannula placed in the left ventricle. The carotid arteries were removed and six cross-sections were cut (each 6 μm thick) from the approximate mid portion of the artery, with three of the sections stained with hematoxylin/eosin to demarcate cell types. The remaining three sections were stained with aldehyde fuchsin and counterstained with Van Gieson's solution to demarcate the internal elastic lamina. The sections were photographed under low power, blindly videodigitized and stored in the image analysis system (Mipron,

Kontron Electronics, Eching, Germany) in a 512 x 512 matrix with eight-bit gray scale, with the 12-field of view. Circulation, 92, pp. 1230-35 (1995). The media, neointima and the vessel wall were traced carefully and the ratios between neointima and media calculated. Circulation. 92, pp. 1230-35 (1995).

Assessment of Smooth Muscle Cell Proliferation Rate

The vascular balloon injury was performed in 1 1 animals as above described to assess the effect of systemic 8-Cl-cAMP administration on SMC proliferation. In 6 rats 8- Cl-cAMP was administered at the time of injury and 3 days later, and in 5 rats, as a control group, a saline solution was administered. Seven days after the balloon injury, the arteries were removed and immunohistochemistry for proliferating cell nuclear antigen (PCNA) was performed. (Figure 1).

Immunohistochemistry

The cells stimulated to enter the cell cycle within the neointima and the media were identified by immunohistochemistry for proliferating cell nuclear antigen (PCNA). The slides were deparaffinized and pretreated with Antigen Pretreatment Solution (Biogenics) for five minutes. The slides were then incubated with 10 % normal horse serum followed by incubation with mouse antihuman PCNA antibody (DAKO), 1 :40 dilution for 60 minutes. Biotinylated horse antimouse immunoglobulin G was used as the secondary antibody, and the detection system was a Vectastain Elite ABC kit (Vector Laboratories). Slides were counterstained slightly with hematoxylin. A section of tonsil served as a positive control for each series of immunohistochemical staining for PCNA. A PCNA index was defined as the number of PCNA positive cells divided by the sum of PCNA positive and negative cells and are expressed as a percentage. J. Am. Coll. Cardiol. , 24, pp. 1398-1405 (1994); Artherosclerosis, 117, pp. 83-96 (1995). The effects of the anesthesia and of the surgical procedure (without the balloon injury) on SMC proliferation were also assessed on an additional 7 rats.

Statistical Analysis

All data are shown as mean + SEM. Statistical analysis between groups was performed by analysis of variance (ANOVA) and by uiilizing an unpaired "t" test using a Systat program. The Tukey's test was applied to the data to compare single mean values. A p value of O.05 was considered significant. Annu. Rev. Cell. Biol, 2, pp. 391-419 (1986); Curr. Opin. Cell Biol, 5(2), pp. 269-273 (1993).

8-Cl-cAMP Systemic Effects

No differences in arterial pressure and heart rate were found between the sham- operated animals and the experimental groups. In addition, blood pressure and heart rate were comparable in the different experimental groups. (Figure 2). No renal function was associated with 8-Cl-cAMP administration. The 8-Cl-cAMP did not affect the renal histological features (data not shown).

Effects of 8-Cl-cAMP Administration on Neointima Formation

In the sham-operated animals not subjected to vascular injury, no neointima formation was detected and the endothelium was found intact 14 days after surgery in both carotid arteries of all animals. A reproducible neointima formation was found 14 days after balloon injury in the control group (neointima = 0.212 + 0.018 mm2, neointima/media ratio = 1.315 ± 0.090).

In the group of animals treated with systemic administration of 8-Cl-cAMP there was observed a significant reduction of both neointima (0J05 + 0.017 mm2, pO.Ol vs. control) and neointima/media ratio (0.819 + 0J43 mm , p<0.01 vs. control). (Figure 3)

It was previously demonstrated that the local delivery of 8-Br-cAMP in the pluronic gel significantly reduced the neointima formation after vascular balloon injury (-54 %). Similar results on neointima formation were obtained with systemic administration of 8-Cl-cAMP (-50.71 %; NS vs. 8-Br-cAMP group).

Quantification of Smooth Muscle Cell Proliferation

Seven days after balloon injury, it was observed that there was a reduction of proliferation rate of medial SMCs in 8-Cl-cAMP-treated group (12.61 + 1.11 % vs. 8.47 + 1.3 %; p< 0.05). The effect of 8-Cl-cAMP on proliferation rate in the neointima was less potent ( from 21.52 ± 2.81 % to 17.56 ± 3.82 % NS). Thus, specific embodiments and applications of treatment with 8-Cl-cAMP to lessen restenosis have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims.

Claims

CLAIMSWhat is claimed is:
1. In a mammal having been previously treated for stenosis in a blood vessel , a method of lessening restenosis comprising administering to the mammal an effective amount of 8-Cl-cAMP or a compound that metabolizes to 8-Cl-cAMP.
2. The method according to claim 1, wherein the stenosis is characterized by arteriosclerosis.
3. The method according to claim 1, wherein the stenosis is characterized by artherosclerosis.
4. The method according to claim 1, wherein the blood vessel comprises a coronary artery.
5. The method according to claim 1 , wherein the step of having been previously treated for stenosis comprises cardiac catheterization.
6. The method according to claim 1 , wherein the step of having been previously treated for stenosis comprises angioplasty.
7. The method according to claim 1, wherein the step of having been previously treated for stenosis comprises atherectomy.
8. The method according to claim 1 , wherein the step of having been previously treated for stenosis comprises transmyocardial revascularization.
9. The method according to claim 1 , wherein the step of having been previously treated for stenosis comprises use of a stent.
10. The method according to claim 1, wherein the step of having been previously treated for stenosis comprises a coronary artery bypass graft.
11. The method according to claim 1 , wherein the step of administering comprises intravenous administration.
12. The method according to claim 1 , wherein the step of administering comprises administering 0J0 to 6.0 mg/kg/day.
13. A method of affecting pathological stenosis in a blood vessel having a smooth muscle tissue;
treating the blood vessel by reducing the pathological stenosis in a manner that distresses the smooth muscle tissue; and
administering to the blood vessel an amount of an active constituent comprising 8- Cl-cAMP or a compound that metabolizes to 8-Cl-cAMP, in a dosage that inhibits restenosis in the blood vessel.
14. The method according to claim 13, wherein administering comprises passing a fluid in the blood vessel having a concentration of OJ╬╝ to 5.0 ╬╝M of the active constituent.
PCT/US1999/000143 1998-01-15 1999-01-13 Use of 8-cl-camp in prevention of restenosis of arterial walls WO1999036074A1 (en)

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