EP0722334A1

EP0722334A1 - Therapeutic treatment for inhibiting blood vessel blockage using a polypeptide

Info

Publication number: EP0722334A1
Application number: EP94927421A
Authority: EP
Inventors: Daniel Burleigh
Original assignee: Mallinckrodt Medical Inc
Current assignee: Mallinckrodt Inc
Priority date: 1993-09-14
Filing date: 1994-09-14
Publication date: 1996-07-24
Also published as: AU7687194A; WO1995007711A1; JPH09502726A

Abstract

Compositions and methods of use thereof suitable for administration to a warm-blooded animal comprising a polypeptide or a derivative thereof capable of administration to an animal to produce reliable cellular flexibility and extracellular matrix remodeling in areas of vessel blockage to allow for therapeutic treatment with lessened vascular smooth muscle cell damage.

Description

THERAPEUTIC TREATMENT FOR INHIBITING BLOOD VESSEL BLOCKAGE USING A POLYPEPTIDE

FIELD OF THE INVENTION

This invention relates generally to a novel use of a polypeptide hormone for therapeutic use, and more particularly, to novel methods of using the hormone Relaxin, and to pharmaceutical compositions comprising the hormone Relaxin for novel therapeutic use in preventing or minimizing arterial cell injury and proliferation during and following angioplasty procedures.

BACKGROUND OF THE INVENTION

Balloon angioplasty, atherectorry, rotorary ablation and similar therapeutic techniques, collectively called angioplasty, are used to improve circulation in vivo and are finding ever-increasing application in therapeutic cardiology. Specifically, balloon angioplasty procedures involve the introduction of a balloon-type catheter into the narrowed portion of an artery. The narrowing of the artery may be caused by different factors but most commonly is caused by a build-up of "atherosclerotic plaque", and proliferation of intimal and neointimal vascular smooth muscle cells. Once the catheter is positioned in the narrowed portion of the artery, the balloon portion of the catheter is inflated. The inflation of the balloon within the narrowed area of the artery is intended to restore the diameter of the blood vessel thus improving circulation.

Often, however, following a balloon angioplasty therapeutic procedure or similar therapeutic technique, patients experience a re-narrowing or restenosis, of the artery. Approximately 30-40% of patients will show a 50% reduction in postoperative luminal diameter within six months after having undergone the angioplasty therapeutic treatment. Restenosis is of considerable concern since it requires further intervention therapeutics, and its effects may be life threatening.

Therefore, the need for a suitable method of therapeutic treatment to prevent or minimize cell damage and restenosis following balloon angioplasty or similar therapeutic techniques which currently may cause vascular injury is of significant importance. It is an object of the present invention to meet this need.

SUMMARY OF THE INVENTION

The present invention discloses a novel method of using a hormone, Relaxin, in balloon-type catheters for therapeutic treatment to prevent or minimize vascular restenosis. Restenosis is a recurrent stenosis, i.e., a narrowing or stricture of a duct or canal. Restenosis and the development of atheromatous lesions (the reason for the procedure in the first place) are thought to share several common pathological elements such as the accumulation of monocytes and macrophages at the area of injury or inflammation and the proliferation of vascular smooth muscle. Growth factors which induce this proliferation of vascular smooth muscle cells thus causing restenosis, may arise from initial platelet lysis and/or from many other cellular elements, including the monocytes and macrophages which infiltrate the injured area in response to the inflammatory stimuli. The stimuli which give rise to the proliferation of smooth muscle and other cells are thought to be triggered in part by the trauma of the procedure itself. Thus the greater the severity of the procedure needed to open the artery, the greater the probability of a more vigorous proliferative process and attendant restenosis.

The hormone Relaxin is a small globular protein of the insulin super-family. Among its many actions, Relaxin acts upon smooth muscle cells to suppress contraction in response to other stimuli, and to relax contractile elements within the cell, allowing stretching beyond limits that normally would cause cellular injury and tissue damage in the absence of the hormone. Indeed, an important physiological function of Relaxin is to cause the relaxation of smooth muscle cells within the cervix, allowing parturition without injury to the mother. Relaxin also acts to induce remodeling of the extracellular matrix, increasing elasticity of tissue in the area of its action, and increasing the limits of stretching and strain this tissue may be subjected to without cell disruption and destruction. In accordance with the present invention, local or systemic treatment of arterial atherosclerotic lesions with Relaxin prior to or concurrent with angioplasty procedures prevents or minimizes tissue damage, and consequent inflammation and restenosis responses.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, a balloon-type catheter such as a balloon infusion catheter, hydrogel catheter, or stent, is coated or filled with a therapeutically effective amount of a composition containing the hormone Relaxin, or a Relaxin containing composition may otherwise be incorporated into the particular delivery system of choice. Relaxin is a small globular . protein comprising two polypeptide chains linked together with disulfide bridges which have the same disposition as those in insulin, and a tertiary structure closely resembling that of insulin. The amino acid sequence for human Relaxin is as follows;

A-chain:

B-chain:

wherein A represents Alanine, C represents Cysteine, D represents Aspartic Acid, E represents Glutamic Acid, F represents Phenylalanine, G represents Glycine, H represents Histidine, I represents Isoleucine, K represents Lysine, L represents Leucine, M represents Methionine, N represents Asparagine, P represents Proline, Q represents Glutamine, R represents Arginine, S represents Serine, T represents Threonine, V represents Valine, W represents Tryptophan, X represents an unspecified or variable amino acid, and Y represents Tyrosine.

Relaxin acts by binding to specific cell surface receptors on those target cells which possess them. Relaxin has been shown to bind specifically to an increasingly diverse group of target cells. Examples of such target cells possessing Relaxin specific receptors include uterine myometrial cells, cervical smooth muscle cells, connective tissue fibroblasts of the pubic symphasis ligaments, intestinal epithelium, brain, and vascular smooth muscle cells. The surface receptors themselves are transmembrane proteins, and the binding of Relaxin thereto triggers a sequence of transduction events within the target cell which mediates pleiotropic actions. Relaxin receptors from rat ovary and rat brain have identical binding and biochemical properties as that of the human receptors indicating that the Relaxin receptor most likely is the same in all target cells. However, to date, Relaxin receptors have not been cloned and sequenced although biochemical evidence suggests that these receptors are symmetrical heterotetramers, incorporating an intracellular tyrosine kinase activity, and are probably members of the insulin - insulin like growth factor receptor family. This means that they are expected to act through the receptor tyrosine kinase transduction cascade.

Relaxin also acts as a tissue remodeling hormone. This means that Relaxin induces the synthesis and secretion of extracellular enzymes which catalyze the degradation and resorption of extracellular matrix proteins. Relaxin induces the replacement of the collagen matrix by elastin and loosens and increases the elasticity of target tissues. Relaxin also increases the degree tissues can be stressed and distended without cellular injury or disruption. Relaxin's specific binding, and tissue responsiveness to Relaxin have been observed in arterial vascular smooth muscle cells as described in G.N. Stemmerman, L. Tashima and F.C. Greenwood, Is Relaxin A Hormone Of The Human Gastrointestinal Epithelium?; Abstract #533, 75th Annual Meeting of the Endocrine Society, Las Vegas, NV 6/9-12/93.

Also to be noted are the protective effects of systemic estrogens against atherosclerosis and atherogenesis which have long been observed, and are felt to be responsible for the differentiated rates of clinically significant coronary artery disease in men and women. It is believed that based on current studies of the present invention that at least part of this estrogen protective effect may be due to induction of paracrine synthesis of Relaxin and its receptors in the vascular smooth muscle cells or other vascular cells.

The observation of Relaxin binding and effects on vascular smooth muscle cells allows an expectation of Relaxin efficacy in restenosis as follows: local delivery of Relaxin to hyperplastic medial and intimal smooth muscle cells within an atherosclerotic lesion, prior to or concurrent with angioplasty, will cause loosening of the extracellular matrix of the lesion, decreasing the trauma, cellular damage, and consequent inflammatory responses which trigger and drive restenosis. Continued controlled local delivery at the lesion site after angioplasty will promote a normal rather than hyperplastic wound-healing response, allowing a better outcome for the interventional procedure.

The differences in rate and extent of restenosis which has been observed in different patients after Percutaneous Transluminal Coronary Angioplasty (PTCA) may result in part from differences in endogenous paracrine Relaxin secretion, an action which may occur naturally in the lesions present in these patients; resulting in the wide spectrum of restenosis pathology observed during the recovery process.

In the present invention, a therapeutically effective amount of the Relaxin polypeptide, a Relaxin mimic, or a molecule having similar receptor binding specificity, or a gene specifying coding or specifying the same may be administered to a warm-blooded animal .in vivo using a local delivery device incorporated into a balloon infusion catheter or a like delivery device of choice for delivery to the particular target site to thus minimize cell damage and subsequent restenosis which would require further interventional therapeutic action and could possibly be life-threatening.

The above-described unique characteristics of the present invention make the Relaxin polypeptide and its derivatives very attractive for therapeutic purposes at sites of stenosis to prevent vascular injury.

As previously noted, the preferred embodiment of the present invention is the peptide, polypeptide or protein Relaxin or derivatives thereof used alone or in combination to prevent cellular damage during invasive therapeutic treatment of vascular stenosis.

The present invention is described in still greater detail in the illustrative examples which follow:

Example 1: Demonstration of Relaxin Specific Binding to Receptors on Porcine Coronary Artery Smooth Muscle Cells In Monolayer Culture.

Porcine Coronary Artery Smooth Muscle Cells are grown to confluent monolayers in six (6) well cluster plates of 35 mm diameter (9.62 cm2) wells. The wells are then washed with binding buffer, e.g., 10 mM HEPES, 5mM KH₂P0₄, 5mM MgCl₂, 150 mM NaCl, 1.0% (w/v) BSA (bovine serum albumin, RIA grade), 10 ug/ml Gentamycin, pH 7.4, and stabilized in the binding buffer for approximately 30 minutes at 22° C. The wells are then incubated in triplicate with 1.0 ml of binding buffer containing 125-I-Relaxin, alone or with a specified dose of unlabeled Relaxin and incubated for approximately 30 minutes at 22° C. After incubation, the binding buffer is removed, and the wells are washed twice with 1 ml portions of ice-cold binding buffer. The Smooth Muscle Cells in each well are then solubilized by dissolving the cells in 1.0 ml of solubilizing solution, e.g., 2.0 N NaOH in distilled water, which is transferred, with a 1.0 ml wash of the same stabilizing solution to a 12 x 75 mm counting tubes. Tubes are then counted in a gamma counter, and the Relaxin binding affinity and capacity are determined from this data.

Example 2; Immunohistochemical Detection of Specifically Bound Relaxin On Porcine Coronary Artery Smooth Muscle Cells.

Porcine Coronary Artery Smooth Muscle Cells (pcSMC) are grown to confluent monolayers in complete culture medium (Dulbecco's Modified Eagle's Medium (DMEM) /10% FBS) on slide chambers. The slide chambers are washed with binding buffer, and stabilized in the same buffer for 30 minutes at 25° C. (Binding Buffer: 10 mM HEPES, 5 mM KH₂P0₄, 5mM MgCl₂, 150 mM NaCl, 1.0% (w/v) BSA., 10 ug/ml Gentamycin, pH 7.4). The slide chambers are then incubated in binding buffer containing 1.0 ug/ml Relaxin, for 30 minutes at 25° C. Then the slide chambers are washed two times with phosphate buffered saline (PBS) containing 0.2% BSA, and fixed for 20 minutes, at 25° C, in 4% paraformaldehyde in PBS, followed by two washings with PBS. The fixative is quenched by incubation for 10 minutes at 25° C, in 50 mM NH₄C1 in PBS and the slides are then washed twice with PBS and air-dried. In some of the slide chambers, cell membranes are permeabilized by incubation for 10 minutes, at 25° C, with 0.2% Triton x-100 in PBS, followed by two washings with PBS. The slide chambers are then incubated for 20 minutes at 25° C in blocking buffer, PBS containing 2.0% BSA, followed by three washings with PBS containing 0.2% BSA. The slide chambers are then incubated with the primary antibody (rabbit anti-porcine relaxin) in binding buffer for 30 minutes at 25° C. As a competitive control, porcine relaxin at 1.0 ug/ml is added to some of the slide chambers during this incubation. All slide chambers are then washed three times with PBS containing 0.2% BSA. The slide chambers are then incubated with the secondary antibody (fluorescein-conjugated goat anti-rabbit IgG) in binding buffer for 30 minutes at 25° C. All slide chambers are then washed twice with PBS containing 0.2% BSA., once with PBS, and once with distilled water. The slides are then mounted with cover glasses and observed by epi- illumination fluorescence microscopy to detect specifically bound Relaxin.

Examp e 3; Relaxin Inhibition of Restenosis in the Rat Carotid Injury Model.

Rats are placed under anesthesia and one carotid artery is exposed by an incision in the neck. A wound is then made in a marked neck segment of the rat carotid using either: a. Inflation of a balloon catheter in the marked segment, or B. repeated passage of a roughened length of suture material through the lumen of the marked segment.

In the test animals, an appropriate dose, preferably between from 10.0 ng to 1.0 g of Relaxin in solution or suspension is delivered to the wounded arterial segment by appropriate means. In the control animals, vehicle is similarly delivered.

After an appropriate time, test and control animals are sacrificed and recovery/healing and restenosis response are assessed by histochemical and biochemical methods to show Relaxin inhibition of restenosis. After the Relaxin hormone, polypeptide or like mimic having similar binding activity, is isolated or prepared as described in the Examples, the compound is may be used alone or with a pharmaceutically acceptable carrier in a method of performing therapy. The preferred therapeutic procedure involves injecting or administering, for example by means of a balloon injector catheter, or other catheter-based delivery device, to a warm-blooded animal a therapeutically effective amount, usually within the range of 10.0 ng to 1.0 g of the present invention. Pharmaceutically acceptable carriers for such therapeutic use include those that are suitable for injection or administration to a warm-blooded animal such as aqueous buffer solutions, e.g. tris (hydroxymethyl)aminomethane (and its salts) , chloride phosphate, citrate, bicarbonate, etc., sterile water for injection, physiological saline, and balanced ionic solutions containing chloride and/or bicarbonate salts of normal blood plasma cations such as Ca⁺, Na⁺, K⁺ and Mg²⁺. Other suitable buffer solutions are described in Remington's Practice of Pharmacy, 11th edition, for example on page 170. The carriers may additionally contain a chelating agent(s), e.g. a small amount of ethylenediaminetetraacetic acid (EDTA) , or other pharmaceutically acceptable chelating agents.

Pharmaceutically acceptable polymer-based controlled release delivery systems may also be used such as polylactic acid and derivatives thereof and/or polyethylene glylcol and its derivatives.

The concentration of the Relaxin peptide and the pharmaceutically acceptable carrier, for example in an aqueous medium, varies with the particular field of use.

A sufficient amount of Relaxin is present in the pharmaceutically acceptable carrier in the present invention when satisfactory effects on areas of stenosis are achievable or satisfactory therapeutic results (decreased USMC hyperplasin as compound insulin controls) are achievable, usually within the range of the expected effective dose range, i.e., 10.0 ng to 1.0 mg.

The Relaxin composition is to be administered to the warm-blooded animals so that the composition remains resident in the vessel wall for about 24 to 48 hours, although shorter and longer residence periods may be acceptable.

The Relaxin compound(s) of the present invention or derivatives thereof, prepared and used as described herein, provide means of jLn vivo therapeutic treatment for areas of vascular stenosis.

After consideration of the above specification, it will be appreciated that many improvements and modifications in the details may be made without departing from the spirit and scope of the invention. It is to be understood, therefore, that the invention is in no way limited, except as defined by the appended claims.

Claims

What is claimed is:

1. A composition suitable for administration to a war - blooded animal comprising a Relaxin hormone or a derivative thereof, or a gene capable of specifying same to reduce vascular restenosis following one or more therapeutic vascular procedure(s) .

2. A composition suitable for administration to a warm¬ blooded animal comprising a Relaxin hormone or a vascular smooth muscle cell relaxation promoting derivative thereof, or a vascular smooth muscle cell relaxation promoting gene capable of specifying same to promote vascular smooth muscle cell relaxation prior to, during, following or during and following one or more therapeutic vascular procedure(s) .

3. A composition suitable for administration to a warm- blooded animal comprising a Relaxin hormone or a vascular cell damage reducing derivative thereof, or a vascular cell damage reducing gene capable of specifying same to reduce vascular cell damage during, following or during and following one or more therapeutic vascular procedure(s) .

4. A composition suitable for administration to a warm¬ blooded animal comprising a Relaxin hormone or a vascular cell inflammation reducing derivative thereof, or a vascular cell inflammation reducing gene capable of specifying same to reduce inflammation during, following or during and following one or more therapeutic vascular procedure(s) .

5. A composition suitable for administration to a warm- blooded animal comprising a Relaxin hormone or a smooth muscle cell proliferation reducing derivative thereof, or a smooth muscle cell proliferation reducing gene capable of specifying same to reduce smooth muscle cell proliferation during, following or during and following one or more therapeutic vascular procedure(s) .

6. A composition suitable for administration to a warm¬ blooded animal comprising a Relaxin hormone or a tissue remodeling promoter derivative thereof, or a tissue remodeling promoter gene capable of specifying same to promote normal tissue remodeling rather than pathological response to injury during lesion healing processes in coronary arteries and other vessels.

7. A composition suitable for administration to a warm¬ blooded animal comprising a Relaxin hormone or an extracellular matrix remodelling promoter derivative thereof, or an extracellular matrix remodeling promoter gene capable of specifying same to promote extracellular matrix remodeling upon in vivo administration for a reduction in vascular cell damage during, following or during and following one or more therapeutic vascular procedure(s) .

8. A method of performing a therapeutic procedure comprising administering to a warm-blooded animal a therapeutically-effective amount of a Relaxin hormone, or a vascular restenosis reducing derivative thereof, or a vascular restenosis reducing gene capable of specifying same to reduce vascular restenosis following one or more therapeutic vascular procedure(s) .

9. A method of performing a therapeutic procedure comprising administering to a warm-blooded animal a therapeutically-effective amount of a Relaxin hormone, or a vascular cell relaxation promoter derivative thereof, or a vascular cell relaxation promoter gene capable of specifying same to promote vascular cell smooth muscle cell relaxation prior to, during, following or prior to, during and following one or more therapeutic vascular procedure(s) .

10. A method of performing a therapeutic procedure comprising administering to a warm-blooded animal a therapeutically-effective amount of a Relaxin hormone, or a vascular cell damage reducing derivative thereof, or a vascular cell damage reducing gene capable of specifying same to reduce vascular cell damage during, following or during and following one or more therapeutic vascular procedure(s) .

11. A method of performing a therapeutic procedure comprising administering to a warm-blooded animal a therapeutically-effective amount of a Relaxin hormone, or an inflammation reducing derivative thereof, or an inflammation reducing gene capable of specifying same to reduce inflammation during, following or during and following one or more therapeutic vascular procedure(s) .

12. A method of performing a therapeutic procedure comprising administering to a warm-blooded animal a therapeutically-effective amount of a Relaxin hormone, or a cell proliferation reducing derivative thereof, or a cell proliferation reducing gene capable of specifying same to reduce smooth muscle cell proliferation during, following or during and following one or more therapeutic vascular procedure(s) .

13. A method of performing a therapeutic procedure comprising administering to a warm-blooded animal a therapeutically-effective amount of a Relaxin hormone, or a vascular cell damage reducing derivative thereof, or a vascular cell damage reducing gene capable of specifying same to promote extracellular matrix remodeling upon in vivo administration for a reduction in vascular cell damage during, following or during and following one or more therapeutic vascular procedure(s) .

14. A method of performing a therapeutic procedure comprising administering to a warm-blooded animal a therapeutically-effective amount of a Relaxin hormone, or a tissue remodeling promoter derivative thereof, or a tissue remodeling promoter gene capable of specifying same to promote normal tissue remodeling rather than pathological response to injury during lesion healing processes in coronary arteries and other vessels.