WO2012138043A2

WO2012138043A2 - Therapeutic composition containing endothelin as an active component

Info

Publication number: WO2012138043A2
Application number: PCT/KR2011/010320
Authority: WO
Inventors: Eun Ju Lee; Hyo Soo Kim
Original assignee: Seoul National University Hospital
Priority date: 2011-04-04
Filing date: 2011-12-29
Publication date: 2012-10-11
Also published as: WO2012138043A3; KR101418941B1; KR20120112894A

Abstract

The present invention relates to a composition for preventing or treating ischemic disease comprising endothelin or a nucleotide sequence encoding endothelin as an active ingredient, and more particularly to a composition for preventing or treating ischemic heart disease, myocardial infarction, angina pectoris, lower limb artery ischemia, distal limb ischemia and ischemic cerebrovascular disease, wherein the composition is administered by intramuscular injection. According to the present invention, administering endothelin-1 by intramuscular injection may efficiently inhibit the progress of ischemia, fibrosis and tissue necrosis, and induce angiogenesis, such that it is promising for preventing or treating the ischemic disease.

Description

THERAPEUTIC COMPOSITION CONTAINING ENDOTHELIN AS AN ACTIVE

COMPONENT

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to a composition for preventing or treating ischemic disease comprising endothelin or a nucleotide sequence encoding endothelin as an active ingredient, and more particularly to a composition for preventing or treating ischemic heart disease, myocardial infarction, angina pectoris, lower limb artery ischemia, distal limb ischemia and ischemic cerebrovascular disease, wherein the composition is administered by intramuscular injection.

BACKGROUND OF TECHNIQUE

Endothelin (ET) is a 21-amino acid vasoconstricting peptide produced primarily in endothelial cells of the blood vessel, endothelin comprises two S-S bonds per a molecule. It is produced by converting preproendothelin to ET that is catalyzed by the ET-converting enzyme, endothelin-1 was firstly isolated from pig endothelial cells in 1988. In most mammals, the ET family includes three distinct isopeptides, ET-1, ET-2, and ET-3.

Endothelin causes transient vasodilation and subsequently sustained vasoconstriction. While the three isopeptides have similar effects on transient vasodilation, ET-3 has negligent effects compared with the other isopeptides on vasoconstriction. The effect of ET-3 on vasoconstriction is only about one hundredth of those of ET-1 and ET-2. The ET receptor has been reported to have two subtypes. ETA and ETB involve in vasoconstriction and vasodilation, respectively. Endothelin is bound to its receptor present in vascular smooth muscle to cause vasoconstriction (Nature, 332:411, 1988; FEBS Letters, 231:440, 1988). The production of endothelin- 1 is promoted by various factors such as thrombin, angiotensin II, vasopressin, TGF -β, TNF-α, hypoxia and oxidized LDL, and suppressed by DERF (endothelium-derived relaxing factor, nitric oxide).

Endothelin affect significantly on both a blood pressure and cardiac output in body. Intravenously bolus-administered endothelin (0.1-3 nmol/kg) to mice results in dose- dependent temporary decompression (lasting from 0.5 to 2 minutes), followed by continuous elevation of arterial blood pressure in a dose-dependent manner.

Endothelin is likely to primarily affect the renal vascular layer. Endothelin causes a long-lasting significant decrease in the renal blood flow in association with a significant decrease in GFR, urinary volume, and excretion of urinary sodium and potassium. Endothelin may exhibit sustained anti-urinary sodium effects even when peptides of urinary sodium in the arteries are significantly increased. Furthermore, endothelin stimulates the activity of plasma renin. These findings demonstrate that ET involves in control of kidney function and various renal dysfunction such as acute renal failure, cyclosporin nephrotoxicity, radiation contras-induced renal failure and chronic renal dysfunction.

The cerebral vascular system has been shown to be highly sensitive for both vasodilation and vasoconstriction effects by entothelin. Therefore, ET may serve as a crucial regulator to control cerebrovascular spasm causing serious subarachnoid hemorrhage. ET also involves in severe apnea and ischemic lesions affecting directly the central nervous system, suggesting that ET may contribute to induction of myocardial infarction and neurogenic death.

In addition, endothelin has been studied in myocardial ischemia (Nichols et.

Al, Br .. JPharm., 99:597, 1989; Clozed and Clozel, Circ. Res., 65:1193, 1989), coronary vasospasm (Fukuda et al., r. Pharm. 165:301, 1989; L'scher, Circ, 83:701, 1991), cardiac insufficiency, proliferation of vascular smooth muscle cell (Takagi, Biochem & Biophys. Res. Commun, 168:537, 1990; Bobek et al. , Am. J. Physiol. 258: .408, 1990) and atherosclerosis (Nakaki et al., Biophys. Res. Commun. 158:880, 1989; Lerman et al., New Eng. J. Med, 325:997, 1991).

Endothelin is closely related to circulatory disorders such as hypertension, ischemia, vascular spasm and angin and its level is increased in these disorders (see WO 1996/19455). As described above, endothelin is the cause of in cardiovascular diseases. Accordingly, there have been various attempts to treat these diseases by either inhibiting the production of endothelin or using antagonists against endothelin to suppress effects of endothelin (U.S. Pat. No. 5622971). However, there is no attempt to use endothelin per se for treatment of ischemic disease.

Throughout this application, various patents and publications are referenced, and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION

Under those circumstances, the present inventors have made intensive researches to provide more effective therapy for ischemic diseases. As results, the present inventors have discovered that endothelin-1 intramuscularly injected to ischemia animal models shows treatment efficacies on ischemic symptoms, addressing that endothelin-1 is a promising drug candidate for ischemic diseases.

Accordingly, it is an object of this invention to provide a composition for preventing or treating ischemic disease. It is another object of this invention to provide a method for preventing or treating ischemic disease.

Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 represents time-dependent schematic view of electrocardiogram (ECG) levels after treatment with ET-1 in ischemic cardiovascular rat model.

FIG. 2 shows results of echocardiography in ischemic cardiovascular rat model. Panel (a) corresponds to LVFS (Left ventricular end-Fractional Shortening) and panel (b) to LVEF (Left ventricular end-Ejection fraction).

FIG. 3c- shows staining results of cardiovascular fibrosis with Masson's

Trichrome (MT) in ischemic cardiovascular rat model treated with endothelin-1.

FIG. 4 shows appearance for necrosis on the 14^th day after injection with ET-1 to Limb Ischemia mouse model.

FIG. 5 represents results of LDPI (Laser Doppler Perfusion Imaging) and naked eye-observation of blood vessel formation on 14^th day after injection with ET- 1 to Limb Ischemia mouse model.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of this invention, there is provided a composition for preventing or treating an ischemic disease comprising endothelin or a nucleotide sequence encoding endothelin as an active ingredient.

The present inventors have made intensive researches to provide more effective therapy for ischemic diseases. As results, the present inventors have discovered that endothelin-1 intramuscularly injected to ischemia animal models shows excellent treatment efficacies on ischemic symptoms, addressing that endothelin-1 is a promising drug candidate for ischemic diseases.

"Endohelin" which is used in the present invention is a peptide of 21 amino acid residues, produced in vascular endothelial cells, and is known as a vasoconstrictior peptide. Most mammals have isopeptides of endothelin-1, endothelin-2 and endothelin-3. The three kinds of endothelin isopeptides have similar functions and effects with respect to transient vasodilation and continuous vasoconstriction.

The term "ischemic disease" as used herein, refers to one of the vascular diseases caused by a local blood deficiency in which blood supply into tissues is stanched due to vessel stenosis, contraction, thrombus, embolism, etc., resulting in cell damages. The ischemic diseases prevented or treated by the composition of this invention include, but not limited to, ischemic heart disease, myocardial infarction, angina pectoris, lower limb artery ischemia, distal limb ischemia and ischemic cerebrovascular disease. Preferably, the ischemic diseases prevented or treated by the composition of this invention is ischemic heart disease or lower limb artery ischemia.

According to the present invention, the present inventor has observed that intramuscular injection of endothelin-1 leads to alleviation of ischemic symptoms thus to treat various ischemic diseases, which is contrary to the previous report.

The terms "treat, treating, treatment" as used herein, refers to ameliorating one or more clinical indicia of disease activity in a patient having a pathologic condition involving ischemic disease.

The term "nucleotide" as used herein, refers to a deoxyribonucleotide or ribonucleotide polymer encoding endothelin, including known analogs of natural nucleotides unless otherwise indicated It would be obvious to the skilled artisan that the nucleotide sequences used in this invention are not limited to natural nucleotides of endothelin gene which are illustrative and their biological equivalents may be also used in this invention for preventing or treating an ischemic disease. In this regard, the sequence variations should be construed to be covered by the present invention.

For nucleotides, the variations may be purely genetic, i.e., ones that do not result in changes in the protein product. This includes nucleic acids that contain functionally equivalent codons, or codons that encode the same amino acid, such as six codons for arginine or serine, or codons that encode biologically equivalent amino acids.

Considering biologically equivalent variations described hereinabove, the nucleic acid molecule of this invention may encompass sequences having substantial identity to them. Sequences having the substantial identity show at least 80%, more preferably at least 90%, most preferably at least 95% similarity to the nucleic acid molecule of this invention, as measured using one of the sequence comparison algorithms. Methods of alignment of sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J. Mol. Bio. 48:443(1970); Pearson and Lipman, Methods in Moi. Biol. 24: 307-31(1988); Higgins and Sharp, Gene 73:237-44(1988); Higgins and Sharp, CABIOS 5: 151-3(1989) Corpet et al., Nuc. Acids Res. 16:10881-90(1988) Huang et al., Comp. Appl. BioSci. 8:155-65(1992) and Pearson et al., Meth. Mol. Biol. 24:307-31(1994). The NCBI Basic Local Alignment Search Tool (BLAST) [Altschul et al., J. Mol. Biol. 215:403- 10(1990)] is available from several sources, including the National Center for Biological Information (NBCI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blasm, blastx, tblastn and tblastx.

The composition of this invention may be provided as a pharmaceutical composition comprising a pharmaceutically effective amount of the polypeptide or the nucleotide of this invention.

The term "pharmaceutically effective amount" as used herein, refers to an amount enough to show and accomplish efficacies and activities for preventing, alleviating, treating ischemic disease.

The pharmaceutical composition of this invention includes a pharmaceutically acceptable carrier besides the active ingredient compound. The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention, which is commonly used in pharmaceutical formulations, but is not limited to, includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methyl hydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils. The pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995). The terms "administration" or "administering" as used herein, refer to the process by which the composition of this invention is delivered to a subject for therapeutic purposes.

The pharmaceutical composition according to the present invention may be administered orally or parenterally, and preferably, administered parenterally. For parenteral administration, it may be administered intravenously, subcutaneously, intramusculerly, intraperitoneally, transdermal^ or intra-articularly. Most preferably, the composition of this invention is administered intramuscularly. A suitable dosage amount of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, pathogenic state, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition. Preferably, pharmaceutical composition of the present invention may be administered with a daily dosage of 0.001-10000 mg/kg (body weight).

According to the conventional techniques known to those skilled in the art, the pharmaceutical composition according to the present invention may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms including a unit dose form and a multi-dose form. Non-limiting examples of the formulations include, but not limited to, a solution, a suspension or an emulsion in oil or aqueous medium, an elixir, a powder, a granule, a tablet and a capsule, and may further comprise a dispersion agent or a stabilizer.

According to a preferred embodiment, the endothelin of this invention is selected from the group consisting of endothelin-1, endothelin-2 and endothelin-3.

Endothelin-1, endothelin-2 and endothelin-3 are isopeptides present in most mammals and have similar functions and effects on transient vasodilation and continuous vasoconstriction.

In another aspect of this invention, there is provided a method for preventing or treating ischemic disease, which comprises administering to a subject the composition of this invention.

The term "subject" as used herein, refers to an animal, preferably a human, to whom treatment with the compositions of this invention is provided.

As the common descriptions regarding the pharmaceutical composition of this invention and administration thereof are mentioned above, they are omitted herein to avoid excessive overlaps.

The features and advantages of the present invention will be summarized as follows:

(a) The present invention provides a composition for preventing or treating ischemic disease and a method for preventing or treating ischemic disease.

(b) The present invention accomplish the excellent efficacies on inhibiting the progress of ischemia, fibrosis and tissue necrosis, and induce angiogenesis, therefore may be effectively used for preventing or treating ischemic diseases.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES

EXAMPLE 1: Effect of ET-1 on ischemic cardiovascular rat model

Ischemic cardiovascular rat model was used to evaluate for in vivo activity of ET-1 administrated to cardiac muscle. The rat model was established by occlusion of the coronary arteries of the cardiac vessel to induce ischemic states.

Sprague Dawley rats (12-weeks old, body weight 240-290g, Orient Bio, Korea) were intraperitoneally administered with 100 mg/kg ketamine and 10 mg/kg xylazine to induce anesthesia. Then, the rats was subjected to endotracheal intubation and ventilated on a Harvard Rodent Respirator (Model 683, Harvard Apparatus, USA). The 4^th intercostal space was exposed by incision and the pericardium was incised. Afterwards, myocardium (including left coronary) between right ventricular outflow tract and left atrial appendage was ligated using a 6-0 silk suture in accordance with a shoe string tie method, inducing myocardial infarction.

FIG. 1 shows time-dependent schematic view of Example 1. On the 4^th day after establishment of ischemic cardiovascular rat model, electrocardiogram (ECG) was measured at the base line. Then, on the 7^th day, ET-1 (0.025 pg/60 μΙ/rat and 0.25 pg/60 μΙ/rat) was intramuscularly injected to cardiac muscle using an insulin syringe. 3-weeks later, ECG was measured to evaluate whether the conditions of the disease in the rat model was decreased.

A control group paired with an endothelin-1 treatment group in ischemic cardiovascular rat model was designed as administration of PBS. Each of these groups included five or more rats with induced-ischemic cardiovascular disease.

FIG. 2 shows results of echocardiography. LVFS (Left ventricular end- Fractional Shortening) is defined as LVEDD-LVESD/LVEDD and LVEF (Left ventricular end-Ejection fraction) is defined as LVEDD²-LVESD²/LVEDD².

LVEDD indicates left ventricular end-diastolic dimension and LVESD indicates left ventricular endsystolic dimension.

The higher values of LVFS and LVEF indicate better amelioration of the disease. The values of LVFS and LVEF in the ET-1 treatment group in a dose of 0.25 pg/60 μΙ/rat were significantly increased compared with those of the ET-1 treatment group in a dose of with 0.025 pg/60 pl/rat.

Where the heart is exposed to ischemia, cardiac wall is usually weakened due to its fibrosis. It also results in loss of cardiac motion and expansion of cardiac volume. In addition, the ischemic condition is responsible for decrease in LVFS and LVEF. Interestingly, the administration of ET-1 significantly reduced pathological conditions including weakness of cardiac wall, loss of cardiac motion and expansion of cardiac volume even though the heart is exposed to ischemia. These are could be also observed in FIG. 3 showing histochemical staining results for fibrosis in cardiac tissues.

The development of fibrosis in cardiac tissues was analyzed. The chest of the rat model was incised and a diastolic cardiac arrest was induced by injection of saturated KCI into right atrium. Following the cardiac arrest, 18 G medicut was placed in the abdominal aorta and a heart perfusion was performed with a physiological saline under 100 mmHg of pressure over 5 min, followed by fixation using 10% formalin.

The heart was excised and fixed using 10% buffered formalin solution for 24 hr. The fixed tissue was sectioned and embedded in paraffin to prepare slides. The prepared slide was stained with Masson's Trichrome (MT) for histologic evaluation: of cardicac tissue for fibrosis. The MT stains connective tissues and collagen fibers as fibrosis markers in a blue color.

FIG; 3 shows results of fibrosis staining. The blue-stained regions represent fibrosis development. The wider the blue-stained region and the thinner the cardiac wall, the less the amelioration of the disease is.

EXAMPLE 2: Effect of ET-1 on limb ischemia nude mouse model

Limb ischemic mouse model was used to evaluate for in vivo activity of ET-1 intramuscularly administrated to cardiac muscle.

The skin of 8-10 weeks old nude mice (male, Orient Bio, Korea) was incised along the left femoral artery and then the blood vessel of the external iliac artery origin was isolated, followed by ligation with 3-0 silk suture. Then, it was also detached to the bifurcation region of the femoral artery and popliteal artery and the femoral artery and vein were ligated, followed by cutting off the blood vessel between them to produce a limb ischemic mouse model.

On the first day after establishment of the limb ischemic mouse model, 0.25 pg/60 μΙ/mouse of ET-1 or vehicle (control) was intramuscularly injected. After 14 days, LDPI (Laser Doppler Perfusion Imaging) and observation of blood vessel were carried out to analyze recovery of blood flow indicating amelioration of the disease. The control group is vehicle-injected mouse.

As shown in FIG. 4, necrosis in the limb was shown to be delayed on the 14^th day of treatment with ET-1 in the mouse model than the control group.

In addition, FIG. 5 shows that analysis result for LDPI (Laser Doppler Perfusion Imaging), control group was not observed in blood flow of limb vessels (yellow arrow) while ETl treatment group was improved in blood flow of limb vessels (red arrow). There is almost similar to normal control group.

The blood flow of limb vessels (yellow arrow) was not observed in the control group (yellow arrow) as analyzed by LDPI (Laser Doppler Perfusion Imaging) and the limb vessels were not observed in the control group by naked eye observation (see FIG. 5). In contrast, the ET-1 treatment group shows improved blood flow of limb vessels (red arrow) and its angiogenic condition is enhanced to the extent similar to the normal control group with no ischemic damage (see FIG. 5).

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is Claimed is:

1. A composition for preventing or treating ischemic disease comprising endothelin or a nucleotide sequence encoding endothelin as an active ingredient.

2. The composition according to claim 1, wherein the composition is administered by intramuscular injection.

3. The composition according to claim 1, wherein the endothelin is selected from the group consisting of endothelin-1, endothelin-2 and endothelin-3.

4. The composition according to claim 1, wherein the ischemic disease is selected from the group consisting of ischemic heart disease, myocardial infarction, angina pectoris, lower limb artery ischemia, distal limb ischemia andiUiischemic cerebrovascular disease.

5. A method for preventing or treating ischemic disease, which comprises administering to a subject the composition according to any one of claims 1 to 4.