WO2010083611A1 - Method for the prevention and treatment of diastolic dysfunction employing an apolipoproteina1 (apoa1) mimetic peptide/phospholipid complex - Google Patents

Abstract

A method of treating a diastolic dysfunction in a mammal comprising administering a therapeutically effective amount of a reverse lipid transport agonist to said mammal. The most preferred agonist is an Apolipoprotein-A1 (APO-A1) mimetic peptide/phospholipid complex.

Description

TITLE OF THE INVENTION

METHOD FOR THE PREVENTION AND TREATMENT OF DIASTOLIC

DYSFUNCTION EMPLOYING AN APOLIPOPROTEINA1 (APOA1) MIMETIC

PEPTIDE/PHOSPHOLIPID COMPLEX

This application claims priority from US Provisional Patent Applications Serial

Number 61/202,051 filed January 23, 2009 and 61/202,191 filed February 5, 2009.

FIELD OF THE INVENTION

[001] The present invention relates to the general field of medical methods and compounds and is particularly concerned with a method and compound for the prevention and treatment of diastolic dysfunction.

BACKGROUND OF THE INVENTION

[002] Diastolic dysfunction is a condition caused by an abnormal filling of the heart during diastole. This condition can cause heart failure, pulmonary edema and many other incapacitating and possibly life-threatening consequences. There is no effective and side-effect free treatment suitable for all patients for this condition. Hence, there exists a need for a new treatment of diastolic dysfunction.

[003] An object of the present invention is therefore to provide a novel treatment of diastolic dysfunction.

SUMMARY OF THE INVENTION

[004] In a broad aspect, the invention provides a method for preventing of treating a diastolic dysfunction in a subject, the method comprising administering to a subject in need of preventing or treating a diastolic dysfunction a therapeutically effective amount of a reverse lipid transport agonist to prevent or treat said diastolic dysfunction. For example, the lipid transport agonist is a reverse cholesterol transport agonist.

[005] In some embodiments of the invention, the diastolic dysfunction is a ventricular diastolic dysfunction, a left ventricular diastolic dysfunction, or any other diastolic dysfunction.

[006] In some embodiments of the invention, the reverse lipid transport agonist is selected from the group consisting of: an HDL, a peptide with HDL-like physiological effects, a peptide with HDL-like physiological effects complexed to a lipid, an HDL-mimetic agent, a CETP modulator, an SRB1 modulator, an LXR/RXR agonist, an ABCA1 agonist, a PPAR agonist and an Apolipoprotein A-I (APOA1) mimetic peptide/phospholipid complex.

[007] In this latter case, administering the APOA1 mimetic peptide/phospholipid complex may include injecting the APOA1 mimetic peptide/phospholipid complex in the subject. Examples of dosages in this case are of from about 1 μg to about 10 g per kg body weight of the subject, about 1 mg to about .5 g per kg body weight of the subject, and about 25 mg per kg body weight of the subject.

[008] For example, the APOA1 mimetic peptide has the sequence of SEQ ID NO: 1 found herein below, and the APOA1 mimetic peptide may be complexed with egg sphingomyelin and 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). [009] In some embodiments, the subject is a mammal, for example a human.

[001O] A method of controlling a diastolic dysfunction in a subject, the method comprising providing, in a subject in need of controlling a diastolic dysfunction, an increased amount of reverse cholesterol transport for controlling the diastolic dysfunction..

[0011] In yet another broad aspect, the invention provides a method for preventing or reversing diastolic dysfunction, the method comprising administering to a patient in need thereof a reverse lipid transport agonist.

[0012] In yet another broad aspect, the invention provides a method for controlling a diastolic dysfunction in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a reverse lipid transport agonist.

[0013] For example, controlling the diastolic dysfunction may include reducing a rate of progression of the diastolic dysfunction, or reversing, at least in part, the diastolic dysfunction.

[0014] In yet another broad aspect, the invention provides the use of a reverse lipid transport agonist for controlling diastolic dysfunction in a subject.

[0015] In yet another broad aspect, the invention provides the use of a reverse lipid transport agonist for the manufacture of a pharmaceutical composition of matter for controlling diastolic dysfunction in a subject. [0016] In yet another broad aspect, the invention provides a method of preventing or treating a diastolic dysfunction in a subject, the method comprising administering, to a subject in need of preventing or treating a diastolic dysfunction, a therapeutically effective amount of a an Apolipoprotein A-I (APOA1) mimetic peptide/phospholipid complex to prevent or treat said diastolic dysfunction.

[0017] In a variant, the method comprises the administration of a therapeutically effective amount of a compound, referred to hereinafter as compound A, that mimics biologic properties of Apolipoprotein A-I (APOA1). Compound A and other suitable compounds are described in US Patent Nos. 6,287,590, issued Sep. 11 , 2001 , and 6,506,799, issued Jan. 14, 2003, which are hereby incorporated by reference in their entirety. Indeed, it is believed that in view of current knowledge regarding the action of the compounds and molecules described in these Patents, results similar to those presented herein are obtainable with these compounds and molecules.

[0018] An experimental study was performed to determine if APOA1 mimetic peptide infusions could induce reduction of diastolic dysfunction, and more specifically, left ventricular diastolic dysfunction.

[0019] Experimental approach: Twelve New-Zealand White male rabbits received a cholesterol-enriched diet and vitamin D₂ until significant aortic valve stenosis was detected by echocardiography. The enriched diet was then stopped to mimic cholesterol-lowering therapy and animals were randomized to receive saline (control group, π=6) or an APOA1 mimetic peptide (treated group, n=6), 3 times per week for 2 weeks. Before sacrifice, left ventricular diastolic dysfunction was studied using transthoracic echocardiography and classified either as normal, mild, moderate or severe dysfunction based on established criteria.

[0020] Results: At the end of the treatment, left ventricular diastolic filling patterns were distributed differently among groups (P=0.018). Left ventricular diastolic dysfunction (DD) was attenuated by APOA1 mimetic peptide infusions (33.3% of normal DD and 66.6% of mild DD vs. 66.6% of mild DD and 33.3% of severe DD for control rabbits).

[0021] Conclusions and implications: Infusions of APOA1 mimetic peptide lead to reduction of left ventricular diastolic dysfunction in a hypercholesterolemic rabbit model. Treatment of diastolic dysfunction is a new application for HDL-based therapies.

[0022] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In the appended drawings:

[0024] The only Figure illustrates the effect of the proposed treatment by comparing the distribution of diastolic dysfunction severity in control (upper panel) and treated (in lower panel) subjects as a function of time.

DETAILED DESCRIPTION [0025] In this document:

[0026] The term "treating" or "treatment" of a state, disease, disorder or condition includes:

[0027] (1) preventing or delaying the appearance of clinical symptoms of the state, disease, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disease, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disease, disorder or condition;

[0028] (2) inhibiting the state, disease, disorder or condition, i.e., arresting or reducing the development of the state, disease, disorder or condition or at least one clinical or subclinical symptom thereof; or

[0029] (3) relieving the state, disease, disorder or condition, i.e., causing regression of the state, disease, disorder or condition or at least one of its clinical or subclinical symptoms.

[0030] The benefit to a subject receiving treatment is either statistically significant or at least perceptible to the subject or to the physician.

[0031] The term "subject" includes mammals (especially humans) and other animals, such as domestic animals (e.g., household pets including cats and dogs) and non-domestic animals (such as wildlife).

[0032] A "therapeutically effective amount" means the amount of a compound that, when administered to a subject for treating a state, disease, disorder or condition, is sufficient to effect such treatment. The "therapeutically effective amount" will vary depending on the compound, the state, disease, disorder or condition and its severity and the age, weight, physical condition and responsiveness of the subject receiving treatment.

[0033] Pharmaceutical Compositions

[0034] The pharmaceutical composition of the present invention comprises at least one compound of the present invention and a pharmaceutically acceptable excipient (such as a pharmaceutically acceptable carrier or diluent). Preferably, the pharmaceutical composition comprises a therapeutically effective amount of the compound(s) of the present invention. The compound of the present invention may be associated with a pharmaceutically acceptable excipient (such as a carrier or a diluent) or be diluted by a carrier, or enclosed within a carrier which can be in the form of a capsule, sachet, paper or other container.

[0035] Examples of suitable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin, lactose, terra alba, sucrose, dextrin, magnesium carbonate, sugar, cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hydroxymethylcellulose and polyvinylpyrrolidone.

[0036] The carrier or diluent may include a sustained release material, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. [0037] The pharmaceutical composition may also include one or more pharmaceutically acceptable auxiliary agents, wetting agents, emulsifying agents, suspending agents, preserving agents, salts for influencing osmotic pressure, buffers, sweetening agents, flavoring agents, colorants, or any combination of the foregoing. The pharmaceutical composition of the invention may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the subject by employing procedures known in the art.

[0038] The pharmaceutical compositions of the present invention may be prepared by conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy, 20th Ed., 2003 (Lippincott Williams & Wilkins). For example, the active compound can be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier, which may be in the form of an ampoule, capsule, sachet, paper, or other container. When the carrier serves as a diluent, it may be a solid, semi-solid, or liquid material that acts as a vehicle, excipient, or medium for the active compound. The active compound can be adsorbed on a granular solid container, for example, in a sachet.

[0039] The pharmaceutical compositions may be in conventional forms, for example, capsules, tablets, aerosols, solutions, suspensions or products for topical application.

[0040] The route of administration may be any route which effectively transports the active compound of the invention to the appropriate or desired site of action. Suitable routes of administration include, but are not limited to, oral, nasal, pulmonary, buccal, subdermal, intradermal, transdermal, parenteral, rectal, depot, subcutaneous, intravenous, intra urethra I, intramuscular, intranasal, ophthalmic (such as with an ophthalmic solution) or topical (such as with a topical ointment).

[0041] The experiments described herein below show that a strategy directed to increasing the efficiency of the reverse lipid transport mechanism through the use of a suitable compound is a possible approach in the treatment and prevention of diastolic dysfunction.

[0042] The compound used to test this hypothesis, hereinafter referred to as Compound A, is a lipoprotein that mimics biologic properties of apolipoprotein A-I (APOA1). This type of compound, an APOA1 mimetic or agonist, is described in further detail in U.S. Patent No. 6,376,464 titled "Lipid complexes of APO A-1 agonist compounds," issued to Dasseux et al. on April 23, 2002. This document is hereby incorporated by reference in its entirety.

[0043] Briefly, these compounds include peptides, or analogues thereof, which are capable of forming amphipathic alpha-helices in the presence of lipids and which mimic the activity of APOA1. They are therefore referred-to as APOA1 agonists. The agonists have as their main feature a "core" peptide composed of 15 to 29 amino acid residues, preferably 22 amino acid residues, or an analogue thereof wherein at least one amide linkage in the peptide is replaced with a substituted amide, an isostere of an amide or an amide mimetic.

[0044] These APOA1 agonists are based, in part, on the discovery that altering certain amino acid residues in the primary sequence of the 22-mer consensus sequence disclosed in Venkatachalapathi et al., 1991 , MoI. Conformation and Biol. Interactions, Indian Acad. Sci. B: 585-596 (PVLDEFREKLNEELEALKQKLK; hereinafter "Seg rest's consensus 22-mer" or "consensus 22-mer") that were thought to be critical for activity, yields synthetic peptides which exhibit activities that approach, or in some embodiments even exceed, the activity of native APOA1. It was discovered that replacing three charged amino acid residues in Segrest's consensus 22-mer peptide (Glu-5, Lys-9 and Glu-13) with a hydrophobic Leu residue provides peptides that mimic the structural and functional properties of APOA1 to a degree that was unprecedented in the art.

[0045] Based on their known biological activity and structures, it is believed that other compounds, such as the compounds presented in the above-mentioned U.S. Patent No. 6,376,464, will show effects similar to Compound A.

[0046] In addition, while the reverse lipid transport agonist function of Compound A, and of other related compounds, is hypothesized to be related to improvements in the treatment and prevention of diastolic dysfunction, other aspects of Compound A are also hypothesized to play a role in the beneficial effects of Compound A and related compounds. For example, the effect of the APOA1 mimetic could be through other functions of HDL-related therapies, such as decreased inflammation or improved endothelial function. For example, the phospholipid composition of the mimetic may have its importance in the antiinflammatory action.

[0047] Example

[0048] Methods

[0049] Animals and experiments [0050] Animal care and procedures complied with the Canadian Council on Animal Care guidelines and were approved by the institutional ethics committee for animal research.

[0051] Twelve male New-Zealand White rabbits (2.7-3.0 kg, aged 12-13 weeks) were fed with a 0.5% cholesterol-enriched diet (Harlan, Indianapolis, Indiana, USA) plus vitamin D₂ (50000 IU per day; Sigma, Markham, Canada) in the drinking water until significant AVS, as defined by a >10% decrease of aortic valve area or of the transvalvular velocities ratio (V₁ZV₂), could be detected by echocardiography (as described in Busseuil D, Shi Y, Mecteau M, Brand G, Kemaleguen AE, Thorin E, Latour JG, Rheaume E, Tardif JC (2008). Regression of aortic valve stenosis by ApoA-l mimetic peptide infusions in rabbits. Brit J Pharm 154(4):765-73, the contents of which are hereby incorporated by reference in its entirety).

[0052] The animals then returned to a standard diet (without vitamin D₂) to mimic cholesterol-lowering therapy and were randomly assigned to receive either saline

(control group, n=6) or the APOA1 mimetic peptide (treated group, n=6). Rabbits were given injections through the marginal ear vein of saline or of the APOA1 mimetic peptide (25 mg/kg) complexed with phospholipids, 3 times per week for 2 weeks. Echocardiograms were performed serially (see Echocardiography Methods), including every 3 to 4 days throughout the randomized treatment period. Two days after their last infusion, the animals underwent a final echocardiogram and were sacrificed.

[0053] APOA1 mimetic peptide [0054] The APOA1 mimetic peptide (Compound A) of sequence: H-Pro-Val-Leu- Asp-Leu-Phe-Arg-Glu-Leu-Leu-Asn-Glu-Leu-Leu-Glu-Ala-Leu-Lys-Gln-Lys-Leu- Lys-OH (SEQ ID NO 1) was synthesized by Polypeptide Laboratories (Torrance, CA, USA), and purity assessed by high performance liquid chromatography and mass spectral analysis was greater than 98%. The peptide was complexed with egg sphingomyelin and 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids. Alabaster, AL, USA) in a 1 :1 :1 weight ratio by mixing the components in saline and performing multiple heating and cooling cycles until the solution appeared perfectly clear. Fresh solution was prepared every week under sterile conditions and kept at 4°C.

[0055] Echocardiography

[0056] Transthoracic echocardiographic studies were performed at baseline, on a weekly basis starting at 8 weeks of hypercholesterolemic diet until significant AVS developed, and then after 4, 7, 10 and 14 days of APOA 1 mimetic peptide or saline control treatments. Studies were carried out with a phased-array probe 1OS (4.5 ~ 11.5 Megahertz) and a Vivid 7 Dimension system (GE Healthcare Ultrasound, Horten, Norway). Intra-muscular injections of ketamine (45 mg/kg) and midazolam (0.75 mg/kg) were used for sedation.

[0057] Left ventricular (LV) M-mode spectrum was obtained in parasternal long- axis view to measure LV diameters at both end cardiac diastole (LVDd) and systole (LVDs). LV fractional shortening was calculated as (LVDd - LVDs) / LVDd x 100%. Teicholz method was employed to calculate LV volumes and LV ejection fraction (EF). Pulsed wave Doppler was used to evaluate transmitral flow (TMF) and pulmonary venous flow (PVF) in apical 4-chamber view. TMF was used to measure the peak velocities during early filling (E) and atrial filling (A) and to calculate the E/A ratio. PVF was used to measure the systolic flow (S), diastolic flow (D) and reversed atrial flow (Ar). LV basal lateral peak systolic velocities (Sm) and mitral annulus velocities during early filling (Em) and atrial filling (Am) were derived by tissue Doppler imaging (TDI). The time intervals from the end of Am to the beginning of Em (b), and from the beginning to the end of Sm (a) were also measured on lateral wall TDI.

[0058] Left ventricular diastolic dysfunction (LVDD) was classified according to published criteria (Khouri et a/., 2004). To further evaluate LVDD, left atrium (LA) M-mode spectrum was obtained in parasternal long-axis view at the aortic valve level and LA dimensions were measured in both end cardiac diastole and systole. LA fractional shortening was calculated as (systolic dimension - diastolic dimension) / systolic dimension x 100%. The average of 3 consecutive cardiac cycles was used for each measurement.

[0059] All echocardiographic imaging and measurements were performed throughout the protocol by the same experienced investigator blinded to randomized treatment assignment.

[0060] Statistical analyses

[0061] Diastolic dysfunction classification was compared across groups using chi- square test. All analyses were done with SAS version 9.1 (SAS Institute Inc., Cary, NC, USA) and conducted at the 0.05 significance level.

[0062] Results [0063] With Reference to the only Figure, the distribution of the pattern of DD classification evolved differently in the control and treated groups. Whereas severe DD appeared in some control animals after 7 days of treatment, no moderate nor severe DD could be detected in treated animals.

[0064] At the end of the treatment, LV diastolic filling patterns were distributed differently among groups (P=O.018). Left ventricular diastolic dysfunction (DD) was attenuated by APOA1 mimetic peptide infusions (33.3% of normal DD and 66.6% of mild DD vs. 66.6% of mild DD and 33.3% of severe DD for control rabbits).

[0065] Conclusion

[0066] Infusions of an APOA1 mimetic peptide lead to reduction of left ventricular diastolic dysfunction in a hypercholesterolemic rabbit model. Treatment of diastolic dysfunction may represent a new application for HDL-based therapies.

[0067] This example suggests that similar results are obtainable in humans using any suitable HDL-based therapy, such as for example one or more infusion(s) or bolus(es) of HDL or peptide (with or without lipids) with HDL-like effects, orally administered HDL-mimetic agents, and/or the administration of cholesteryl ester transfer protein (CETP) modulators, or scavenger receptor class B, member 1 (SRB1) modulators or liver X receptor (LXR)/retinoid X receptor (RXR) agonists, or ATP-binding cassette transporter-1 (ABCA1) agonists, or peroxisome proliferator- activated receptor (PPAR) agonists, among others.

[0068] While the experiments described herein concerned the regulation of diastolic dysfunction, one of ordinary skilled in the art will readily appreciate that these experiments may be predictive of biological effects in humans or other mammals and/or may serve as models for use of the present invention in humans or other mammals for any other similar cardiac dysfunction.

[0069]Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of preventing or treating a diastolic dysfunction in a subject, said method comprising administering, to a subject in need of preventing or treating a diastolic dysfunction, a therapeutically effective amount of a reverse lipid transport agonist to prevent or treat said diastolic dysfunction.

2. The method as defined in claim 1 , wherein said reverse lipid transport agonist is a reverse cholesterol transport agonist.

3. The method as defined in claim 1 , wherein said diastolic dysfunction is a ventricular diastolic dysfunction.

4. The method as defined in claim 1 , wherein said diastolic dysfunction is a left ventricular diastolic dysfunction.

5. The method as defined in claim 1 , wherein said reverse lipid transport agonist is selected from the group consisting of a high density lipoprotein (HDL), a peptide with HDL-like physiological effects, a peptide with HDL- like physiological effects complexed to a lipid, an HDL-mimetic agent, a cholesteryl ester transfer protein (CETP) modulator, a scavenger receptor class B, member 1 (SRB1) modulator, a liver X receptor / retinoid X receptor (LXR/RXR) agonist, an ATP-binding cassette transporter-1 (ABCA1) agonist and a peroxisome proliferator-activated receptor (PPAR) agonist.

6. The method as defined in claim 1 , wherein said reverse lipid transport agonist is an Apolipoprotein A-I (APOA1) mimetic peptide/phospholipid complex.

7. The method as defined in claim 6, wherein administering said APOA1 mimetic peptide/phospholipid complex includes injecting said APOA1 mimetic peptide/phospholipid complex in said subject.

8. The method as defined in claim 7, wherein said APOA1 mimetic peptide/phospholipid complex is injected at a dosage of from about 1 μg to about 10 g per kg body weight of said subject.

9. The method as defined in claim 8, wherein said APOA1 mimetic peptide/phospholipid complex is injected at a dosage of from about 1 mg to about .5 g per kg body weight of said subject.

10. The method as defined in claim 9, wherein said APOA1 mimetic peptide/phospholipid complex is injected a dosage of about 25 mg per kg body weight of said subject.

11. The method as defined in claim 6, wherein a APOA1 mimetic peptide of said APOA1 mimetic peptide/phospholipid complex has a sequence set forth in SEQ ID NO: 1.

12. The method as defined in claim 6, wherein a APOA1 mimetic peptide of said APOA1 mimetic peptide/phospholipid complex is complexed with egg sphingomyelin and 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC).

13. The method as defined in claim 1 , wherein said reverse lipid transport agonist comprises a polypeptide having a sequence set forth in SEQ ID NO: 1.

14. The method as defined in claim 1 , wherein said subject is a mammal.

15. The method as defined in claim 14, wherein said subject is a human.

16. A method of controlling a diastolic dysfunction in a subject, said method comprising providing, in a subject in need of controlling a diastolic dysfunction, an increased amount of reverse cholesterol transport for controlling said diastolic dysfunction.

17. The method as defined in claim 16, wherein controlling said diastolic dysfunction includes reducing a rate of progression of said diastolic dysfunction.

18. The method as defined in claim 16, wherein controlling said diastolic dysfunction includes reversing, at least in part, said diastolic dysfunction.

19. Use of a reverse lipid transport agonist for controlling diastolic dysfunction in a subject.

20. Use of a reverse lipid transport agonist for the manufacture of a pharmaceutical composition of matter for controlling diastolic dysfunction in a subject.

21. A method of controlling a diastolic dysfunction in a subject, said method comprising administering, to a subject in need of controlling a diastolic dysfunction, a therapeutically effective amount of a reverse lipid transport agonist for controlling said diastolic dysfunction.

22.A method of preventing or treating a diastolic dysfunction in a subject, said method comprising administering, to a subject in need of preventing or treating a diastolic dysfunction, a therapeutically effective amount of a an Apolipoprotein A-I (APOA1) mimetic peptide/phospholipid complex to prevent or treat said diastolic dysfunction.

23. Use of an Apolipoprotein A-I (AP0A1) mimetic peptide/phospholipid complex for controlling diastolic dysfunction in a subject.

24. Use of an Apolipoprotein A-I (APOA1) mimetic peptide/phospholipid complex for the manufacture of a pharmaceutical composition of matter for controlling diastolic dysfunction in a subject.