WO1999066930A1 - Compositions and methods for treating elevated blood cholesterol - Google Patents
Compositions and methods for treating elevated blood cholesterol Download PDFInfo
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- WO1999066930A1 WO1999066930A1 PCT/US1999/013888 US9913888W WO9966930A1 WO 1999066930 A1 WO1999066930 A1 WO 1999066930A1 US 9913888 W US9913888 W US 9913888W WO 9966930 A1 WO9966930 A1 WO 9966930A1
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- coa reductase
- asp
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- reductase inhibitor
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- 0 *C(C[C@](C1)O)OC1O Chemical compound *C(C[C@](C1)O)OC1O 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/005—Enzyme inhibitors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/55—Protease inhibitors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Definitions
- the present invention relates to compositions and methods for treating elevated blood cholesterol in a mammal while counteracting potential adverse side effects such as myopathy.
- the compositions useful herein comprise the combination of a pharmaceutically effective amount of a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (hereafter "HMG-CoA reductase inhibitor”) and a caspase inhibitor to a mammal in need thereof.
- HMG-CoA reductase inhibitor 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor
- LDL low density lipoprotein
- HDL high density lipoprotein
- this therapy is not easy to administer or tolerate and was therefore often unsuccessful except in specialist lipid clinics.
- the fibrates produce a moderate reduction in LDL cholesterol accompanied by increased HDL cholesterol and a substantial reduction in triglycerides, and because they are well tolerated these drugs have been more widely used.
- Probucol produces only a small reduction in LDL cholesterol and also reduces HDL cholesterol, which, because of the strong inverse relationship between HDL cholesterol level and CHD risk, is generally considered undesirable.
- lovastatin the first inhibitor of HMG-CoA reductase to become available for prescription in 1987, for the first time physicians were able to obtain large reductions in plasma cholesterol with very few adverse effects.
- lovastatin, simvastatin and pravastatin all members of the HMG-CoA reductase inhibitor class, slow the progression of atherosclerotic lesions in the coronary and carotid arteries.
- Simvastatin and pravastatin have also been shown to reduce the risk of coronary heart disease events, and in the case of simvastatin a highly significant reduction in the risk of coronary death and total mortality has been shown by the Scandinavian Simvastatin Survival Study. This study also provided some evidence for a reduction in cerebro vascular events.
- HMG-CoA reductase inhibitors can cause potentially adverse side effects such as myopathy and related disorders in a small percentage of patients.
- Myopathy is characterized by muscle pain and weakness.
- Caspases belong to a broad class of enzymes known as proteases, which are enzymes that hydrolyze peptide bonds. Specfically, caspases are cysteine proteases that preferentially target and cleave peptide sequences having an aspartic acid moiety. Caspases are believed to be involved in the normal cell turnover process and are mediators of apoptosis. See Nicholson and Thornberry, 1997, "Caspases: killer proteases", TIBS , vol.
- caspase inhibitors can block the apoptosis, i.e. programmed cell death, that can be induced by HMG-CoA reductase inhibitors.
- caspase inhibitors have not previously been investigated either in vitro or in vivo for their ability to mitigate the potentially adverse myopathy side effects that can be associated with HMG-CoA reductase inhibitor therapy for treating or preventing elevated blood cholesterol.
- HMG-CoA reductase inhibitor therapy for treating or preventing elevated blood cholesterol.
- CoA reductase inhibitor and a caspase inhibitor is effective for treating or preventing elevated blood cholesterol while mitigating the potentially adverse myopathy side effects that can be associated with the therapy.
- the combination has the advantage of providing increased safety and better patient compliance, which should maximize therapeutic efficacy.
- the caspase inhibitor blocks the potentially harmful effect of the HMG-CoA reductase inhibitor on muscle cells.
- the caspase inhibitor is believed to interfere with apoptosis which can potentially be induced in muscle cells by the HMG-CoA reductase inhibitor. It is an object of the present invention to provide compositions comprising the combination of an HMG-CoA reductase inhibitor and a caspase inhibitor.
- the present invention relates to a pharmaceutical composition comprising an HMG-CoA reductase inhibitor and a caspase inhibitor.
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising a pharmaceutically-effective amount of an HMG-CoA reductase inhibitor and an amount of a caspase inhibitor effective to counteract HMG-CoA reductase-associated myopathy.
- the present invention relates to a method for treating or preventing elevated blood cholesterol in a mammal in need thereof comprising administering an HMG-CoA reductase inhibitor and a caspase inhibitor. In further embodiments, the present invention relates to a method for treating or preventing elevated blood cholesterol in a mammal in need thereof comprising sequentially administering a caspase inhibitor and an HMG-CoA reductase inhibitor.
- the present invention relates to the use of a composition in the manufacture of a medicament for treating or preventing elevated blood cholesterol in a mammal in need thereof, said composition comprising an HMG- CoA reductase inhibitor and a caspase inhibitor.
- the present invention relates to the use of a composition comprising an HMG-CoA reductase inhibitor and a caspase inhibitor for treating or preventing elevated blood cholesterol in a mammal in need thereof. All percentages and ratios used herein, unless otherwise indicated, are by weight.
- the invention hereof can comprise, consist of, or consist essentially of the essential as well as optional ingredients, components, and methods described herein.
- FIGURE Figure 1 shows that activation of Mstl cleavage by 10 ⁇ M lovastatin is blocked by the caspase inhibitors Z-Asp-Glu-Val-Asp-FMK, Z-Val-Ala-Asp-FMK, or Z-Tyr-Val-Ala-Asp-FMK.
- Osteoclast like cells are purified from cocultures by sequential treatment of culture dishes with collagenase and EDT A. Cells are then treated for 17 hours with lovastatin, and cell lysates are analyzed by an in-gel kinase assay using myelin basic protein as a substrate. Lane 1 is a no-treatment control.
- Lane 2 shows treatment with 20 ⁇ M Z-Asp-Glu-Val-Asp-FMK.
- Lane 3 shows treatment with 20 ⁇ M Z-Val-Ala-Asp-FMK.
- Lane 4 shows treatment with 20 ⁇ M Z-Tyr-Val- Ala-Asp-FMK.
- Lane 5 shows treatment with 10 ⁇ M lovastatin.
- Lane 6 shows treatment with the combination of 10 ⁇ M lovastatin and 20 ⁇ M Z-Asp-Glu-Val-Asp- FMK.
- Lane 7 shows treatment with the combination of 10 ⁇ M lovastatin and 20 ⁇ M Z-VAl-Ala-Asp-FMK.
- Lane 8 shows treatment with the combination of 10 ⁇ M lovastatin and 20 ⁇ M Z-Tyr-Val-Ala-Asp-FMK.
- the present invention relates to compositions and methods for treating or preventing elevated blood cholesterol in a mammal in need of such treatment, while counteracting the occurence of adverse myopathy effects.
- the compositions comprise a pharmaceutically effective amount of an HMG-CoA reductase inhibitor and a pharmaceutically effective amount of a caspase inhibitor.
- pharmaceutically effective amount means that amount of the HMG-CoA reductase inhibitor or caspase inhibitor that will elicit the desired therapeutic effect or response or provide the desired benefit when administered in accordance with the desired treatment regimen.
- a prefered pharmaceutically effective amount of the HMG-CoA reductase inhibitor is an amount that is effective for treating or preventing elevated blood cholesterol.
- a preferred pharmaceutically effective amount of the caspase inhibitor is an amount that will block or mitigate the occurrence of adverse myopathy effects, while not blocking, or only minimally blocking, the therapeutic blood cholesterol effects of the HMG-CoA reductase inhibitor.
- counteracting the occurence of adverse myopathy effects means preventing, decreasing, or lessening the occurrence of unwanted muscular effects, relative to treatment with a HMG-CoA reductase inhibitor alone.
- the term "until the desired therapeutic effect is achieved and/or maintained”, as used herein, means that the therapeutic agent or agents are continuously administered, according to the dosing schedule chosen, up to the time that the clinical or medical effect sought for the disease or condition being treated is observed by the clinician or researcher.
- the pharmaceutical composition is continuously administered until the desired change in blood cholesterol is observed. In such instances, achieving a decrease in blood cholesterol is a desried objective.
- the pharmaceutical composition is continuously administered for as long as necessary to prevent the undesired condition. In such instances, maintenance of blood cholesterol level is often an objective as well as prevention of or reducing the risk of developing atherosclerotic disease or cardiovascular disorders such as heart attack and stroke.
- compositions of the present invention are provided.
- compositions of the present invention comprise a pharmaceutically effective amount of an HMG-CoA reductase inhibitor and a pharmaceutically effective amount of a caspase inhibitor. These compositions are useful for treating or preventing elevated blood cholesterol in a mammal in need thereof while counteracting the potentially adverse effects, such as myopathy, that can be associated with the administration of the HMG-CoA reductase inhibitor.
- compositions herein comprise a compound which inhibits the enzyme, HMG-CoA reductase.
- Compounds which have inhibitory activity for HMG- CoA reductase can be readily identified by using assays well-known in the art. See U.S. Patent No. 4,231,938, to Monoghan et al., issued November 4, 1980 and U.S. Patent No. 5,354,772, to Kathawal, issued October 11, 1994, both of which are incorporated by reference herein in their entirety.
- HMG-CoA reductase inhibitors examples include but are not limited to lovastatin (MEVACOR®; see U.S. Patent No. 4,231,938, already cited above and incorporated by reference herein), simvastatin
- HMG-CoA reductase inhibitor is intended to include all pharmaceutically acceptable lactone and open acid (that is where the lactone ring is opened to form the free acid), as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefor the use of such lactone, open acid, salt, and ester forms is included within the scope of this invention.
- the HMG-CoA reductase inhibitor is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, mevastatin, and the pharmaceutically acceptable lactones, open acids, salts, and esters thereof, and mixtures thereof.
- the HMG-CoA reductase inhibitor is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, and the pharmaceutically acceptable lactones, open acids, salts, and esters thereof, and mixtures thereof. More preferably, the HMG-CoA reductase inhibitor is selected from the group consisting of lovastatin, simvastatin, and the pharmaceutically acceptable lactones, open acids, salts, and esters thereof, and mixtures thereof.
- HMG-CoA reductase inhibitors can be represented by the chemical formula
- Z is selected from the group consisting of:
- Rl is C1-C10 alkyl
- R2 is selected from the group consisting of C1-C3 alkyl, hydroxy, oxo, and C1-C3 hydroxy substituted alkyl,
- R3 is selected from the group consisting of hydrogen, hydroxy, C1-C3 alkyl, and Ci-
- C3 hydroxy substituted alkyl, a, b, c, and d are all single bonds, or a and c are double bonds, or b and d are double bonds, or one of a, b, c, and d is a double bond, and n is 0, 1, or 2; b)
- X is selected from the group consisting of N[CH(CH3)2] and CH(CH2)3CH3
- R4 and R5 are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, C1 -C4 alkyl, C1-C4 alkoxy, and trifluoromethyl
- R6, R7, R8, and R9 are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, C1-C4 alkyl, and C1-C4 alkoxy.
- pharmaceutically acceptable salts as used herein in referring to the HMG-CoA reductase inhibitors shall mean non-toxic salts of the compounds employed in this invention which are generally prepared by reacting the free acid with a suitable organic or inorganic base.
- salt forms of HMG-CoA reductase inhibitors include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, ole
- esters as used herein in referring to the HMG-CoA reductase inhibitors is used in its standard meaning to denote the condensation product of a carboxylic acid and an alcohol. Ester derivatives of the described compounds can function as prodrugs which, when absorbed into the bloodstream of a warm-blooded animal, can cleave in such a manner as to release the drug form and permit the drug to afford improved therapeutic efficacy.
- lactones is used herein in referring to the HMG-CoA reductase inhibitors is used in its standard meaning to denote a cyclic condensation product of a carboxylic acid and an alcohol, i.e. a cyclic ester.
- open acid is used herein in referring to the HMG-CoA reductase inhibitors to denote that the lactone ring is open, i.e. uncyclized, to form the free acid. It is recognized that mixtures of two or more HMG-CoA reductase inhibitors can be utilized.
- the dosage regimen utilizing a HMG-CoA reductase inhibitor is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt or ester thereof employed. A consideration of these factors is well within the purview of the ordinarily skilled clinician for the purpose of determining the therapeutically effective or prophylactically effective dosage amounts needed to prevent, counter, or arrest the progress of the condition.
- patient includes mammals, especially humans. Administering of the drug or drugs to the patient includes both self-administration and administration to the patient by another person.
- the precise dosage of the HMG-CoA reductase inhibitor will vary with the dosing schedule, the particular compound chosen, the age, size, sex and condition of the mammal or human, the nature and severity of the disorder to be treated, and other relevant medical and physical factors. Thus, a precise pharmaceutically effective amount cannot be specified in advance and can be readily determined by the caregiver or clinician. Appropriate amounts can be determined by routine experimentation from animal models and human clinical studies.
- the amounts of the HMG-CoA reductase inhibitor can be the same or similar to those amounts which are employed for anti- hypercholesterolemic treatment and which are described in the Physicians' Desk Reference (PDR), 52 nd Ed. of the PDR, 1998 (Medical Economics Co), which is incorporated by reference herein in its entirety.
- the doses can be the same or similar to those amounts which are known in the art.
- the HMG-CoA reductase inhibitors can be administered via a wide variety of routes including oral administration, intravenous administration, intranasal administration, injections, ocular administration, and the like.
- a preferred route of delivery is oral administration.
- Oral dosage amounts of the HMG-CoA reductase inhibitor are from about 1 to 200 mg/day, and more preferably from about 5 to 160 mg/day. However, dosage amounts will vary depending on the potency of the specific HMG-CoA reductase inhibitor used as well as other factors as noted above.
- An HMG-CoA reductase inhibitor which has sufficiently greater potency may be given in sub- milligram daily dosages.
- the HMG-CoA reductase inhibitor may be administered from 1 to 4 times per day, and preferably once per day.
- the daily dosage amount for simvastatin can be selected from 5 mg, 10 mg, 20 mg, 40 mg, and 80 mg; for lovastatin, 10 mg, 20 mg, 40 mg and 80 mg; for fluvastatin sodium, 20 mg, 40 mg and 80 mg; for pravastatin sodium, 10 mg, 20 mg, and 40 mg; and for atorvastatin calcium, 10 mg, 20 mg, and 40 mg.
- compositions of the present invention comprise a pharmaceutically effective amount of a caspase inhibitor.
- the caspase inhibitors useful herein are generally relatively short aspartic acid- containing peptides, although non-peptide inhibitors are also intended as being within the scope of the present invention.
- “relatively short”, as used herein means that the peptides typically contain from about 3 to about 5 amino acids in length.
- “aspartic acid-containing” is meant that these peptides comprise at least one aspartic acid moiety, preferably at the carboxy-terminal end. These peptides are preferably blocked at both the amino and carboxy terminal ends with blocking groups.
- caspase inhibitors useful herein can be represented by the following chemical formula
- (ATBG) is an amino terminal blocking group
- (AA) is an amino acid moiety
- "Asp” is aspartic acid moiety
- (CTBG) is a carboxy terminal blocking group
- n is an integer from about 2 to about 4.
- the amino acid (AA) can be selected from any of the naturally occurring amino acids, the D-enantiomers of the naturally-occurring amino acids (for example, D-alanine), and non-naturally occurring amino acids (for example, e.g., 3-aminopropionic acid and N-methyl glycine).
- the (ATBG) and (CTBG) moities are selected from any of the blocking groups that are well known to peptide chemists of ordinary skill in the art.
- Nonlimiting examples of (ATBG) moieties are bezyloxycarbonyl group (also known as the cbz or Z group) and the t-butoxycarbonyl group (also known as the boc group), and the acyl group.
- Nonlimiting examples of (CTBG) moieties are alkyl groups (for example methyl and ethyl esters), the benzyl group, and the fluoromethyl keto group [which is abbreviated as (OMe)-CH2F or FMK].
- Nonlimiting examples of caspase inhibitors useful herein are disclosed in U.S. Patent No. 5,210, 272, to Palmer, issued May 11, 1993, U.S. Patent 5,101,068, to Palmer et al., issued March 31, 1992, and U.S. Patent No. 4,518,528, to Rasnick, issued May 21, 1985, which are all incorporated by reference herein in their entirety.
- Preferred caspase inhibitors useful herein are selected from the group consisting of Z-Val-Ala-Asp-FMK (which has a molecular weight of about 468), Z- Asp-Glu-Val- Asp-FMK (which has a molecular weight of about 668), and Z-Tyr-Val- Ala-Asp-FMK (which has a molecular weight of about 630) MW 630.
- Z-Val-Ala-Asp-FMK which has a molecular weight of about 468
- Z- Asp-Glu-Val- Asp-FMK which has a molecular weight of about 668
- Z-Tyr-Val- Ala-Asp-FMK which has a molecular weight of about 630
- the precise dosage of the caspase inhibitor will vary with the dosing schedule, the particular compound chosen, the age, size, sex and condition of the mammal or human, the nature and severity of the disorder to be treated, and other relevant medical and physical factors. Thus, a precise pharmaceutically effective amount cannot be specified in advance and can be readily determined by the caregiver or clinician. Appropriate amounts can be determined by routine experimentation from animal models and human clinical studies. Generally, an appropriate amount is chosen to counteract the potentially adverse myopathy effects of the HMG-CoA reductase inhibitor. The amount should be below that level which will inhibit the desired bone resorption inhibiting effect of the nitrogen-contianing bisphosphonate.
- an effective oral dose of the caspase inhibitor is typically chosen so as to provide a local concentration in the esophagus from about 1 ⁇ M to about 100 ⁇ M, preferably about 10 ⁇ M, although other ranges can be used.
- Nonlimiting exemplary doses are about 1 ug/kg to about 100 ug/kg, preferably about 10 ug/kg, for a human subject.
- human doses which can be administered are generally in the range of about 0.1 mg/day to about 10 mg/day, preferably from about 0.25 mg/day to about 5 mg/day, and more preferably from about 0.5 mg/day to about 1.5 mg/day.
- a typical nonlimiting dosage amount would be about 0.75 mg/day.
- the pharmaceutical compositions herein comprise from about 0.1 mg to about 10 mg, preferably from about 0.25 mg to about 5 mg, and more preferably from about 0.5 mg to about 1.5 mg of the caspase inhibitor.
- a typical nonlimiting amount for is about 0.75 mg.
- Other components of the pharmaceutical compositions are generally in the range of about 0.1 mg/day to about 10 mg/day, preferably from about 0.25 mg/day to about 5 mg/day, and more preferably from about 0.5 mg/day to about 1.5 mg/day.
- Other components of the pharmaceutical compositions are generally in the range of about 0.1 mg/day to about 10 mg/day, preferably from about 0.25 mg/day to about 5 mg/day, and more preferably from about
- the HMG-CoA reductase inhibitor and the caspase inhibitor are typically administered in admixture with suitable pharmaceutical diluents, excipients, or carriers, collectively referred to herein as "carrier materials”, suitably selected with respect to oral administration, i.e. tablets, capsules, elixirs, syrups, powders, and the like, and consistent with conventional pharmaceutical practices.
- carrier materials suitably selected with respect to oral administration, i.e. tablets, capsules, elixirs, syrups, powders, and the like, and consistent with conventional pharmaceutical practices.
- the active ingredient can be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, croscarmellose sodium and the like;
- an oral, non-toxic, pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, croscarmellose sodium and the like
- the oral drug components can be combined with any oral, non- toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
- suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated.
- Suitable binders can include starch, gelatin, natural sugars such a glucose, anhydrous lactose, free-flow lactose, beta-lactose, and corn sweeteners, natural and synthetic gums, such as acacia, guar, tragacanth or sodium alginate, carboxymethyl cellulose, polyethylene glycol, waxes, and the like.
- Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
- the compounds used in the present method can also be coupled with soluble polymers as targetable drug carriers.
- Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropyl-methacrylamide, and the like.
- the present invention comprises methods for treating or preventing elevated blood cholesterol in mammals.
- the mammal is a human.
- compositions and methods of the present invention are administered and caried out until the desired therapeutic effect is achieved.
- the HMG-CoA reductase inhibitor and the caspase inhibitor are generally administered concurrently.
- the HMG-CoA reductase inhibitor and the caspase inhibitor can be adminsitered sequentially.
- the caspase inhibitor is administered first.
- Bone marrow cells are harvested from 6-week-old male Balb/C mice by flushing marrow spaces of freshly isolated long bones (tibiae and femora) with ⁇ -MEM (minimal essential media) containing penicillin/streptomycin (100 I.U./ml of each and 20 mM Hepes buffer). The bone marrow cells are suspended in ⁇ -MEM and the cells are filtered through an approximately 70 ⁇ m cell strainer. The filtrate is centrifuged at about 300 x g for about 7 minutes. The resulting pellet is resuspended in ⁇ -MEM supplemented with fetal calf serum (10 % v/v) and 10 nM 1, 25-(OH)2 vitamin D3.
- ⁇ -MEM minimal essential media
- penicillin/streptomycin 100 I.U./ml of each and 20 mM Hepes buffer
- the bone marrow cells are suspended in ⁇ -MEM and the cells are filtered through an approximately 70
- osteoblastic MB 1.8 cells are added to sub-confluent monolayers of osteoblastic MB 1.8 cells in cell culture plates and cultured for 7 days at 37DC in the presence of 5% CO2. Culture media is replenished ever other day. Fusion of the osteoclast precursor cells from bone marrow (with each other) to form multinucleated osteoclast-like cells typically occurs after about 7 days. Osteoclast-like cells are enriched by sequential treatment with collagenase (1 mg/mL in phosphate buffered saline) for one hour at 37°C and EDTA (0.2 g/L in phosphate buffered saline) for 20 min at 37°C.
- Non-adherent cells are rinsed away by washing with phosphate buffered saline.
- Osteoclast-like cells which are resistant to the sequential treatments are present at about 95% purity and are maintained in ⁇ -MEM supplemented with fetal calf serum (10 % v/v), 10 nM 1,25- (OH)2 vitamin D3, macrophage-colony-stimulating factor (5 ng/mL).
- the compounds to be evaluated are prepared as a solution of the desired concentration in ⁇ -MEM.
- examples of compounds that can be evaluated include HMG-CoA reductase inhibitorts such as lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, cerivastsin, mevastatin, and the pharmaceutically acceptable salts, esters, and lactones thereof, as well as compounds that block the effects of these HMG-CoA reductase inhibitors, such as caspase inhibitors such as Z-Val-Ala-Asp- FMK, Z-Asp-Glu-Val-Asp-FMK , and Z-Tyr-Val- Ala-Asp-FMK.
- HMG-CoA reductase inhibitorts such as lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, cerivastsin, mevastatin, and the pharmaceutically acceptable salt
- Combinations of compounds can also be evaluated.
- the solutions of the compounds to be evaluated are added to the cultures for a time period of 17-24 hours.
- No treatment controls (controls not treated with comcpounds) are prepared by adding equivalent volumes of ⁇ -MEM to the control dishes.
- HEPES N-(2- hydroxyethyl)piperazine-N'-(2-ethansulfonic acid) or Tris buffer containing the following: ⁇ -glycerophosphate (50 mM); Na3VO4 (ImM); NaF (ImM); Microcystin LR (1 ⁇ M); leupeptin (10 ⁇ g/ml); aprotinin (10 ⁇ g/ml); phenylmethyl sulfonylfluoride (1 mM).
- HEPES N-(2- hydroxyethyl)piperazine-N'-(2-ethansulfonic acid
- Protein concentrations are determined for each lysate and 5-20 ⁇ g are loaded into each lane of a SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel containing Myelin Basic Protein, or another kinase substrate, which has been polymerized into the gel at a concentration between 50-400 ⁇ g/ml.
- SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- Myelin Basic Protein or another kinase substrate, which has been polymerized into the gel at a concentration between 50-400 ⁇ g/ml.
- Molecular weight standards are also loaded into one or more lanes of the gels.
- In-gel kinase assays are run according to a standard procedure based on Kameshita and Fujisawa, 1989 (Anal. Biochem. 183:139-143) and of Gotoh et al., 1990 (Eur
- the proteins are electrophoresed in the above gels.
- the gels are then successively soaked in 50 mM HEPES, pH 7.6; 5 mM 2-mercaptoethanol and each of the following (for each wash): (a) 20% isopropanol; (b) no additions; (c) urea (6 M); (d) Urea (3 M); (e) Urea (0.75 M); and Tween 20 (0.05% vokvol).
- Kinase reactions are then run by first soaking the gels in 20 mM HEPES, pH 7.6; 20 mM MgCl2; 2 mM DTT and then in the same buffer containing 0.02 M ATP (non-radioactive) with ca. 1000 cpm/pmol 32p. ⁇ -ATP.
- the gels are then washed six times with 5% trichloroacetic acid and 1% pyrophosphate.
- the gels are then stained with Coomassie brilliant blue dye (0.125%) in 50% methanol, 10% acetic acid; destained with 30% methanol, 10% acetic acid; soaked in 2% glycerol; and dried using a gel dryer.
- the gels are then exposed to autoradiography film for times ranging from several hours to weeks.
- the bands observed in the autoradiographs representing the gels reflect kinase activities.
- Mst 1 (apparent molecular weight about 59 kDa)
- Mst 2 (apparent molecular weight about 60 kDa)
- a 34 kDa Mst kinase fragment are observed and identified by their migration as compared to the migration of molecular weight standards.
- the band intensities on the autoradiography film are quantitated by densitometry and comparisons between bands from untreated controls and bands from echistatin-treated cells provide the basis for the analyses.
- All the ingredients except magnesium stearate are blended together in a suitable mixer.
- the powder mixture is then granulated with adequate quantities of granulating solvent(s), e.g. water.
- the wet granulated mass is dried in a suitable dryer.
- the dried granulation is sized through a suitable screen.
- the sized granulation is mixed with magnesium stearate before tableting.
- the tablets may be coated if deemed necessary.
- Additional ingredients that may be added to the above include suitable color and mixtures of colors.
- the composition is useful for treating or preventing elevated blood cholesterol.
- the simvastatin is replaced by a HMG-CoA reductase inhibitor selected from lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, or mevastatin, and the caspase inhibitor is replaced by Z-Asp-Glu-Val- Asp-FMK or Z-Tyr-Val- Ala- Asp-FMK.
- a HMG-CoA reductase inhibitor selected from lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, or mevastatin
- the caspase inhibitor is replaced by Z-Asp-Glu-Val- Asp-FMK or Z-Tyr-Val- Ala- Asp-FMK.
- the ingredients are combined and blended together and are compressed using conventional tableting techniques.
- composition is useful for treating or preventing elevated blood cholesterol.
- the simvastatin is replaced by a HMG-CoA reductase inhibitor selected from lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, or mevastatin, and the caspase inhibitor is replaced by Z-Asp-Glu-Val- Asp-FMK or Z-Tyr-Val- Ala- Asp-FMK.
- a HMG-CoA reductase inhibitor selected from lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, or mevastatin
- the caspase inhibitor is replaced by Z-Asp-Glu-Val- Asp-FMK or Z-Tyr-Val- Ala- Asp-FMK.
- the dry ingredients are combined and blended together and encapsulated in a gelatin coating using standard manufacturing techniques.
- the composition is useful for treating or preventing elevated blood cholesterol.
- the simvastatin is replaced by a HMG-CoA reductase inhibitor selected from lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, or mevastatin, and the caspase inhibitor is replaced by Z-Asp-Glu-Val- Asp-FMK or Z-Tyr-Val- Ala- Asp-FMK.
- a HMG-CoA reductase inhibitor selected from lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, or mevastatin
- the caspase inhibitor is replaced by Z-Asp-Glu-Val- Asp-FMK or Z-Tyr-Val- Ala- Asp-FMK.
- An oral suspension is prepared by combining the ingredients using standard formulation techniques.
- composition is useful for treating or preventing elevated blood cholesterol.
- the simvastatin is replaced by a HMG-CoA reductase inhibitor selected from lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, or mevastatin, and the caspase inhibitor is replaced by Z-Asp-Glu-Val- Asp-FMK or Z-Tyr-Val- Ala-Asp-FMK.
- a HMG-CoA reductase inhibitor selected from lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, or mevastatin
- the caspase inhibitor is replaced by Z-Asp-Glu-Val- Asp-FMK or Z-Tyr-Val- Ala-Asp-FMK.
- the ingredients are combined using standard formulation techniques.
- composition is useful for treating or preventing elevated blood cholesterol.
- the simvastatin is replaced by a HMG-CoA reductase inhibitor selected from lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, or mevastatin, and the caspase inhibitor is replaced by Z-Asp-Glu-Val- Asp-FMK or Z-Tyr-Val- Ala- Asp-FMK.
- a HMG-CoA reductase inhibitor selected from lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, or mevastatin
- the caspase inhibitor is replaced by Z-Asp-Glu-Val- Asp-FMK or Z-Tyr-Val- Ala- Asp-FMK.
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002336192A CA2336192A1 (en) | 1998-06-24 | 1999-06-21 | Compositions and methods for treating elevated blood cholesterol |
JP2000555616A JP2002518449A (en) | 1998-06-24 | 1999-06-21 | Compositions and methods for treating high blood cholesterol |
AU46990/99A AU4699099A (en) | 1998-06-24 | 1999-06-21 | Compositions and methods for treating elevated blood cholesterol |
EP99930452A EP1089732A4 (en) | 1998-06-24 | 1999-06-21 | Compositions and methods for treating elevated blood cholesterol |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US9040498P | 1998-06-24 | 1998-06-24 | |
US60/090,404 | 1998-06-24 | ||
GB9817174.7 | 1998-08-06 | ||
GBGB9817174.7A GB9817174D0 (en) | 1998-08-06 | 1998-08-06 | Compositions and methods for treating elevated blood cholesterol |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999066930A1 true WO1999066930A1 (en) | 1999-12-29 |
Family
ID=26314177
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1999/013888 WO1999066930A1 (en) | 1998-06-24 | 1999-06-21 | Compositions and methods for treating elevated blood cholesterol |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1089732A4 (en) |
JP (1) | JP2002518449A (en) |
AU (1) | AU4699099A (en) |
CA (1) | CA2336192A1 (en) |
WO (1) | WO1999066930A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6492170B1 (en) | 2000-09-11 | 2002-12-10 | Isis Pharmaceuticals, Inc. | Antisense modulation of caspase 9 expression |
US6566135B1 (en) | 2000-10-04 | 2003-05-20 | Isis Pharmaceuticals, Inc. | Antisense modulation of caspase 6 expression |
US7229982B2 (en) | 2002-07-26 | 2007-06-12 | Moore William D | Pharmaceutical formulation |
JP2008189684A (en) * | 2000-10-23 | 2008-08-21 | Daiichi Sankyo Co Ltd | Composition for improving lipid in blood |
-
1999
- 1999-06-21 AU AU46990/99A patent/AU4699099A/en not_active Abandoned
- 1999-06-21 CA CA002336192A patent/CA2336192A1/en not_active Abandoned
- 1999-06-21 WO PCT/US1999/013888 patent/WO1999066930A1/en not_active Application Discontinuation
- 1999-06-21 EP EP99930452A patent/EP1089732A4/en not_active Withdrawn
- 1999-06-21 JP JP2000555616A patent/JP2002518449A/en not_active Withdrawn
Non-Patent Citations (4)
Title |
---|
BIOLOGICAL ABSTRACTS, 1 January 1900, Philadelphia, PA, US; abstract no. 1999:112431, ITAL M, ET AL: "PRENYLTRANSFERASE INHIBITORS INDUCE APOPTOSIS IN PROLIFERATING THYROID CELLS THROUGH A P53-INDEPENDENT, CRMA-SENSITIVE, AND CASPASE 3-LIKE PROTEASE-DEPENDENT MECHANISM" XP002949846 * |
DATABASE STN CAPLUS 1 January 1900 (1900-01-01), VILLA P, KAUFMANN S H, EARNSHAW W C: "CASPASES AND CASPASE INHIBITORS", XP002949845, Database accession no. 1997:713669 * |
KROON A A, STALENHOEF A F H: "LDL-CHOLESTEROL LOWERING AND ATHEROSCLEROSIS - CLINICAL BENEFIT ANDPOSSIBLE MECHANISMS: AN UPDATE", NETHERLANDS JOURNAL OF MEDICINE., ELSEVIER SCIENCE BV., AMSTERDAM., NL, vol. 51, 1 January 1997 (1997-01-01), NL, pages 16 - 27, XP002924473, ISSN: 0300-2977, DOI: 10.1016/S0300-2977(97)00031-4 * |
See also references of EP1089732A4 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6492170B1 (en) | 2000-09-11 | 2002-12-10 | Isis Pharmaceuticals, Inc. | Antisense modulation of caspase 9 expression |
US6566135B1 (en) | 2000-10-04 | 2003-05-20 | Isis Pharmaceuticals, Inc. | Antisense modulation of caspase 6 expression |
JP2008189684A (en) * | 2000-10-23 | 2008-08-21 | Daiichi Sankyo Co Ltd | Composition for improving lipid in blood |
US7229982B2 (en) | 2002-07-26 | 2007-06-12 | Moore William D | Pharmaceutical formulation |
US7718643B2 (en) | 2002-07-26 | 2010-05-18 | Schering Corporation | Pharmaceutical formulation |
Also Published As
Publication number | Publication date |
---|---|
JP2002518449A (en) | 2002-06-25 |
AU4699099A (en) | 2000-01-10 |
EP1089732A4 (en) | 2001-09-19 |
CA2336192A1 (en) | 1999-12-29 |
EP1089732A1 (en) | 2001-04-11 |
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