JP2001500125A - Methods for lowering cholesterol levels - Google Patents

Abstract

  (57) [Summary] Disclosed are methods for reducing blood cholesterol levels in mammals and humans by administering IL-10. Preferred dosages and methods of administration are also disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION Methods for Lowering Cholesterol Levels Background of the Invention The present invention relates to lowering serum cholesterol levels in mammals, especially humans. Elevated serum cholesterol levels are well recognized as a risk factor for heart disease. That is, the higher the serum cholesterol level, especially low density lipoprotein cholesterol (LDL-C), the more the patient may develop heart disease. After a patient has already developed heart disease and has experienced a heart attack, the higher the serum cholesterol level, the more likely that the patient will have another heart attack. Thus, there is a well-recognized advantage to lowering serum cholesterol levels. There are many compounds available by formula for lowering serum cholesterol levels. Presumably, HMGCoA reductase inhibitors (eg, lovastatin, pravastatin, simvastatin, and fluvast atin) are most commonly used. Although these compounds have some effect in lowering serum cholesterol, they do not always achieve the desired level, and they may cause undesirable side effects (e.g., , Muscle necrosis and hyperkalemia). The present invention lowers serum cholesterol using well-tolerated active ingredients. Furthermore, in its preferred aspects, the present invention is believed to achieve significantly lower serum cholesterol levels than HMGCoA reductase inhibitors. SUMMARY OF THE INVENTION The present invention can be summarized as a method for lowering serum cholesterol in a mammal, especially a human, by administering an effective amount of interleukin 10 (IL-10). Preferably, IL-10 is administered to a mammal diagnosed with elevated serum cholesterol levels (hypercholesterolemia) or a mammal that has already developed atherosclerotic heart or vascular disease. You. Preferably, the mammal to be treated is a human and the IL-10 is recombinant human IL-10 (rh IL-10). A currently preferred method of administration is by subcutaneous injection of about 1-100 mcg / kg of patient weight. More preferably, IL-10 is administered daily in an amount of 1-15 mcg / kg for at least 3 days. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the mean serum cholesterol levels for patients who received 8 mcg / kg rhIL-10 subcutaneously for 7 consecutive days and a control group that received a placebo. FIG. 2 shows the same data as FIG. 1 as fractional cholesterol levels using an initial screening level equal to 1. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for administering to a mammal (eg, a mammal having hypercholesterolemia or nor mocholesterolemia) by administering an effective amount of IL-10 to lower serum cholesterol. Methods for lowering blood cholesterol levels are provided. Elevated cholesterol levels can be associated with cardiovascular disease (eg, atherosclerosis), cerebrovascular disease (stroke), and peripheral vascular disease. Interleukin 10, or IL-10, as used herein, is a compound of the mature (ie, lacking secretory leader sequence) IL-10 disclosed in (a) International Application Publication No. WO 91/003249. And is defined as a protein having an amino acid sequence substantially identical to that of (b) and having a common biological activity with native IL-10. For purposes of the present invention, glycosylated IL-10 (eg, IL-10 produced in eukaryotic cells such as yeast or CHO cells) and non-glycosylated IL-10 (eg, chemically synthesized IL-10). Or IL-10) produced in E. coli are both equivalent and can be used interchangeably. Also included are muteins and other analogs (including viral IL-10) that maintain the biological activity of IL-10. IL-10 suitable for use in the present invention can be obtained from a number of sources. For example, it can be isolated from the culture medium of activated T cells capable of secreting proteins. Further, IL-10 or an active fragment thereof can be chemically synthesized using standard techniques known in the art. See, for example, Merrifield, 1986, Science 233: 341-347 and Atherton et al., Solid Phase Peptide Synthesls, A Practical Approach, 1989, IRL Press, Oxford. Preferably, the protein or polypeptide is obtained by recombinant techniques using an isolated nucleic acid encoding an IL-10 polypeptide. General methods of molecular biology are described, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York and Ausubel et al. Described by Current Protocols in Molecular Biology, Green / Wiley, New York (1987 and periodic addendum). Suitable sequences can be obtained from either genomic or cDNA libraries using standard techniques. DNA constructs encoding IL-10 can also be prepared synthetically using established standard methods, eg, an automated DNA synthesizer, then purified, annealed, ligated, and cloned into an appropriate vector. Can be done. Atherton et al., 1989. Polymerase chain reaction (PCR) technology can be used. See, for example, PCR Protocols: A Guide to Methods and Applications, 1990, Innis et al. (Eds.), Academic Press, New York. The DNA construct may include the entire native sequence of IL-10 or a homolog thereof. The term "homologue" is intended to indicate a naturally occurring variant of the DNA sequence encoding IL-10 or a variant or fragment produced by modification of the DNA sequence. Examples of suitable alterations in the DNA sequence are nucleotide substitutions that do not result in another amino acid sequence, or nucleotide substitutions that result in a different amino acid sequence and, thus, likely different protein structures. Other examples of possible modifications include insertion of one or several nucleotides into the sequence, addition of one or several nucleotides to either end of the sequence, or within one end or sequence of the sequence. Deletion of one or several nucleotides. Any homologous DNA sequence encoding a protein that exhibits IL-10 activity similar to that of the native protein (eg, with respect to inhibiting T cell proliferation or lowering cholesterol) may be used for use in the claimed invention. Intended. As described above, the nucleotide sequences used to transfect host cells can be modified to obtain IL-10 muteins and fragments having various desired properties. Such modified IL-10 can be altered from a naturally occurring sequence at the primary level, for example, by amino acid insertions, substitutions, deletions, and fusions. Preferably, the amino acid substitutions are conservative (ie, a basic amino acid residue is replaced with another basic amino acid residue, etc.). These modifications can be used in many combinations to produce the final modified protein chain. Amino acid sequence variants include increasing serum half-life, facilitating purification or preparation, improving therapeutic efficacy, and reducing the severity or onset of side effects during therapeutic use It can be adjusted to take into account various purposes. Amino acid sequence variants are usually certain variants that are not found in nature, while other variants are post-translational variants (eg, glycosylation variants or polyethylene glycol (PEG)). Protein, etc.). Such variants can be used in the present invention as long as they maintain the biological activity of IL-10. Instead of human IL-10, viral IL-10 or mouse IL-10, or IL-10 from some other mammalian species may be used, but preferably for human treatment, human IL-10 Is used. Most preferably, the IL-10 used is recombinant human IL-10 (rhIL-10). Recombinant production of human IL-10 is described in US Pat. No. 5,231,012. Preparation of human IL-10 and mouse IL-10 is described in International Application Publication No. WO 91/00349. The cloning and expression of the virus IL-10 (BCRFI protein) from Epstein-Barr virus is disclosed in Moore et al. (Science 248: 1230, 1990) and described in EP 0 506 836. Administration of IL-10 is preferably parenteral, by intraperitoneal, intravenous, subcutaneous or intramuscular injection, or infusion, or by other acceptable systemic methods. Most preferred is administration by subcutaneous injection. Alternatively, IL-10 can be administered by an implantable or injectable drug delivery system. See, for example, Urquhart et al., 1984, Ann Rev. Pharacol. See Toxicol 24: 199; Ed. Lewis, 1981, Controlled Release of Pesticides and Pharmacuticals, Plenum Press, New York, New York; U.S. Patent Nos. 3,773,919 and 3,270,960. Oral administration can also be performed using well-known formulations that protect IL-10 from gastrointestinal proteases. Compositions useful for parenteral administration of such drugs are well known. See, for example, Remington's Pharmaceutical Science, 11th Edition, 1990, Mack Publishing Co., Easton, PA. For parenteral administration, IL-10 is typically provided as a lyophilized powder, the preferred formulation of which is given in the examples. The powder is reconstituted in sterile water for injection. The powder may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, such as buffers and preservatives. IL-10 can be formulated in a purified form substantially free of aggregates and other source proteins at concentrations ranging from about 25 mcg / ml to 2 mg / ml. If desired, any of the well-known carrier proteins such as human serum albumin can also be added. Other injectable forms (solutions, suspensions, emulsions) can be used in connection with the pharmaceutical carrier. Examples of such carriers are normal saline, Ringer's solution, dextrose solution, and Hanks' solution. Non-aqueous carriers such as fixed oils and ethyl oleate can also be used. IL-10 can also be delivered by standard gene therapy techniques, including, for example, direct DNA injection into tissues, use of recombinant viral vectors or phospholipids, and implantation of transfected cells. See, for example, Rosenberg, 1992, J.A. Clin. On col. See 10: 180. An effective amount of IL-10 is defined as any amount that significantly lowers cholesterol levels. A decrease in cholesterol by at least 5% is considered significant. More preferably, cholesterol levels are reduced by 20-50%. The dosage, frequency and duration of administration will vary depending on factors such as cholesterol levels (eg, the severity of cholesterol elevation), age of the patient, nutrition, and the like. Usually, initially, the administration is daily and may be continued periodically throughout the life of the patient. Dosage and frequency can be determined during the initial screening of cholesterol levels in reducing cholesterol levels and the magnitude of the effect of IL-10. The dosage is intended to reduce cholesterol levels to acceptable levels. This is currently considered to be less than about 200 milligrams per deciliter of serum. More aggressive cholesterol, especially LDL, for patients at high risk (eg, previous evidence of established coronary artery disease, smokers, low HDL-C (high density lipoprotein cholesterol) levels, diabetes, etc.) -C is guaranteed to decrease. Currently preferred dosages of IL-10 for practicing the present invention are 1-100 mcg / kg, more preferably 1-15 mcg / kg, administered daily for at least 3 days (more preferably, at least 7 days). is there. Other dosing schedules (eg, every other day, once every three days, etc.) are also considered effective. A currently preferred method of administration is by subcutaneous injection. However, if an orally effective IL-10 formulation of IL-10 is to be completed, oral administration is more convenient. It may be useful to administer IL-10 in combination with other pharmaceutically active compounds to complement the cholesterol lowering effect of IL-10. For example, it may be another cholesterol lowering agent [eg, lovastatin available from Merck, Inc., Rahway, NJ (1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8- [2- (Tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl) -ethyl] -1-naphthalenyl 2-methylbutanoate, (US Pat. No. 4,231,938), or other HMG-CoA reductase inhibitors Gemfibrozil (5- (2,5-dimethylphenoxy) -2,2-dimethylpentanoic acid) available from Parke-Davis, Inc., Ann Arbor, Michigan; available from Squibb, Inc., Princeton New Jersey Pravastatin; niacin; nicotinic acid and its derivatives; cholestyramine; other anion exchange resins that bind cholesterol; other cholesterol absorption inhibitors; or bile acids]. To reduce cholesterol levels, which can be associated with the acute onset of heart disease such as myocardial infarction, IL-10 can be treated with a thrombolytic agent [eg, tissue plasminogen activator (tpA) (eg, US Pat. No. 4,370,417, supra). No. 4,752,603; UK Patent No. 2, 119,804; PCT Patent Applications Nos. 87/05934, 87/04722, 84/01786; Australian Patent Application No. 55514/86; EPO Patent Application No. 227,462. Nos. 234,051, 238,304 and 174,835, and tPA is commercially available from Genetech, Inc., South San Francisco, California); Eminase (Beech am Inc., Bristol) And Streptokinase (e.g., materials disclosed in European Patent Applications Nos. 248,227, 28,489; and Burroughs-Wellcome, Inc., Research Triangle, Commercially available from North Carolina Or a combination of such thrombolytic agents (see, for example, European Patent Applications Nos. 91,240 and 28,489 for streptokinase / tPA complexes). obtain. These references are incorporated herein by reference to provide examples of other cholesterol-lowering and thrombolytic agents that may be used in combination with IL-10 in certain embodiments of the present invention. The specific cholesterol lowering agents and thrombolytic agents described above are merely examples of such agents known to those skilled in the art that can be used in the practice of the present invention. It is believed that administering IL-10 in combination with another cholesterol-lowering agent may allow significantly less use of the other agent, thereby eliminating or reducing the undesirable side effects of the other agent. Administration of one dose can be intravenous, intranasal, parenteral, oral, subcutaneous, intramuscular, topical, transdermal, or any other acceptable method. IL-10 can be administered in many conventional dosage forms. Parenteral preparations include sterile solutions or suspensions. Inhaled administration may be in the form of a nasal or buccal spray (ie, via an inhaler). Topical dosage forms may be creams, ointments, lotions, transdermal devices such as conventional reservoir or matrix patch or iontophoresis devices. The formulations and pharmaceutical compositions contemplated by the above dosage forms can be prepared using conventional techniques, using conventional pharmaceutically acceptable excipients and additives. Preferably, IL-10 is administered via the subcutaneous route. The solution to be administered can be reconstituted from a lyophilized powder and can further include preservatives, buffers, dispersants and the like. Preferably, IL-10 is reconstituted in a medium commonly used for intravenous injection (eg, sterile water, without preservatives). Preferably, the maximum concentration of IL-10 should not exceed 2,000 micrograms per milliliter. Administration can be accomplished by continuous intravenous or subcutaneous infusion, or by intravenous injection. For continuous infusion, a daily dose may be added to normal saline or other solution, and the solution may be infused by a mechanical pump or by gravity. The following examples demonstrate the effect of administration of human recombinant IL-10 (rhIL-10) on serum cholesterol levels in healthy human volunteers. EXAMPLES Example 1: Daily administration for 7 days A group of patients with an initial serum cholesterol level between 150-160 mg / dl was dosed daily with 8 mcg of IL-10 / kg body weight for 7 days. IL-10 was administered by subcutaneous injection. IL-10 was initially supplied as a lyophilized powder prepared from the following formulation. ^{* On} reconstitution, 1.0 ml volume ^** Sublimation during lyophilization The lyophilized powder in each vial was reconstituted with 1.0 ml of sterile water for injection. The results are shown in FIGS. In a second group of patients having an average initial serum cholesterol level between 130-140 mg / dl, the placebo was the same as the first group except that the injected composition did not contain any IL-10 Injected with therapy. The results of this control experiment are also shown in FIGS. It is immediately apparent that IL-10 causes a rapid and very significant reduction in serum cholesterol levels. Future experiments are designed to determine which type of cholesterol--LDL cholesterol (so-called bad cholesterol) or HDL cholesterol (so-called good cholesterol)-is most affected by IL-10. Future experiments should also determine the duration of the decrease in cholesterol levels, and whether follow-up measurements of cholesterol and further dosing of IL-10 are required. Example 2: Effect of a single dose of IL-10 A randomized, third-party blind, escalating single dose, placebo control experiment was performed at the following doses. Placebo (18 participants) 1, 2.5, 5, 10, 25, and 50 mcg / kg IL-10 (6 participants at each dosage level) The results are shown in the table below. Percent of baseline (pre-treatment) cholesterol levels Cholesterol concentration by treatment (mcg / ml) Change from baseline (pre-treatment) cholesterol level (mg / dl) Based on the above, it is clear that IL-10 has a surprising effect of significantly lowering serum cholesterol levels in human patients.

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Claims (1)

[Claims]   1. Preparation of a medicament for lowering serum cholesterol levels in mammals Use of IL-10 for building.   2. Admixing an effective amount of IL-10 with a pharmaceutically acceptable carrier Pharmaceutical compositions for lowering serum cholesterol levels in mammals Method for the manufacture of.   3. Serum in a mammal comprising IL-10 and a pharmaceutically acceptable carrier Pharmaceutical composition capable of lowering cholesterol levels.   4. The IL-10 is selected from the group consisting of a cholesterol lowering agent and a thrombolytic agent 4. The composition of any of claims 1 to 3, wherein the composition is administered in combination with at least one other drug to be administered. A method, use, or pharmaceutical composition according to any one of the preceding claims.   5. The method according to any one of claims 1 to 4, wherein the IL-10 is a human. Or pharmaceutical compositions.