MXPA98008947A - Method to treat the inflamac - Google Patents

Abstract

The use of IL-10 and a steroid is described for preparing a medicament for the treatment or prevention of an inflammatory disease, in particular septic shock and inflammatory conditions caused by autoimmune diseases, inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, uveitis and psoriasis. Also provided are pharmaceutical compositions and kits comprising IL-10 plus at least one steroid, eg, betamethasone or its derivatives.

Description

METHOD TO TREAT INFLAMMATION FIELD OF THE INVENTION This invention relates to a method for treating inflammatory conditions or diseases related to inflammation, including septic shock.

BACKGROUND OF THE INVENTION One of the mechanisms by which the immune system normally regulated includes the production of proteins called cytokines. Cytokines mediate numerous immune / inflammatory responses. Many cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), interferon gamma (IFN-α), interleukin-6 (IL-6), are produced by stimulated monocytes / macrophages and have been involved in some changes inflammatory, immunological, hematological, and metabolic produced during infection and tissue damage (see, eg, Hart et al., Proc. Nati. Acad. Sci. 86: 3803 (1989)). Septic shock is one of the examples of a state of the disease characterized by the production of inflammatory cytokines. It is a condition often fatal that usually results from gram-negative bacteremia. Despite the use of potent antibiotics and intensive care, there is still a high mortality rate due to sepsis as well as cases of gram-negative bacteremia resulting in septic shock (See, eg, Ziegler et al., New Eng. J. Med. 324: 429 (1991), Bone et al., New Eng. J. Med. 317: 653 (1987), and Kreger et al., J. Med. 68: (1980)). Approximately 100,000-300,000 cases of sepsis caused by gram negative bacteremia are reported per year, with results of 30.0Ó0 to 100,000 estimated deaths. (Wolff, New Eng. J. Med. 324: 486 (1991)). Sepsis 9,082 JJB requires early treatment, since the patient's condition deteriorates rapidly. It is an important cause of morbidity and mortality in hospitalized patients. Symptoms of septic shock include fever or hypothermia, tachycardia, tachypnea, hypotension, peripheral hypoperfusion, or systemic toxicity. (Ziegler et al., Supra). On the current treatments under investigation for septic shock are the administration of antibodies directed against tumor necrosis factor (TNF-a), interleukin-1 (IL-1) and liposaccharides (LPS). Unfortunately, these treatments have disappointed in clinical judgments, showing inconsistent or insignificant results (See eg Gibaldi, Pharmacotherapy (United States) 13: 302 (1993) and Colletti et al., Crit. Nurs. North Am.%: 345 ( 1993)) There is therefore a need for an effective treatment for inflammatory conditions, especially septic shock. Among other inflammatory conditions treatable or foreseeable by this invention are those caused by auto-immune diseases that exhibit inflammation as a symptom such as inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, uveitis, psoriasis, etc.

Summary of the Invention. This invention completes the need to provide a method for treating or preventing inflammatory conditions in mammals comprising administering an effective amount of a combination of IL-10 and at least one steroid to a mammal in need of such treatment. In particular, this invention provides a method for treating or preventing septic shock, or an autoimmune disease such as inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, uveitis or psoriasis, in mammals comprising administering an effective amount of a combination of IL-10 and at least one steroid to a mammal in need of such treatment. This invention is also directed to the use of a combination of IL-10 and a steroid for the manufacture of a treatment of an inflammatory disease in mammals. Another aspect of the invention is a pharmaceutical composition comprising a combination of IL-10 and at least one steroid, and a physiologically acceptable carrier. Still another aspect of this invention is a kit for treating or preventing an inflammatory condition in a mammal comprising in combination an effective amount of IL-10 added with a pharmaceutical carrier and an effective amount of a steroid added with a pharmaceutical carrier.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the live effect of the recombinant IL-10 and / or the steroid betamethasone phosphate on LPS (lipopolysaccharide) -induced serum IL1 in mice primed with C.Parvum. The data marked with double asterisks indicate P < 0.05 compared with PBS Tween / MSA. Figure 2 illustrates the in vivo effect of the recombinant IL-10 and / or the steroid betamethasone phosphate on LPS (lipopolysaccharide) induced serum IL-6 in mice primed with C.Parvum. The data marked with double asterisks indicate P < 0.01 compared with PBS / Tween / MSA. Figure 3 illustrates the live effect of the recombinant IL-10 and / or the steroid betamethasone phosphate on LPS (lipopolysaccharide) -induced serum TNF-a in mice primed with C.Parvum. The data marked with double asterisks indicate P < 0.01 compared with PBS / Tween / MSA. Figure 4 illustrates the in vivo effect of the recombinant IL-10 and / or the steroid betamethasone phosphate on LPS (lipopolysaccharide) induced serum IFN-? in mice primed with C.Parvum. The data marked with double asterisks indicate P = O.01 compared with PBS / Tween / MSA.

Figure 5 illustrates the result of another experiment for the main inhibition of four serum cytokines in mice treated with betamethasone phosphate and human recombinant IL-10 in combination (the present invention), IL-10 only, and betamethasone phosphate only.

DETAILED DESCRIPTION OF THE INVENTION All references cited herein are hereby incorporated by reference in their entirety. As used herein, "interleukin-10" or "IL-10" is defined as a protein that (a) has a complete amino acid sequence of IL-10 (eg, lacking a guiding secretory sequence) as developed in US patent No. 5,231,012 and (b) has biological activity that is common in natural IL-10.

Also included are the muteins and other analogues, including the protein of Epstein Barr BCRF1 virus (viral IL-10), which retains the biological activity of IL-10.

IL-10 useful for use in this invention can be obtained from an activated conditioned culture medium of cells secreting protein, and purified by standard means. Additionally, IL-10, or fragments -actives thereof, can be chemically synthesized using standard techniques known in the art. See Marrifield, Science 233: 341 (1986) and Atherton et al. Solid Phase Peptide Synthesis: A Practical Approach, 1989, I.R.L. Oxford Press See also U.S. Patent No. 5,231,012. Preferably, the protein or polypeptide is obtained by recombinant techniques using isolated nucleic acencoding the IL-10 polypeptide. General methods of molecular biology are described, eg. by Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, New York, 2nd Ed., 1989, and by Ausubel et al. (eds) Currents Protocols ¡n Molecular Biblogy, Green?? Ooley, New York (1987 and periodic supplements). The appropriate dryings can be obtained using standard techniques from both genomic and cDNA libraries. Polymerase chain reaction (PCR) techniques can be used. See for example PCR Protocols: A Guide to Methods and Applications, 1990, Innis et al. (Ed.) Academic Press, New York, New York. Libraries are constructed of nucleic acextracted from appropriate cells. See, for example U.S. Patent do not. 5,231,012, which develops recombinant methods of IL-10 production. Useful gene sequences can be found, eg. in several sequence databases, eg, GenBank and BMPL or nucleic acid and PIR and Swis-Prot for protein, and / or Intelligenetics, Mountain View, California, or The Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison Wisconsin. Clones comprising sequences encoding human IL-10 have been deposited with the American Type Culture Collection (ATCC), Rockville, Maryland, under accession numbers 68191 and 18192. Identification of other clones harboring the sequences encoding IL-10 it is carried out either by nucleic acid hybridization or by immunological detection of the encoded protein if the expression vector is used. Oligonucleotide tests based on the deposited sequences described in US Patent no. 5,231,012 are particularly useful. Oligonucleotide test sequences can also be prepared from conserved regions of related genes in other species. Alternatively, degenerate tests based on the amino acid sequence of IL-10 can be used. Standard methods can be used to produce prokaryotic, mammalian, yeast or insect cell lines that express large amounts of polypeptide. Exemplary E. Coli strains useful for expression and cloning include W3110 (ATCC Bi, 27325), X1776 (ATCC No. 31244). X2282, 'RR1 (ATCC Mp / 31343). Exemplary mammalian cell lines include COS-7 cells, mouse L cells and CHP. See Sambrook (1989), supra and Ausubel et al., 1987 suplements, supra. Various expression vectors can be used to express DNA encoding IL-10. Conventional vectors used for the expression of recombinant proteins in prokaryotic or eukaryotic cells can be used. Preferred vectors include pcD vectors described by Okayar? A et al., Mol.

Cell. Biol. 3: 280 (1983); and Takebe et al. Mol. Cell. Biol. 8: 466 (1988). Other SV40-based mammalian expression vectors include those described in Kaufman et al., Mol. Cell. Biol. 2: 1304 (1982) and US Patent No. 4,675,285, These SV40-based vectors are particularly useful in COS-7 monkey cells (ATCC No. CRL 1651), as well as in other mammalian cells such as cells. L of mouse. See, also Pouwels et al., (1989 and supplements) Cloning Vectors: A Laboratory Manual, Elsevier, New York. IL-10 can be produced in soluble form, as a secreted product of transformed or transfected yeast, mammalian or insect cells. The peptides can be purified by standard methods that are known in the art. For example, purification steps may include precipitation with ammonium sulfate, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography, and type. See Methods in Enzymology Purification Principles and Practices (Springer-Verlag, New York, 1982). Alternatively, IL-10 can be produced in an insoluble form, such as aggregates or inclusion bodies. IL-10 in such form is purified by standard procedures that are well known in the art. Examples of purification steps include the separation of the inclusion bodies, of the destroyed cells by centrifugation, and then solubilizing the inclusion bodies with caotropic agent and reducing agent so that the peptide assumes a conformation - biologically active For specifications of these procedures, see, eg. Winkler et al., Biochemistry 25: 4041 (1986), Winkler et al., Bio / Technology 3: 9923 (1985); Koths et al., And US Patent No. 4,569,790. The nucleotide sequences used to transfect host cells can be modified using standard techniques to make IL-10 or fragments thereof with a variety of desired properties. Such modified IL-10 can vary from naturally occuring sequences to primary structure levels, e.g. by amino acids, insertions, substitutions, eliminations or mergers. These modifications can be used in a number of combinations to produce the final modified protein chain. The sequence of amino acid variants can be prepared with several objectives in mind, including increasing the serum half-life, facilitating purification or preparation, improving therapeutic efficacy, and decreasing the severity or occurrence of side effects during therapeutic use. The amino acid sequence variants are usually predetermined variants not found in nature, although others may be post-translational variants. Such variants can be used in this invention as long as they retain the biological activity of IL-10. Modifications of polypeptide coding sequences can be easily achieved by a variety of techniques, such as site-directed mutagenesis (Gillman et al., Gene 8:81 (1987)). Many modifications are evaluated by routine selection in a test useful for the desired characteristics. For example, U.S. Patent No. 5,231,012 discloses a number of in vitro assays useful for measuring the activity of IL-10. Preferably, human IL-10 is used for the treatment of humans, although viral IL-10 can possibly be used. More preferably, the IL-10 used is human recombinant IL-10. The preparation of human IL-10 has been described in US Pat. No. 5, 231,012. The cloning and expression of viral IL-10 (BCRF1 protein) of the Epstein Barr Virus has been described by Moore et al., Science 248: 1230 (1990). When we refer to IL-10, active fragments thereof, analogs and homologs are included. Active fragments, analogs and homologs of IL-10 include those proteins, polypeptides or peptides possessing one or more characteristic activities of IL-10. Examples of IL-10 activity include the inhibition or substantial reduction of the level of IL-2, lymphotoxin, IL-3, or GM-CSF. The activity of IL-10 also includes inhibition of cytokine production by activated macrophages, e.g. IL-1, IL-6, and TNF-a. For examples of the processes and assays for determining IL-10 activity, see U.S. Patent No. 5,231,012. This patent also provides proteins having IL-10 activity and production of said proteins that include synthetic and recombinant techniques. Useful steroids for use in this invention include glucocorticoids. Among the preferred glucocorticoids are prednisone, dexamethasone, fluticasone, betamethasone, and other steroids and derivatives thereof. In the examples that follow, the steroid used was betamethasone phosphate in a saline buffer. Betamethasone derivatives are commercially available from Schering Corporation, Kenilworth, New Jersey. In the methods of the present invention IL-10 is preferably combined in the same composition with the steroid. However, the combination of IL-10 plus steroid can be carried out by any means of co-administration. The co-administration can be sequential or simultaneous. "Co-administration" generally means that the multiple (two or more) therapies are present in the container during a specified time interval. Typically, if a second agent is administered - Within the half-life of the first agent, the two agents are considered as coadministered. The invention further provides a method for predicting a predisposition of a mammal for the development of an inflammatory condition, characterized by lower than optimal levels of IL-10, which comprises assaying a sample taken from a mammal for an IL-10 level. Lower than optimal IL-10 levels include undetectable amounts. A detectable level can be compared to a normal level of IL-10. Alternatively, one can assay for inflammatory mediators such as IL-1, IL-6, TNF-a and IFN-g by the use of commercially available equipment. Overproduction of one of these mediators may indicate that insufficient quantities of IL-10 are available. Preferably, blood is the source of the sample. The method allows the prediction of the predisposition of a number of inflammatory conditions, such as inflammatory bowel disease, rheumatoid arthritis, or psoriasis. To 'prepare pharmaceutical compositions including IL-10 and a steroid, the IL-10 and the steroid are added with a pharmaceutically acceptable carrier or excipients which are preferably inert. A pharmaceutical vehicle can be any compatible substance useful for the distribution of the polypeptide in the patient. The preparation of said pharmaceutical compositions is known in the art; see, e.g., Remington's Pharmaceutical Sciences, and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984). The proportion of IL-10, steroid and additive can be varied over a wide range while both are present in therapeutically effective amounts. On a per dose basis, the amount of IL-10 will preferably be in the range of about 0.5 to 15 μgfkg of body weight, and the amount of steroid may be in the range of about 0.5 to 50 mg, more preferably 2 to 12 and most preferred 5 to 12 mg. The compositions can be ingested orally or injected into the body. Formulations for oral use include compounds to protect the protease polypeptides that occur in the gastrointestinal tract. The injections are usually intramuscular, subcutaneous, intradermal or intravenous. Alternatively, intramuscular injection or other routes may be used in the appropriate circumstances. When administered parenterally, the compositions can be formulated in the form of an injectable unit dose (solution, suspension, emulsion) in association with a pharmaceutical carrier. For example, IL-10 and steroid can be administered in aqueous vehicles such as water. saline or buffer vehicles with or without various additives and / or dilution agents.

Examples of useful vehicles are normal saline, Ringer's solution, dextrose solution, and Hank's solution. Non-aqueous vehicles such as fixed oils and ethyl oleate can also be used. A preferred vehicle is 5% dextrose / saline. The vehicle may contain minor amounts of additives such as substances that improve isotonicity and chemical stability, e.g. buffers and condoms. However, IL-10 in the composition is preferably formulated in purified form substantially free of aggregates and other proteins. In addition, it should be noted that a suspension, such as a suspension of zinc, can be prepared to include a polypeptide.

Such suspension may be useful for subcutaneous (SQ) or intramuscular injection (IM) As used herein, the phrase "a therapeutically effective amount" means an amount sufficient to lessen a symptom or a sign of a given inflammatory condition. For example, in the case of an inflammatory condition, the signs and symptoms include one or more pains, swelling, redness and other signs and we feel well known for inflammation. In the particular case of septic shock, the signs and symptoms are one or more of hypertension, microthrombus, organic failure, plasma loss due to vascular permeability, as well as other well-known signs and symptoms of septic shock The term prevention of inflammatory conditions (such as septic shock) as used herein can be defined by the following parameters Certain circumstances predispose individuals to develop inflammatory conditions such as septic shock, for example, candidates for abdominal surgery, or any situation that may cause the rupture or laceration of the intestines (eg rupture of the appendix) that can cause a loss of consciousness. intestinal microflora in the abdominal cavity Other examples include bullet wounds, victims of automobile accidents with abdominal trauma, and the type of administration of IL-10 plus at least one steroid to such high-risk individuals to develop such an inflammatory condition. as a septic shock prior to the start of the symptoms can improve such symptoms masters, preventing the real beginning of the full manifestations of the disease. Typical mammals that can be treated using the methods of the present invention include companion animals such as dogs and cats, and primates, including humans. Preferably, IL-10 derived from species of the target animal will be used. An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method, route, and administration dose and the severity of side effects. The determination of the proper dose is made by a clinician using the parameters known in the art. Generally, the dose begins with a somewhat smaller amount than the optimal dose and is increased by small increments thereafter until the desired or optimum effect is achieved. (See generally The Merck Manual §269"Pharmacokinetics and Drug Administration"). The preferred total daily dose of IL-10 and steroid is selected from a range of approximately 0.5 to 9 mg of an injectable steroid, 2.5 to 50 mg of an orally administered steroid, and 2 to 15 μg / kg of body weight. IL-10 body The dosages are in a program that performs the desired treatment and can be periodic over a short or long term. The daily infusion ratio can be varied based on monitoring of side effects, blood cell counts, and efficacy. See Gillman et al. (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics 8th Ed. Pergamon Press, (1990) Remington's Pharamaceutical Sciences, 17th ed., Mack Publishing Co., Easton Penn .; Avis et al. (Eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications. Dekker, New York; Liebermap et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, New York; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, New York. Preferably, the therapeutically effective amount is a unit dose presented in an ampoule. Alternatively, the therapeutically effective amount may be presented in a vial containing multiple doses or may be offered in some other form. The total daily dose can be given as a single injection, a continuous infusion, or it can be divided into several small doses for intravenous administration or administration by another route such as intramuscular injection. Compositions of the invention can also be introduced into the patient's body by an implant or an injectable drug distribution system, e.g. Urquart et al. Ann. Rev. Pharamácol. Toxicol 24: 199 (1984), Lewis (Ed.) Controlled Reléase of Pesticides and - Pharmaceuticals (Plenum Press, N.Y., 1981), US Patent No. 3,270,960, and of type. In appropriate circumstances, multiple medications can be administered in combination. For example, IL-10 and a combination steroid steroid may be administered in combination in addition to a therapeutically effective dose of one or more additional therapeutically active agents. The broad scope of this invention is better understood with reference to the following examples, which do not attempt to limit the invention to those specifically included.

EXAMPLES Six mice in each group were primed with 0.5 mg of heat killed C.Parvum (I.V. administration) as a test. As a control, one group was treated with phosphate buffered saline (PBS) and Tween 20 (trademark or registered name) 0.5% one hour before the test. As another control, this group was treated with mouse albumin serum (MSA, a protein placebo) at the time of the test. A second group was treated with 0.1 mg / kg betamethasone phosphate in saline buffer for one hour prior to the test. A third group was treated with 1 μg of human recombinant IL-10 (rHulL-10) i.p. at the time of the test. According to this invention another group was treated with 0.1 mg / kg of betamethasone phosphate in p.o. one hour prior to the test and 1 μg rHulL-10 i.p. at the time of the test. Ninety minutes after the test, blood was drawn to each mouse and tested for TNF-α, IL-1, and IL-6 concentrations. Three hours after the test blood was extracted from each mouse and tested for IFN- ?. The results are shown in figures 1 to 4.

It will be noted that in all cases the combination of IL-10 and steroid reduces the amount of agent causing the inflammation by more than the steroid or IL-10 alone. In addition, in this experiment, the combined IL-10 and steroid exerts a synergistic effect in decreasing the concentration of at least one agent causing inflammation, TNF-α. The results of a similar experiment are shown in Figure 5. Based on the above, it is believed that the present invention provides a method of treating or preventing inflammation, including toxic shock, which is more effective than the administration of a steroid or IL-10 separately. Alternatively to or in addition to obtaining more effective treatment, the invention is considered to have the advantage of eliminating or reducing the well-known side effects of steroids, such as liver damage, kidney damage and increased susceptibility to infection. This advantage will result from the use of smaller amounts of estroides or a shorter course of treatment than the additive and / or synergistic combination of IL-10 and a steroid will provide.

Claims (10)

1. -
3. Having thus specially described and determined the nature of the present invention and the manner in which it is to be carried out, it is declared to claim as property and exclusive right: 1.- Use of IL-10 and at least one steroid to prepare a medicine useful for the treatment or prevention of an inflammatory condition in a mammal, which needs such treatment. 2. The use of claim 1 wherein the inflammatory disease is septic shock or an auto-immune disease. 3. The use of any of claims 1 or 2 wherein the inflammatory disease is septic shock.
4. 4. The use of any of claims 1-3 wherein IL-10 is human IL-10.
5. 5. The use of any of claims 1-4 wherein the steroid is a glucocorticoid.
6. 6. The use of any of claims 1-5 wherein the glucocorticoid is betamethasone or a derivative thereof.
7. 7. The use of any of claims 1-6 wherein the IL-10 is administered in a daily dose of 0.5 to 15 μg / kg and the steroid is administered at a daily dose of 0.5 to 50 mg.
8. 8. The use of any of claims 1-7 wherein the steroid is administered by injection at a daily dose of 0.5 to 9 mg.
9. 9. The use of any of claims 1-8 wherein the steroid is administered orally at a daily dose of 2.5 to 50 mg.
10. 10. A kit for treating or preventing the inflammatory condition in a mammal comprising in combination an effective amount of * IL-10 added with a pharmaceutical carrier or and an effective amount of an added steroid with a pharmaceutically acceptable carrier.