Title:  Methods for prevention and treatment of septic shock

United States Patent:  6,291,483

Assignee:  National Institute of Immunology (New Delhi, IN)

Filed:  February 11, 1998

Foreign Application Priority Data:  Feb 14, 1996[IN] (294/96)

Berberine or pharmaceutically acceptable salts thereof either alone or in combination with cyclosporin can be used to treat or prevent graft vs. host disease. Berberine or pharmaceutically acceptable salts thereof can also be used to treat or prevent septic shock syndrome.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a new immunosuppressive therapy comprising use of a protoberberine alkaloid, berberine or its pharmaceutically acceptable salts. Berberine is isolated from a medicinal plant, Berberis aristata and is also commercially available. Berberine and its pharmaceutically acceptable salts can also be produced synthetically (Kametani 1969 and Sainsburg 1969). Another aspect of the invention is an improved immunosuppressive therapy comprising use of berberine or its pharmaceutically acceptable salts and cyclosporin.

The method of treatment of this invention induces selective immunosuppression by functionally inhibiting the response of T lymphocyctes against allogenic or antigenic stimulus and also suppresses the inflammatory response of macrophages to bacterial endotoxin. The present method, therefore, can be used in clinical situations, such as, where a patient is immunocompromised, for example, prior to and/or following bone marrow transplant. The present method can also be used in patients who will undergo organ transplant or who have undergone an organ transplant. The present method can also be used for a patient with an autoimmune disorder especially T cell mediated autoimmune disorders. In the method according to this invention, T lymphocyte activation and/or proliferation is inhibited. It is expected that when berberine or its pharmaceutically acceptable salts is administered according to the method of this invention, IL-2 induced T cell proliferation is inhibited and IFN_gamma. production by T cells following mitogenic, allogenic or antigenic challenge is also inhibited.

The method of the invention can also be used to treat IL-2 and/or IFN_.gamma. mediated autoimmune disorders. The method of this invention can also inhibit TNF mediated pathology in TNF-a sensitive target tissue.

According to another aspect of this invention berberine or its pharmaceutically acceptable salts can be administered to treat or prevent septic shock in immunocompromised or non immunocompromised patients. Berberine or its pharmaceutically acceptable salts can also be used to treat or prevent TNF-a induced septic shock.

The present method of treatment, as described herein appears to be more potent and less toxic than treatment with the known immunosuppressive drugs.

Berberine and its pharmaceutically acceptable salts are commercially available. Berberine may also be extracted and isolated from Berberis aristata, a plant belonging to family Berberidaceae. One method of extracting and isolating berberine from Berberis aristata comprises coarsely grinding the air dried stems/roots of Berberis aristata and subjecting this material to thorough defatting with non-polar solvents like hexane or petroleum ether at 60-80^oC. followed by extraction with 95% ethanol or 95% methanol in water. The alcoholic extract is then concentrated in a water bath and the basic constituents are extracted from the residue preferentially with a 7% aqueous solution of citric acid or acetic acid or dilute HCl/H₂ SO₄. The aqueous acidic solution was treated with Mayer's reagent and filtered. The Mayer's complex (precipitate) so obtained was either directly loaded on a column of Amberlite-IRA 400 (Cl--) (Amberlite is a packing made from asbestos with binders. IRA-400 is an indicator of mesh size and grade equivalent to 20-50 mesh strongly cationic resin) and eluted with methanol, or stirred with Amberlite-IRA 400 (Cl--) and the alkaloid chloride was then filtered from resin, lyophilized, adsorbed on silica gel and eluted on a column of silica with chloroform (CHCl₃):methanol (MeOH) (increasing polarity). CHCl₃ :MeOH (90:10) elutes resulted in a biologically active compound. The compound, appearing as yellowish needles, m.p. 145-147^oC., thus obtained was found to be soluble in ethanol, methanol, water and insoluble in chloroform, benzene and petroleum ether at 60-80^oC. It did not give any test for phenols. The C.I. Mass spectrum of the compound gave MH+ at m/z 337, revealing its molecular weight to be 336. This fact, coupled with the elemental analysis data gave the molecular formula of the compound to be C₂₀ H₁₈ NO₄. The U.V. spectrum of the compound showed absorption maxima at 263 and 345 (log e 4.4 & 4.4) and minima at 250, 305 and 370 nm (log e 4.1, 3.8 and 3.7), the spectral pattern remaining unaffected by addition of an acid. Upon addition of sodium borohydride the compound was decolorized and a distinct shift in its UV pattern was observed, thus showing the native compound to be a quaternary alkaloid in nature. The UV absorption minimum at 305 nm is typical of 2,3,9,10 oxygenated protoberberine alkaloids (Shamma, 1972). On the basis of this data the compound was identified to be berberine and it was further confirmed by a direct comparison with an authentic sample of berberine (m.m.p., Co-TLC an Co-IR). The chemical structure of berberine is given below: ##STR1##

To prevent GVHD and septic shock in patients who will undergo bone marrow or organ transplants, in cases where it is possible, suitable protocols for administration of berberine or its pharmaceutically acceptable salts include administration of berberine or pharmaceutically acceptable salts to the patient who will receive the transplant prior to the transplant. It is preferred that the berberine or its pharmaceutically acceptable salts be administered to the patient at least seven days prior to receiving the transplant. In another embodiment, berberine or its pharmaceutically acceptable salts can be administered to the patient about 24 hours before the bone marrow or organ transplant and continued thereafter. Berberine or its pharmaceutically acceptable salts can also be administered to the patient daily following the transplant or both prior to and after the transplant takes place. Following the transplant, Berberine or its pharmaceutically acceptable salts can be administered for about two weeks up to several months depending on the clinical condition and progress of the patient. It is preferred that berberine or its pharmaceutically acceptable salts be administered immediately after the transplant to protect against bone marrow rejection or organ graft rejection and to protect against the development of septic shock syndrome. In another embodiment of the invention berberine or its pharmaceutically acceptable salts is administered to the organ or marrow donor for at least seven days before the organ or marrow is removed from the donor.

Berberine or its pharmaceutically acceptable salts can also be administered to immunocomprised or immunocompetent patients to treat or prevent septic shock. Berberine or its pharmaceutically salts can also be administered to patients, (either immunocompromised or nonimmunocompromised) suffering from septic shock. Berberine or its pharmaceutically acceptable salts can be administered to patients who have or who will undergo surgery to prevent septic shock. It is preferred that berberine or its pharmaceutically acceptable salts be administered at least 24 hours before the surgery. Berberine or its pharmaceutically acceptable salts can also be administered to patients who have been burned to prevent or treat septic shock. It is preferred that pharmaceutically acceptable salts of berberine be used in the method of this invention. It is more preferred that the salts are berberine sulphate or berberine chloride.

Berberine or its pharmaceutically acceptable salts may be administered via any appropriate dosage routes, specifically, topically, orally, by injection, transdermally, intravenously, intraarterially, intraperitoneally, intrapleurally, subcutaneously, intramuscularly, sublingually, intraepidermally or rectally. The preferred routes of administration are intravenously, intraarterially, orally or topically, Topical preparations include but are not limited to ointments, gels, salves, creams, lotions and sprays. Berberine and its pharmaceutically salts can also be administered in the forms of tablets, granules, powder, capsules, caplets and the like. Berberine salts are water soluble and can be administered in water. Berberine and its pharmaceutically acceptable salts can also be administered in other pharmaceutically acceptable carriers, diluents, solvents and excipients. The daily dose of berberine or its pharmaceutically acceptable salts is in the range of 1 to 20 mg/kg of body weight; preferably 5 to 10 mg/kg of body weight. It is more preferred that the dosage be 10 mg/kg of body weight. The daily dose of berberine or its pharmaceutically acceptable salts can be administered as a single dose or in multiple doses. The same protocol can be used for administration of berberine or its pharmaceutically acceptable salts to organ or marrow donors.

In another aspect of this invention it has also been found that administration of berberine or its pharmaceutically acceptable salts in combination with cyclosporin results in a superior and/or synergistic effect of inhibiting graft vs. host reaction. It is preferred that the drugs be administered together or immediately following one another. If the drugs are not administered together, cyclosporin can be administered and then berberine or its pharmaceutically acceptable salts or vice versa.

The amount of cyclosporin administered can be in the range of 0.1 mg/kg to 15 mg/kg of body weight. It is preferred that the amount of cyclosporin be 1 mg/kg to 10 mg/kg of body weight. It is more preferred that the amount be 10 mg/kg of body weight. The amount of berberine or its pharmaceutically acceptable salts that can be administered is in the range of 1 mg/kg to 15 mg/kg of body weight. It is more preferred that the amount of berberine or its pharmaceutically acceptable salts be in the range of 5 to 10 mg/kg of body weight. It is more preferred that the amount be 10 mg/kg of body weight. The mode of administration and pharmaceutical forms of berberine and its pharmaceutically acceptable salts and cyclosporin are the same as described above for berberine and its pharmaceutically acceptable salts.

Claim 1 of 21 Claims

What is claimed is:

1. A method of treating TNF mediated or induced septic shock comprising suppressing TNF induced effects or inhibiting TNF-mediated effects by administering a therapeutically effective amount of berberine or a pharmaceutically acceptable salt thereof to a patient in need of such treatment.

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