WO1995013082A1 - Composition utilisee en immunotherapie et procede - Google Patents

Composition utilisee en immunotherapie et procede Download PDF

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Publication number
WO1995013082A1
WO1995013082A1 PCT/US1993/010724 US9310724W WO9513082A1 WO 1995013082 A1 WO1995013082 A1 WO 1995013082A1 US 9310724 W US9310724 W US 9310724W WO 9513082 A1 WO9513082 A1 WO 9513082A1
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Prior art keywords
vehicle
extract
composition
ethanol
methylene chloride
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PCT/US1993/010724
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English (en)
Inventor
Tien Wen Tao Wiedmann
Jian Wang
Nathan B. Pliam
Hank C. H. Wuh
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Pharmagenesis, Inc.
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Application filed by Pharmagenesis, Inc. filed Critical Pharmagenesis, Inc.
Priority to AU55947/94A priority Critical patent/AU5594794A/en
Priority to PCT/US1993/010724 priority patent/WO1995013082A1/fr
Publication of WO1995013082A1 publication Critical patent/WO1995013082A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A61K31/36—Compounds containing methylenedioxyphenyl groups, e.g. sesamin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/37—Celastraceae (Staff-tree or Bittersweet family), e.g. tripterygium or spindletree
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention relates to a composition for use in immunotherapy, and to a composition for use in treating transplantation rejection.
  • the immune system functions as the body's major defense against diseases caused by invading organisms. This complex system fights disease by killing invaders such as bacteria, viruses, parasites or cancerous cells while leaving the body's normal tissues unharmed.
  • invaders such as bacteria, viruses, parasites or cancerous cells
  • a second essential feature is memory, the ability to remember a particular foreign invader and to mount an enhanced defensive response when the previously encountered invader returns. The loss of recognition of a particular tissue as self and the subsequent immune response directed against that tissue produce serious illness.
  • An autoimmune disease results from the immune system attacking the body's own organs or tissues, producing a clinical condition associated with the destruction of that tissue.
  • An autoimmune attack directed against the joint lining tissue results in rheumatoid arthritis; an attack against the conducting fibers of the nervous system results in multiple sclerosis.
  • the autoimmune diseases most likely share a common pathogenesis and the need for safe and effective therapy.
  • Rheumatoid arthritis is one of the most common of the autoimmune diseases.
  • Current treatments include three general classes of drugs (Schumacher, 1988) : antiinflammatory agents (aspirin, non-steroidal antiinflammatory drugs and low dose corticosteroids) ; disease-modifying antirheumatic drugs, known as "DMARDs” (antimalarials, gold salts, penicillamine, and sulfasalazine) and immunosuppressive agents (azathioprine, chlorambucil, highdose corticosteroids.
  • DMARDs antimalarials, gold salts, penicillamine, and sulfasalazine
  • immunosuppressive agents azathioprine, chlorambucil, highdose corticosteroids.
  • cyclophosphamide methotrexate, nitrogen mustard, 6- mercaptopurine, vincristine, hydroxyurea, and cyclosporin A
  • Immunosuppressive agents are also used in treating or preventing transplantation rejection.
  • Organ transplantation involving human organ donors and recipients (allogeneic grafts) and more recently, involving non-human primate donors and human recipients (xenogeneic grafts) has received considerable medical and scientific attention during the past two decades (Keown, 1991, Roberts, 1989, Platt, 1990). To a great extent, this effort has been aimed at eliminating, or at least reducing, the problem of rejection of the transplanted organ. In the absence of adequate immunosuppressive therapy, the transplanted organ is destroyed by the host immune system.
  • transplant rejection is mediated by both antibod ⁇ ies and cytotoxic T lymphocytes (CTLs) , with the participation of CD4+ "helper" T cells (Noelle, 1991) .
  • CTLs cytotoxic T lymphocytes
  • foreign class I MHC antigens stimulate CD8+ CTLs
  • foreign class II MHC antigens stimulate CD4+ T cells (Roitt, 1991) .
  • the most commonly used agents for preventing transplant rejection include corticoste ⁇ roids, cytotoxic drugs that specifically inhibit T cell activation such as azathioprine, immunosuppressive drugs such as cyclosporin A, and specific antibodies directed against T lymphocytes or surface receptors that mediate their activation (Briggs, 1991) . All of these drug therapies are limited in effectiveness, in part because the doses needed for effective treatment of transplant rejection may increase the patient's susceptibility to infection by a variety of opportunis ⁇ tic invaders, in part because of direct toxicity. Cyclosporin A, currently the most effective and most commonly used agent, is significantly toxic to the kidney. This nephrotoxicity limits the quantity of drug that can be safely given.
  • the invention includes a composition for use in immunosuppression therapy in a mammalian subject.
  • the vehicle is composed of an ethanol extract from the root xylem of Tripterygium wilfordii and, carried in the vehicle, an immunosuppressant drug, particularly cyclosporin A, FK506, azathioprine, rapamycin, mycophenolic acid, or a glucocorticoid.
  • the composition has an increased immunosuppressive activity with respect to a composition containing either the immunosuppressant drug or vehicle alone, allowing greater immunosuppression activity with reduced toxicity.
  • the composition of claim is used in treating transplantation rejection, and the immunosuppressant drug is cyclosporin A.
  • the vehicle may be a relatively unpurified ethanol extract, or may be purified to remove components which do not contribute to the potentiating effect of the vehicle.
  • the vehicle is composed of plant components which are (a) extractable from T. wilfordii root xylem by ethanol, (b) further extractable from ethanol:water (2:1) by methylene chloride; and (c) further retained on silica gel in 100% methylene chloride.
  • the vehicle is further refined to include plant components which are (a') further eluted from silica gel by methylene chloride:methanol 95:5; and (b') further contained in the intermediate fractions which are eluted from a silica gel column by elution with methylene chloride:methanol 97:3.
  • a plant-extract vehicle of the type for the manufacture of a medicament for use in treating transplantation rejection in a mammalian subject is also disclosed.
  • Fig. 1 is a flow diagram of a method for preparing a Tripterygium wilfordii (TW) ethanol extract vehicle, in accordance with the invention, also showing additional purification methods that may be used to achieve more purified extract vehicles, for use in the composition of the invention;
  • TW Tripterygium wilfordii
  • Fig. 2A is a thin layer chromatogram of 1:1,000 TW extract vehicle (Lane A), 1:5,000 extract vehicle (Lane B) and purified 1:10,000 TW extract vehicle (Lane C) ;
  • Fig. 2B is a proton NMR spectrum of the purified TW extract vehicle;
  • Fig. 3 shows the suppressive effect of purified TW extract vehicle on production of IL-lb, TNF-alpha, IL-2, and IL-6 by human peripheral blood lymphocytes in culture;
  • Fig. 4 is a plot of transplant survival time for untreated animals (open triangles) , and animals treated with TW extract vehicle alone (solid diamonds) , cyclosporin A alone (open circles) , and cyclosporin A in a TW extract vehicle;
  • Fig. 5 is a plot of transplant survival time for untreated animals (squares) , and animals treated with purified TW extract vehicle by oral administration (circles) or intraperitoneal administration
  • Immunosuppression therapy refers to treatment of a mammalian subject, including a human subject, to suppress an autoimmune response or an immune response against a transplanted allogeneic or xenogeneic tissue or organ.
  • an "ethanol extract of the root xylem from T. wilfordii” refers to a composition containing ethanol- soluble components extracted from the root xylem of T. wilfordii ⁇ such a composition may include water soluble components that are also soluble in ethanol.
  • the extract may be relatively unpurified, or may exist in progressively more purified form, as described below. For example, water-soluble components and components which bind to silica gel in the presence of chloroform may be removed from the extract.
  • Patentiated immunosuppression activity refers to enhanced efficacy in immunosuppression therapy, as demonstrated by an enhanced therapeutic effect at a given immunosuppressant drug dose, or equivalent therapeutic effect at a reduced immunosuppressant drug dose.
  • the extract vehicle of the invention is obtained from the root xylem of Tripterygium wilfordii (TW) , a medicinal plant which is grown in the Fujiang province and other southern provinces of China. Plant material can be obtained easily in China.
  • TW Tripterygium wilfordii
  • Fig. 1 One method of preparation of a TW ethanol extract is illustrated in Fig. 1, and detailed in Example 1. Briefly, dried plant material is ground into a crude powder and then extracted by boiling in 95% ethanol with refluxing. The ethanol is removed and the extraction typically repeated twice. The resulting extracts are then combined and the ethanol removed (for example, by evaporation or heat-assisted evaporation. About 10 g dry ethanol extract is usually recovered per kg dry weight of plant material. This simple ethanol extract represents one preparation useful in the practice of the treatment method of the present invention.
  • This vehicle is composed of plant components which are extractable from T. wilfordii root xylem by ethanol.
  • the ethanol extract vehicle from above may be further purified, to remove components, for example, which do not contribute to the potentiation of immunosuppressant drug.
  • An exemplary purification method is shown in Fig. 1, and detailed in Example 2. Briefly, the ethanol extract from above is filtered and Volume is reduced under vacuum. The resulting syrup is diluted with water. Chloroform-soluble components are extracted by the adding chloroform, separating the non-aqueous-phase material, and discarding the aqueous-phase components. The chloroform extract can be concentrated, e.g., by evaporation, and applied to a silica gel column.
  • the extract material is then eluted successively with chloroform and chloroform:methanol (95:5) andwith chloroform:methanol (90:10), at a yield of about 1 g extract material per 20 g of original ethanol extract, corresponding to about 100 g dry weight starting plant material.
  • This partial purified extract vehicle is referred to as a 1:1000 TW extract vehicle.
  • the vehicle includes components which are further extractable from ethanol:water (2:1) by methylene chloride; further retained on silica gel in 100% methylene chloride; and further eluted from silica gel by methylene chloride:methanol 95:5.
  • the 1:1000 extract vehicle can be further purified (Example 2) by application to a silica gel column and elution with methylenechloride:methanol (97:3). Typically, six fractions are collected with the first and last of the six fractions to be discarded. The four intermediate eluted fractions are combined, with a yield of about 20 g material per 100 gram 1:1000 extract. The resulting extract is referred to herein as a 1:5000 extract vehicle.
  • the 1:5000 extract vehicle can be further purified by the same procedure, i.e., by elution on silica gel with methylene chloride:methanol (97:3) and collection of the intermediate fractions, with a final yield of about 1 g material per 2 gram 1:5000 extract.
  • This purified extract vehicle is referred to herein as a 1:10,000 extract vehicle, and includes components which are contained in the intermediate fractions which are eluted from a silica gel column by elution with methylene chloride:methanol 97:3.
  • FIG. 2A shows a thin-layer chromatogram of the various extract TW extract vehicles prepared as above.
  • the vehicle inhibited peripheral blood lymphocyte (PBL) proliferation that was stimulated by CD3 antibody.
  • PBL peripheral blood lymphocyte
  • Increasing amounts of purified TW extract vehicle produced dose dependent inhibition, in the concentration range from about 0.3 to 1.25 ⁇ g/ml vehicle components/culture medium.
  • IL-2, and IL-6 was assessed by measuring the concentration of these cytokines in the culture medium of anti-CD3 stimulated and unstimulated human PBLs. Cytokine levels were measured by standard ELISA methods using commercially available kits. Briefly, assay buffer was added to each of the wells of a microtiter plate containing pre-bound anti-cytokine antibody, followed by addition of standard or sample solution, diluted appropriately for the concentration range measured, followed by a second reporter-labeled antibody specific against the anti-cytokine antibody. Details are given in Example 4.
  • Figure 3 shows the levels of IL-lb, TNF-alpha, IL-2, and IL-6 in human PBLs at basal levels (solid bars) , in cells stimulated with anti-CD3 (crosshatched bars) , and in stimulated cells treated with purified TW extract vehicle (shaded bars) .
  • Anti CD3 stimulation resulted in a significant increase in all four cytokines measured.
  • the purified TW extract significantly inhibited the production of TNF-alpha, IL-2, and IL-6, whereas the extract decreased production of IL-lb only slightly. Vehicle was added to the culture at a concentration of 5 ⁇ g/ml culture medium. 3.
  • the vehicle also suppressed, but at higher concentration, the cell-proliferative activity of IL-2 on the IL-2 dependent cell line, HT-2, according to published methods (Watson, 1979) .
  • cytotoxicity of the vehicle was assessed by measuring the effect of the purified extract on the ability of cultured cells to reduce MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) , an index of cellular respiration. This is a sensitive assay for the detection of cytotoxicity (Green, 1984) . Details of the procedure are given in Example 5. In addition, staining with the vital dye trypan blue, which stains dead cells, was also carried out routinely in all of the culture systems described. Toxicity was evaluated in vitro in two different cell culture systems, human PBLs and mouse thymocytes. No vehicle cytotoxicity was observed at a vehicle concentration of 10 mg/ml in PBLs and 3.1 g /ml in mouse thymocytes, the highest concentrations tested in each system.
  • a measure of in vivo immunosuppression is inhibition of cell proliferation in the mixed lymphocyte reaction (MLR) (Bradley; Mishell, 1980) .
  • MLR mixed lymphocyte reaction
  • mice were treated with the TW extract vehicle for 14 days.
  • Spleen cells the "responder” cells
  • the responder cells proliferate in the presence of the allogenic stimulator cells. Irradiation of the stimulator cells renders them unable to proliferate.
  • tritiated thymidine was added to the mixed cell cultures, and incorporation of the labeled nucleotide into DNA was measured as an index of cell proliferation.
  • C3H mice were injected intraperitoneally with either 1 or 10 mg purified TW extract vehicle/Kg body weight. Animals were treated daily for 14 days prior to harvesting the spleen cells. Spleen cells from C3H mice were cultured with irradiated spleen cells from BalbC or C57 Black (C57B1) mice. Irradiated spleen cells from C3H mice served as controls. Irradiated allogeneic spleen cells stimulated C3H cell proliferation 2-4 fold, in comparison with irradiated syngeneic cells. The extract vehicle effectively inhibited the mixed lymphocyte response, and was dose dependent in the range 1-10 mg/Kg animal weight.
  • C3H mice were treated for 14 days with 10 mg purified TW extract vehicle/kg body weight or with diluent alone. Spleen cells were harvested and their response to the mitogens Phytohemagglutinin (PHA) and Concanavalin A (ConA) was assessed. A similar immunosuppressive effect by the vehicle was observed.
  • PHA Phytohemagglutinin
  • ConA Concanavalin A
  • the invention includes a pharmaceutical composition which includes an immunosuppressant drug in a TW extract vehicle of the type described above.
  • the immunosuppressant drug is one of the following: (a) Cyclosporin A or cyclosporin C ("cyclosporin") , a non-polar cyclic oligopeptide;
  • an immunosuppressant glucocorticoid such as prednisone or dexamethasone.
  • the vehicle is a mixture of ethanol-soluble TW plant components, as described above.
  • the proportions of the two components is preferably in the range 0.5 to 10 parts by weight immunosuppressant drug to 1-50 parts by dry weight vehicle components, where the immunosuppressant drug is dissolved or suspended in a liquid form of the vehicle, or mixed with a dried, powdered form of the vehicle.
  • the composition may be administered to a subject orally, transdermally or parenterally, e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection.
  • the pre ⁇ parations may be tablets, granules, powders, capsules or the like.
  • the composition extract is typically formulated with additives, for example, an excipient such as a saccharide or cellulose preparation, a binder such as starch paste or methyl cellulose, a filler, a disintegrator and so on, all being ones usually used in the manufacture of medical preparations.
  • the composition may be prepared as a liquid suspension, emulsion, or syrup, being supplied either in liquid form or a dried form suitable for hydration in water or normal saline.
  • compositions of the present invention may be injected in the form of aqueous solutions, suspensions or oily or aqueous emulsions, such as liposome suspen ⁇ sions.
  • the composition is formulated as a lipid, e-g., triglyceride, or phospholipid suspension, with the extract vehicle components including a liquid carrier, and the immunosuppressant being dissolved or suspended in the lipid phase of the suspension.
  • composition is employed in immunosuppression therapy, in particular, therapy in treating autoimmune disease or transplantation rejection.
  • Table 1 gives a list of autoimmune diseases which are appropriate for immunotherapy.
  • SLE Systemic Lupus erythematosis
  • the patient is given the compound on a periodic basis, e.g., 1-2 times per week at a dosage level sufficient to reduce symptoms and improve patient comfort.
  • the composition may be administered by intravenous injection or by direct injection into the affected joint.
  • the patient may be treated at repeated intervals of at least 24 hours, over a several week period following the onset of symptoms of the disease in the patient.
  • the composition may be administered by oral or parenteral administration, such as IV administration.
  • the dose that is administered is preferably 25-75% of the dose of the immunosuppressant drug that would be administered, when given in the absence of the Tripterygium wilfordii extract vehicle.
  • the amount of vehicle administered is preferably in the range 1 to 25 mg/Kg patient body weight per day, with lower amounts being preferred for parenteral administration, and higher amounts being preferred for oral administration.
  • Parenteral administration may be by injection, e.g., intravenously, intramuscularly, or subcutaneously, inhalation, or uptake via a mucosal membrane.
  • the method is intended particularly for the treatment of rejection of heart, kidney, liver, and bone marrow transplants.
  • the method may also be used in the treatment of graft-versus-host disease, in which transplanted immune cells attack the allogeneic host.
  • Initial treatment is administered perioperatively.
  • the composition may be administered chron ⁇ ically to prevent graft rejection, or in treating acute episodes of late graft rejection.
  • the dose administered is preferably 25-75% of the normal dose of the immunosuppressant drug alone, where the amount of vehicle administered is in the range 1-25 mg dried extract material/Kg body weight.
  • the dose may be increased or decreased appropriately, depending on the response of the patient, and over the period of treatment, the ability of the patient to resist infection.
  • the effective dose of immunosuppressant drug is reduced substantially by its formulation in the TW extract vehicle, allowing higher drug doses to be administered and/or more prolonged treatment without serious side effects.
  • the treatment is typically started soon after the surgical transplantation procedure, and is continued on a daily dosing regimen, for a period of at least several weeks, for treatment of acute transplantation rejection.
  • the patient may be tested periodically for immunosuppression level, e.g., by a mixed lymphocyte reaction involving allogenic lymphocytes, or by biopsying the transplanted tissue.
  • the treatment of transplantation rejection in accordance with the invention is illustrated by the heart transplantation model reported in Example 6.
  • the method involves a well-characterized rat model system (Ono and Lindsey, 1969) in which a transplanted heart is attached to the abdominal great vessels of the recipient animal, and the viability of the transplanted heart is gauged by the heart's ability to beat in the recipient animal.
  • control solution 5% ethanol, lOml/Kg
  • purified extract vehicle oral administration, 10 mg/Kg
  • cyclosporin A intraperitoneal (IP) administration
  • the group treated with ethanol vehicle alone had a mean graft survival time of 6.7 days.
  • mean graft survival increased to 11.8 days.
  • IP administration of cyclosporin A mean graft survival time increased to 46.3 days, and there was a small percentage of grafts surviving at 100 days. The most effective results were observed in treatment with the composition of the invention, where mean graft survival time was 93.5 days, and nearly half of the grafts survived to 100 days.
  • the invention includes treating transplantation rejection by administration to a subject of the TW extract vehicle alone.
  • the vehicle is administered orally, or parenterally, in the doses given above, and employing the dosing schedules given above.
  • Fig. 5 is plotted the effect of the treatment method on allograft survival.
  • the three groups are (a) 5% alcohol solution (squares) , (b) 10 mg/Kg purified TW extract administered daily by oral administration (circles) , and (c) 10 mg/Kg purified TW extract administered daily by intraperitoneal injection (tri- angles) .
  • Fig. 5 Graft survival times are shown in Fig. 5. As seen, the hearts grafts were rejected by the untreated recipient animals in 6.7 days. In the animals treated with purified TW extract by oral administration, the allografts remained viable for 17 days, 3 days follow ⁇ ing discontinuation of treatment. In the animals treated with purified TW extract by intraperitoneal injection, the allografts remained viable for 21.3 days, one week after discontinuation of treatment. Details of the treatment method are given in Example 10. There was not evidence of toxicity in the treated animals. From the foregoing, it can be appreciated how various objects and features of the invention are met.
  • the purified extract material is readily prepared in quantity from root xylem of Tripterygium wilfordii , in a final amount which is about 0.1% of the original ethanol extract.
  • the material can be administered orally and is effective in prolonging transplant survival in mammals, as shown by the treatment method in Example 8. At the same time, the purified extract appears to have low toxicity, allowing long-term treatment with fewer side effects.
  • Tripterygium wilfordii Ethanol Extract Vehicle Tripterygium wilfordii plants were obtained in Fujiangzhou, China. Plants were air dried in sunlight. The root xylem of the plants (300 g) was ground into a crude powder and extracted with 5 volumes (1.5 1) of 95% ethanol, under reflux at 85°C for 4 hours. The filtered xylem powder was then extracted two more times in 3 volumes (900 ml total) of 95% ethanol. The three extracts (total of about 3,3, 1) were combined and the resulting mixture was concentrated at 50°C under vacuum, to about 2% of the original volume, i.e., about 66 ml.
  • TW Extract Vehicle A The CH L, TW Extract Vehicle
  • Example 2 The ethanol extract syrup obtained in Example 1 was then diluted with 33 ml water, filtered through Whatman # 1 filter paper. The filtrate was extracted 4 times (50 ml/extraction) with methylene chloride (CH 2 C1 2 ) .
  • B. The 1:1.000 TW Extract Vehicle
  • the column was washed successively with 100 ml methylene chloride, and 100 ml methylene chloride:methanol (95:5).
  • the fraction which eluted in 95:5 solvent contained about 0.3 g material, and is referred to herein as a 1:1000 extract vehicle.
  • Example 3 Thin-Layer Chromato ⁇ raphv of Purified TX Vehicle
  • a silica gel coated aluminum thin layer chromatography plate (Whatman, catalog # 4420 222).
  • the development solvent was hexane:methylene chloride:methanol in volume ratios of 1:1:0.15.
  • samples were visualized using an ultraviolet lamp and by application of an aerosol of 0.5% vanillin in H j SC ⁇ -ethanol (4:1).
  • TLC profiles of the various TW extracts are shown in Figure 2.
  • Lane A shows the 1:1,000 extract
  • lane B shows the 1:5,000 extract
  • lane C the 1:10,000 extract, herein called the purified TW extract. It can be seen that purification between the 1:1000, 1:5000 and 1:10000 extract vehicles has removed a number of major plant components.
  • the thin layer chromatographic profile showed no alkaloid in the extract, as determined by application of the Dragendorff reagent.
  • the thin layer chromatographic profile showed no alkaloid in the extract, as determined by application of the Dragendorff reagent.
  • PBLs were prepared, incubated and treated as described above.
  • the purified TW extract was used at 5 ⁇ g/ml.
  • Samples of tissue culture medium were col- lected at the end of 24 hours incubation and stored at -70°C prior to assay.
  • Cytokine measurements were carried out using commercially available ELISA assay kits (R&D Systems), in accordance with the supplier's protocols. In brief, 100 ⁇ l of the assay buffer supplied was added to each of the wells of a microtiter plate containing pre-bound anti-cytokine antibody, followed by 100 ⁇ l of standard or sample solution, diluted appropriately for the concentration range measured. All incubations were carried out at 37° or 24°C, per the supplier's protocol. Following two hours incubation, the plates were washed four times with assay buffer, and the second antibody, anti-cytokine-horseradish peroxidase (HRP) , was added to each well in a volume of 200 ⁇ l.
  • HRP anti-cytokine-horseradish peroxidase
  • basal levels of IL-1, TNFa, IL-2 and IL-6 increased markedly (3.8 to 167.3 pg/ml, 30.9 to 654.8 pg/ l, 7.6 to 148.7 pg/ml, and 108.5 to 2646 pg/ml, respectively) with X-35 stimulation.
  • the purified TW extract inhibited this X-35 stimulated increase by 16, 89, 93 and 100%, respectively.
  • the extract most likely inhibits cytokine production, though the decrease in medium cytokine concentration could theoretically result from increased catabolism. Decreased cytokine production may be responsible, at least in part, for the decrease in PBL proliferation in vitro and for the immunosuppressive effect of the extract in vivo .
  • MTT MTT
  • a yellow-colored compound is reduced by mito- chondrial enzymes to form a purple crystalline reduc ⁇ tion product (formazan) , providing an index of cellular respiration as well as a sensitive assay for cytotoxic ⁇ ity (Green) .
  • Cytotoxicity was assessed in both cultured PBLs and thymocytes (Green) .
  • a stock solution of MTT Sigma Chemical Co., St.
  • Heterotopic whole heart transplantation was per- formed according to the standard method (Ono) .
  • the donor (Brown Norway (200-255g) rats, Charles River, Wilmington, MA) and the recipient (Adult male Lewis rats, 225-275g, Charles River) were anesthetized with sodium pentobarbital (40 mg/kg) .
  • the heart graft was removed and stored at 4°C in PhysioSol Irrigation Solution (Abbott Laboratories, N. Chicago, IL) .
  • the ascending aorta and pulmonary artery were transsected, and the vena cava and pulmonary veins were ligated.
  • the recipient abdo- minal aorta and inferior vena cava were exposed through a median abdominal incision.
  • the donor heart aorta and pulmonary artery were anastomosed end-to-side to reci ⁇ pient's infrarenal abdominal aorta and inferior vena cava, respectively, with running 8-0 monofilament nylon suture (Ethilon, Inc., Somerville, NJ) .
  • blood did not enter the left ventricle but rather flowed through the coronary arteries to the right atrium, pulmonary artery and the recipient vena cava.
  • the cold ischemic time of all the cardiac grafts was less than 45 minutes.
  • Graft heartbeat was monitored by abdominal palpation.
  • the period of functional graft survival was measured as the number of days during which cardiac graft contractions could be detected by abdominal palpation. Results were confirmed by direct visualiza ⁇ tion at laparotomy.
  • the treatment methods started on the day prior to surgery and continuing daily until postoperative day 52, or until the end of allograft survival. Each graft recipient was followed until the graft ceased beating. The results are seen in Fig. 4, and are discussed above.
  • the transplantation model described in Part A was employed.
  • the animals (3 animals/group) were treated for 14 days with the extract vehicle (lOmg/kg) or with ethanol solution (5% ethanol, lOml/kg) , starting on the day prior to surgery and then daily.
  • the extract vehicle was administered both orally and by intraperitoneal injection. Each graft recipient was followed until the graft ceased beating. The results are shown in Fig. 5, described above.

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Abstract

L'invention se rapporte à une composition utilisée en thérapie immunossuppressive. Cette composition comprend un médicament immunossuppresseur, telle que la cyclosporine A, dans un excipient composé d'un éthanol extrait du xylème de la racine de Trypterygium wilfordii. Cet excipient est efficace seul, ou en combinaison avec un immunossuppresseur tel que celui décrit ci-dessus, dans le traitement du rejet de transplantation. L'invention se rapporte également à la durée de vie d'une greffe chez des animaux non traités (triangles vides), et chez des animaux traités avec un excipient seulement, constitué d'un extrait de TW (losanges pleins), avec la cyclosporine A seulement (cercles vides) et avec la cyclosporine A contenue dans un excipient constitué d'un extrait de TW.
PCT/US1993/010724 1993-11-08 1993-11-08 Composition utilisee en immunotherapie et procede WO1995013082A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6025391A (en) * 1996-04-12 2000-02-15 Novartis Ag Enteric-coated pharmaceutical compositions of mycophenolate
WO2002078684A2 (fr) * 2001-03-30 2002-10-10 Sangstat Medical Corporation Composes generateurs de monoxyde de carbone pour le traitement de troubles vasculaires, inflammatoires et immunitaires
WO2006012204A2 (fr) * 2004-06-25 2006-02-02 Pharmagenesis, Inc. Procede de traitement de troubles inflammatoires au moyen de composes de triptolide
CN101367862B (zh) * 2008-09-25 2010-10-20 成都普思生物科技有限公司 一种从雷公藤中分离雷公藤甲素的方法
US8617906B2 (en) 2004-10-13 2013-12-31 Pharmagenesis, Inc. Identification and screening of triptolide target molecules
US9528088B2 (en) 2002-06-28 2016-12-27 Life Technologies Corporation Methods for eliminating at least a substantial portion of a clonal antigen-specific memory T cell subpopulation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4005108A (en) * 1973-04-03 1977-01-25 Research Corporation Novel anti-leukemic diterpenoid triepoxides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4005108A (en) * 1973-04-03 1977-01-25 Research Corporation Novel anti-leukemic diterpenoid triepoxides

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6025391A (en) * 1996-04-12 2000-02-15 Novartis Ag Enteric-coated pharmaceutical compositions of mycophenolate
US6172107B1 (en) 1996-04-12 2001-01-09 Novartis Ag Entric-coated pharmaceutical compositions
US6306900B1 (en) 1996-04-12 2001-10-23 Novartis Ag Enteric coated pharmaceutical compositions
WO2002078684A2 (fr) * 2001-03-30 2002-10-10 Sangstat Medical Corporation Composes generateurs de monoxyde de carbone pour le traitement de troubles vasculaires, inflammatoires et immunitaires
WO2002078684A3 (fr) * 2001-03-30 2002-12-19 Sangstat Medical Corp Composes generateurs de monoxyde de carbone pour le traitement de troubles vasculaires, inflammatoires et immunitaires
US9528088B2 (en) 2002-06-28 2016-12-27 Life Technologies Corporation Methods for eliminating at least a substantial portion of a clonal antigen-specific memory T cell subpopulation
WO2006012204A2 (fr) * 2004-06-25 2006-02-02 Pharmagenesis, Inc. Procede de traitement de troubles inflammatoires au moyen de composes de triptolide
WO2006012204A3 (fr) * 2004-06-25 2009-04-09 Pharmagenesis Inc Procede de traitement de troubles inflammatoires au moyen de composes de triptolide
US8617906B2 (en) 2004-10-13 2013-12-31 Pharmagenesis, Inc. Identification and screening of triptolide target molecules
CN101367862B (zh) * 2008-09-25 2010-10-20 成都普思生物科技有限公司 一种从雷公藤中分离雷公藤甲素的方法

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