WO1994026265A1 - Composition de 16-hydroxytriptolide et procede d'immunotherapie - Google Patents

Composition de 16-hydroxytriptolide et procede d'immunotherapie Download PDF

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Publication number
WO1994026265A1
WO1994026265A1 PCT/US1994/004990 US9404990W WO9426265A1 WO 1994026265 A1 WO1994026265 A1 WO 1994026265A1 US 9404990 W US9404990 W US 9404990W WO 9426265 A1 WO9426265 A1 WO 9426265A1
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hydroxytriptolide
composition
treating
rejection
immunosuppressant drug
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PCT/US1994/004990
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English (en)
Inventor
Renling Jin
Tien Wen Wiedmann
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Pharmagenesis, Inc.
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Priority to AU68261/94A priority Critical patent/AU6826194A/en
Publication of WO1994026265A1 publication Critical patent/WO1994026265A1/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
    • A61K31/33—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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • 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
    • 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
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • 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

Definitions

  • the present invention relates to a composition and method for transplantation rejection and immunosuppression.
  • 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.
  • 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.
  • rheumatoid arthritis 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.
  • anti-inflammatory agents aspirin, non-steroidal anti-inflammatory drugs and low dose corticosteroids
  • DMARDs disease-modifying antirheumatic drugs
  • immunosuppressive agents azathioprine, chlorambucil, high dose corticoste ⁇ roids, cyclophosphamide, methotrexate, nitrogen mustard, 6-mercaptopurine, vincristine, hydroxy- urea, and cyclosporin A
  • immunosuppressive agents have also been used in treating or preventing transplantation rejection.
  • Organ transplantation involving human organ donors and human recipients (allogeneic grafts) , and non-human primate donors and human recipients (xenogeneic grafts) has received considerable medical and scientific attention (Roberts, 1989; Platt, 1990; Keown, 1991; Wang, 1991; Hasan, 1992; Murase, 1993). 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 The major targets in transplant rejection are non- self allelic forms of class I and class II major histocompatibility complex (MHC) antigens. Rejec ⁇ tion is mediated by both antibodies and cytotoxic T lymphocytes (CTLs) , with the participation of CD4+ "helper" T cells (Noelle, 1991) . In general, foreign class I MHC antigens stimulate CD8+ CTLs, and foreign class II MHC antigens stimulate CD4+ T cells (Roitt, 1991) .
  • CTLs cytotoxic T lymphocytes
  • foreign class II MHC antigens stimulate CD4+ T cells (Roitt, 1991) .
  • GVHD graft-versus-host disease
  • the most commonly used agents for preventing transplant rejection include corticosteroids, 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; Kennedy, 1983; Storb, 1985; Storb, 1986) . 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 opportunistic invaders, and in part because of direct toxicity and other side effects.
  • 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 physician is frequently forced to administer sub-optimal doses of the drug because of this toxicity.
  • a drug capable of potentiating the action of immunosuppressive agents such as cyclosporin A.
  • a drug would increase the ' efficacy of such immunosuppressive agents and also decrease deleterious side-effects by allowing admin ⁇ istration of lower dosage levels.
  • the invention relates to a purified compound for use in immunosuppressive therapy and referred to herein as 16-hydroxytriptolide, having the structural formula:
  • the invention includes a composition for use in immunosuppression therapy in a mammalian subject.
  • the composition includes a therapeutically effective amount of purified 16- hydroxytriptolide in a pharmaceutically acceptable delivery vehicle, and also carried in the vehicle, an immunosuppressant drug, particularly cyclosporin A, FK506, azathioprine, methotrexate, rapamycin, mycophenolic acid, or a glucocorticoid.
  • the composition has an increased immunosuppressive activity relative to the sum of the effects produced by 16-hydroxytriptolide or immunosuppressant drug used alone, allowing greater immunosuppressive activity with reduced toxicity.
  • the composition is employed in immunosuppressive therapy, particularly in therapy for transplantation rejection and autoimmune disease.
  • the composition is used in treating transplantation rejection.
  • the composition can be used for treating rejection of an allograft or a xenograft, or complications caused by graft-versus-host disease.
  • the composition is used in treating transplantation rejection, and the immunosuppressant drug is cyclosporin A. Also disclosed is an immunosuppression therapy method for use in treating a mammalian subject.
  • the method includes administering to the subject, a pharmaceutically effective amount of (i) purified 16-hydroxytriptolide and (ii) an immunosuppressant drug which is cyclosporin A, FK506, azathioprine, methotrexate, rapamycin, mycophenolic acid, a glucocorticoid, or a combination of two or more of such drugs.
  • an immunosuppressant drug which is cyclosporin A, FK506, azathioprine, methotrexate, rapamycin, mycophenolic acid, a glucocorticoid, or a combination of two or more of such drugs.
  • Fig. 1 show a proposed structure of 16- hydroxytriptolide
  • Fig. 2 shows an electron impact mass spectrum of 16-hydroxytriptolide
  • Fig. 3 shows a fourier transform infrared (IR) spectrum of 16-hydroxytriptolide
  • Fig. 4 shows a ⁇ nuclear magnetic resonance (NMR) spectrum of 16-hydroxytriptolide
  • Fig. 5 shows a proton-decoupled 13 C NMR spectrum of 16-hydroxytriptolide
  • Fig. 6 shows a l H- l u COSY spectrum of 16- hydroxytriptolide
  • Fig. 7 shows a l U- C chemical shift correlation spectrum of 16-hydroxytriptolide
  • Fig. 8 shows inhibition by 16- hydroxytriptolide of human peripheral blood lymphocyte (PBL) proliferation in the presence of anti-CD3 antibody
  • Fig. 9 shows the suppressive effect of 16- hydroxytriptolide on IL-1 stimulation of mouse thymocyte proliferation
  • Fig. 10 shows the suppressive effect of 16- hydroxytriptolide on IL-2 stimulation of HT-2 cell proliferation
  • Figs. 11A and 11B show the effect of 16- hydroxytriptolide on the concentrations of the cytokines IL-6 (Fig. 11A) , TNF ⁇ (Fig. 11A) , and IL-2 (Fig. 11B) in PHA-stimulated human PBL cultures.
  • the invention employs a purified compound which may be isolated from Tripterygium wilfordii .
  • the compound designated "tripterygin” in parent application Ser. No. 08/058,321, is referred to herein as "16-hydroxytriptolide.”
  • a postulated structural formula is shown in Fig. 1.
  • 16-Hydroxytriptolide can be purified from the root xylem of Tripterygium wilfordii , a medicinal plant which is readily available in Fujiangzhou and other southern provinces of China or through commercial sources in the United States.
  • the presence of 16-hydroxytriptolide in the fractions generated at various stages of purifica ⁇ tion can be monitored by use of one or more of the assays described in Examples 3-6 (e.g., the PBL assay in Example 3) .
  • other compounds present in the mixture may also be detectable in the assays, thereby precluding unambiguous distinction of 16-hydroxytriptolide from such other compounds.
  • Example 1 A protocol for isolating purified 16-hydroxy- triptolide is described in Example 1. Briefly, dried plant material is ground into a crude powder and extracted by reflux with a volume of 95% ethanol that is about five times the weight (on a ml/g basis) of the dried plant material. The residual solid is extracted twice more with 95% ethanol, and the three resultant ethanol extracts are combined, filtered and reduced to a concentrated, syrupy extract (to about 2% of the original ethanol volume) . The concentrated extract is diluted by about
  • methylene chloride fractions 50% with water, filtered, and then extracted with several portions of methylene chloride. Following concentration, the collected methylene chloride fractions are applied to a silica gel column and eluted in a series of methylene chloride:methanol mixtures, i.e., mixtures with ratios of 100:0, 97:3, 95:5, and 90:10. The fractions which elute with the 95:5 mixture are combined and concentrated.
  • the resultant concentrate is purified further by silica gel chromatography using a series of hexane:methylene chloride:methanol mixtures, i.e., mixtures having ratios of 1:2:5, 1:2:10, 1:2:15, and 1:2:20.
  • the fractions which elute with the 1:2:15 mixture are collected and concentrated.
  • the resultant concentrate is purified further by silica gel chromatography using a series of hexane: acetone mixtures, i.e., having ratios of 9:1, 8:2, 7:3, and 6:4.
  • the fractions which elute with the 7:3 mixture are combined and concentrated.
  • the concentrate is purified by reversed phase (C-18) high performance liquid chromatography (HPLC) (70% methanol in water as eluant) by passage through two ODS columns as described in Example 1.
  • HPLC high performance liquid chromatography
  • the resultant material is crystallized from methanol to yield the final, purified 16- hydroxytriptolide.
  • Fig. 1 shows a structure and carbon atom numbering scheme for the 16-hydroxytriptolide compound of the invention.
  • the l E- l H COSY spectrum shown at Fig. 6, revealed coupling interactions between protons at C-ll and C-12, between protons at C-15, C-16, and C-17, between protons at C-5, C-6, and C-7, and between protons at C-l and C-2.
  • the purified 16-hydroxytriptolide compound may be administered with the immunosuppressant compound together in the same formulation, or separately in separate formulations. Where the compounds are prepared in separate formulations, the 16-hydroxytriptolide compound and immunosuppressant compound can be administered by different routes if desired.
  • the immunosuppressant drug which is administered with the 16-hydroxytriptolide compound is preferably one of the following:
  • Cyclosporin A or cyclosporin C (“cyclosporin”) , a non-polar cyclic oligopeptide
  • cyclosporin cyclosporin
  • FK506 a fungal macrolide immunosuppressant
  • an immunosuppressant glucocorticoid such as prednisone or dexamethasone.
  • the proportions of the two components (16- hydroxytriptolide and immunosuppressant drug) are preferably in the range of 1:50 to 50:1 by weight.
  • the preparations may be tablets, granules, powders, capsules or the like.
  • the compound 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 compounds 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.
  • the compounds may be injected in the form of aqueous solutions, suspensions or oily or aqueous emulsions, such as liposome suspensions.
  • the compounds are formulated as a lipid formulation, e.g., triglyceride, or phospholipid suspension, with the 16-hydroxytriptolide being dissolved in the lipid phase of the suspension.
  • the purified 16-hydroxytriptolide compound was examined for immunosuppressive activity in a variety of in vitro biological assays.
  • PBL peripheral blood lymphocyte
  • X-35 antibody anti- CD3 monoclonal antibody
  • a solution of purified 16-hydroxytriptolide or sol ⁇ vent alone (control) was added to each culture, at selected concentrations.
  • tritiated thymidine was added to the culture medium, and thymidine incorporation into DNA was assayed, as a measure of DNA synthesis associated with cell proliferation.
  • 16-hydroxytriptolide 8 shows inhibition of peripheral blood lymphocyte proliferation, in the presence of stimulation with anti-CD3 antibody, as a function of the concentration of added 16- hydroxytriptolide.
  • increasing amounts of purified 16-hydroxytriptolide produced dose dependent inhibition of proliferation of both unstimulated and anti-CD3-stimulated PBLs, with substantially complete inhibition occurring at a dose of 2 x 10" 8 M 16-hydroxytriptolide.
  • mice thymocytes in culture were stimulated with IL-1 in the presence of phytohemagglutinin (PHA) and increasing concentrations of purified 16-hydroxytriptolide.
  • PHA phytohemagglutinin
  • the cells were cultured for 72 hours, and during the last four hours, incubated with tritiated thymidine.
  • Thymocyte proliferation was assessed by measurement of radiolabeled thymidine incorporation into DNA.
  • FIG. 9 shows the inhibition of IL-1- stimulated thymocyte proliferation in culture by 16-hydroxytriptolide.
  • IL- 1-stimulated cell proliferation was inhibited maximally by 16-hydroxytriptolide at a con ⁇ centration of about 10" 8 M, with half-maximal inhibition occurring at a concentration of about 3 x 10 "9 M.
  • cytokine levels were measured by standard ELISA methods using commercially available kits, as detailed in Example 6. 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 cytokine concentration measured, followed by a second reporter-labeled antibody specific against the anti-cytokine antibody.
  • 16-hydroxytriptolide inhibited the production of 11-6 and TNF ⁇ .
  • Cytotoxicity Potential cytotoxicity of 16- hydroxytriptolide was assessed by measurement of the effect of 16-hydroxytriptolide on reduction of MTT (3-[4,5-Dimethylthiazol-2-yl] 2,5-diphenyl- tetrazolium bromide) by cultured cells, an index of cellular respiration and a sensitive assay for the detection of cytotoxicity (Green, et al . ) . Toxicity was evaluated in vitro in human PBLs and in mouse thymocytes and expressed as percent of control, as detailed in Example 7. Sodium azide was used as a cytotoxic control. Cytotoxicity was also assessed using the standard method of trypan blue dye staining.
  • 16-hydroxytriptolide showed no significant toxicity at concentrations less than 10 "7 M, the highest concentration range at which the compound produced dose-dependent inhibition of cytokine production or action. Cytotoxicity was observed at higher concentrations. At 0.5 x 10" 6 M, 16-hydroxytriptolide decreased cellular respiration by 29%, and at 2 x 10" 6 M, 16-hydroxy ⁇ triptolide decreased cellular respiration by 39%.
  • the 16-hydroxytriptolide and immunosuppressant compounds of the invention are employed in immunosuppression therapy, particularly in treating transplantation rejection or autoimmune disease.
  • the effective dose of immunosuppressant drug is reduced significantly by co-administration of the drug with the purified 16-hydroxytriptolide compound, allowing higher drug doses to be administered and/or more prolonged treatment while reducing deleterious side effects.
  • Table 3 below gives a list of autoimmune diseases which are appropriate for immunotherapy.
  • the patient is given the 16-hydroxytriptolide and immunosuppressive compounds in a pharmaceutically acceptable vehicle or vehicles on a periodic basis, e.g., 1-2 times per week at a dosage level sufficient to reduce symptoms and improve patient comfort.
  • the 16-hydroxytriptolide and immunosuppressant compounds are administered separately, the compounds may be administered with different dosing schedules as appropriate to maintain the potentiating effect of the 16- hydroxytriptolide compound on the immu- nosuppressant compound.
  • the 16- hydroxytriptolide compound may be given in liquid, tablet or capsule form, at a preferred dose of 0.1 and 2 mg/kg patient body weight per day.
  • the dose may be increased or decreased appropriately depending on the response of the patient, and patient tolerance.
  • a parenteral suspension can be administered by injection, e.g., intravenously, intramuscularly, or subcutaneously, inhalation, or uptake via a mucosal membrane.
  • a dose between about 0.05 and 1 mg 16-hydroxytriptolide/kg body weight per day is preferred, and this level may be increased or decreased appropriately, depending on the conditions of disease, the age of the patient, and the ability of the patient to resist infec ⁇ tion.
  • 16-hydroxy ⁇ triptolide is administered in an amount effective to achieve a serum concentration of 16- hydroxytriptolide of between about 10 "8 and 10 "7 M.
  • the dose of immunosuppressant drug that is administered is preferably 25-75% of the dose that would be administered when given in the absence of the 16-hydroxytriptolide compound, although lower levels of immunosuppressant drug may be administered.
  • the compounds may be administered by intravenous injection or by direct injection into the affected joint, for example.
  • 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 compounds may be administered by oral or parenteral administration, such as intravenous (IV) administration.
  • IV intravenous
  • the method is intended particularly for the treatment of rejection of heart, kidney, liver, and bone marrow transplants. The method is useful in the treatment of xenograft as well as allograft rejection.
  • the method may also be used in the treatment of graft-versus-host disease, in which transplanted immune cells attack the allogeneic host.
  • the composition may be administered chronically to prevent graft rejection, or in treating acute episodes of late graft rejection. Treatment is typically begun perioperatively and is typically continued on a daily dosing regimen, for a period of at least several weeks, for treatment of acute transplantation rejection. During the treatment period, the patient may be tested periodically for immunosuppression level. Biopsy of the transplanted tissue may also be appropriate.
  • Bioassay Fractions produced at various stages in the purification procedure below were assayed to identify fractions containing 16-hydroxytriptolide by one or more of the assays described in Examples 3-6.
  • the concentrated mixture was then diluted with 33 ml water and filtered through Whatman #1 filter paper.
  • the filtrate was extracted 4 times (50 ml/extraction) with methylene chloride.
  • the combined filtrate (about 200 ml) was concentrated, and applied to a 6.5 cm (diameter) x 12 cm column of silica gel (-60-200 mesh) .
  • the column was washed successively with 600 ml methylene chloride, and 1500 ml methylene chloride:methanol (95:5).
  • the fractions which eluted with the methylene chloride:methanol solvent were then concentrated under reduced pressure, yielding about 0.9 g of crude product (concentrate #1).
  • Concentrate #2 was loaded on a 5.5 x 49 cm silica gel column and eluted with the following series of mixtures of hexane:CH 2 C1 2 :methanol as follows: 1:2:5 (2 liters), 1:2:10 (2 liters), 1:2:15 (2 liters), and 1:2:20 (2 liters). Fractions which were eluted by the 1:2:15 mixture were concentrated under reduced pressure, yielding 2.26 g of a concentrate #3.
  • Concentrate #3 was purified by silica gel chromatography (2 cm x 42 cm) using the following series of hexane:acetone mixtures: 9:1 (500 ml), 8:2 (1000 ml), 7:3 (1000 ml), and 6:4 (300 ml). Fractions which eluted with the 7:3 mixture were combined and concentrated, yielding 271 mg of a concentrate #4.
  • Concentrate #4 was purified by reversed phase HPLC on a 20 x 250 mm ODS column (Japanese Analytical, Inc., distributed by DyChrom, Santa Clara, CA) .
  • the mobile phase was 70% methanol in water, at a flow rate of 3 ml/min (100 mg/run) .
  • the elution time of 16-hydroxytriptolide was 3.6 min.
  • 16-Hydroxytriptolide-containing fractions were concentrated under reduced pressure (75°C) and further purified by another ODS HPLC column (10 x 250 mm) using a mobile phase of 70% methanol in water, at a flow rate of l ml/min (20 mg/run) .
  • the elution time of 16-hydroxytriptolide was 2.5 min.
  • Example 2 Physical Characteristics of 16-Hvdroxytriptolide Mass spectrometric analyses of 16- hydroxytriptolide were performed using a VG-ZAB2- EQ mass spectrometer for characterization by fast atom bombardment (FAB) , and a Kratos MS-50 for high resolution mass determination using electron impact (El) ionization for determination of exact mass values.
  • the exact mass found for MH + was 377.160050, in good agreement with the calculated value for C ⁇ j O-, (377.160028).
  • the infrared spectrum Fig.
  • Human peripheral blood lymphocytes were prepared using an established method (Boyum) . Human blood buffy coat samples, approximately 25 ml/donor, were obtained from the Stanford
  • HBSS Hank's balanced salt solution
  • HBSS Hank's balanced salt solution
  • a volume of 25 ml of the cell suspension was then layered onto 15 ml of Ficoll-Pacque (Pharmacia LKB Biotechnology, Inc. , Piscataway, NJ) in a 50 ml conical centrifuge tube. Tubes were centrifuged in a Beckman GPR tabletop centrifuge (GH-3.7 Rotor) at 400 x g for 30 minutes at 15°C.
  • GH-3.7 Rotor Beckman GPR tabletop centrifuge
  • the PBL suspensions at the interfaces were transferred to new 50 ml tubes using a transfer pipette, and the PBL samples were resuspended in a total volume of 45 ml HBSS and centrifuged at 354 x g for 10 minutes at 15°C. Supernatants were discarded. PBL's were resuspended in 10 ml HBSS, combined to make a total of 45 ml HBSS, and centrifuged at 265 x g for 10 minutes at 15°C. The cell pellets were suspended in 10 ml of X-Vivo tissue culture medium (BioWhittaker Inc. , Walkersville, MD) and counted using a hemocytometer. Tissue culture medium was then added to achieve a final cell concentration of 1 x 10 6 cells/ml. Additional dilutions were carried out as required for each assay.
  • X-Vivo tissue culture medium BioWhittaker Inc. , Walkersville, MD
  • Assays were carried out in 96 well sterile tissue culture plates (Costar 3790 and Costar 3595) .
  • a volume of 150 ⁇ l of X-Vivo medium or sterile distilled water was added to the outer wells of the plate to prevent evaporation of medium within the experimental wells.
  • PBL's from 2 different donors were used in parallel in all experiments.
  • a volume of 100 ⁇ l PBL suspension was added to each well using a multichannel pipette. Plates were incubated in an atmosphere of 93% air/7% C0 2 in a tissue culture incubator at 37°C.
  • X-35 AMAC #0178
  • an anti-CD3 surface antigen antibody was used at 5 ng/ml to stimulate PBL proliferation.
  • 16-Hydroxytriptolide was dissolved in ethanol or DMSO (10 mg/ml) and then diluted in sterile X-Vivo tissue culture medium to obtain the final concentrations required for each experiment.
  • 50 ⁇ l of X-Vivo tissue culture medium containing 8 ⁇ Ci/ml [ 3 H]Thymidine 49 Ci/mmol, Amersham, Arlington Heights, IL was added to each tissue culture well.
  • the cells were removed from the tissue culture wells and applied to filter paper using a cell harvester (Brandel, Model MB-24) . The filter paper was dried for one hour under a heat lamp and then cut into l cm discs.
  • 16-hydroxytriptolide inhibited PBL proliferation over a concentration range of 10 "9 to
  • mice were prepared, and the action of IL-1, which stimulates thymocyte proliferation, was measured using standard techniques (O'Gara) .
  • Three to six week old C3H/HeN mice were obtained from Simonsen Laboratories, Gilroy, California and sacrificed by C0 2 inhalation. Thymi were removed, separated from adherent non-thymic tissue, homogenized in Hank's balanced salt solution (Gibco) using a glass homogenizer, and centrifuged at 180 x g for 10 minutes at 15°C.
  • Hank's balanced salt solution Gibco
  • thymocytes were resuspended in RPMI 1640 tissue culture medium (Gibco) containing 50 ⁇ M 2- mercaptoethanol (Fisher, Orangeburg, NY) , 2 mM glutamine (Gibco) , 1 mM sodium pyruvate, non-essential amino acids, penicillin (100 U/ml) streptomycin (100 ⁇ g/ml) , 10% heat-inactivated fetal bovine serum and Phytohemagglutinin (PHA, Pharmacia, final concentration 10 ⁇ g/ml) .
  • Untreated cells showed minimal DNA synthesis (thymidine incorporation 80 cpm/well) .
  • PHA alone stimulated thymidine incorporation 2-3 fold.
  • Treatment with 0.1 ng/ml IL-1 in the presence of PHA resulted in a 60-fold increase.
  • Addition of the 16-hydroxytriptolide resulted in a dose- dependent inhibition of IL-1 stimulation, as shown in Figure 9. Inhibition was measured over a range of about 10" 9 M to 2 x 10" 6 M. Half-maximal inhibition occurred at 2 x 10" 9 M.
  • IL-2 Inhibition of IL-2 Action by 16-Hvdro ⁇ ytriptolide
  • the effect of 16-hydroxytriptolide on the action of IL-2 was assessed by measurement of the compound's ability to inhibit IL-2-stimulated growth of the IL-2 dependent cell line HT-2, a well established biological assay of IL-2 action (O'Gara) .
  • HT-2 cells were cultured in 75 cm 2 (Corning, Corning, NY) tissue culture flasks in RPMI 1640 medium containing 10% fetal bovine serum (Hyclone, Logan, UT) , 50 ⁇ M 2- mercaptoethanol
  • 16-hydroxytriptolide over a concentration range of 10" 9 to 10" 7 M, inhibited IL-2 induced DNA synthesis. Half maximal inhibition occurred at about 3 x 10 "8 M.
  • Human PBLs were prepared as in Example 3. The cells were incubated in the presence and absence of varying concentrations of 16- hydroxytriptolide, in the presence of 10 ⁇ g/ml PHA. Samples of tissue culture medium were collected following 24 hours incubation and stored at -70°C prior to assay.
  • Cytokine measurements were carried out using commercially available ELISA assay kits for cytokines IL-1, IL-2, IL-6, and TNF ⁇ (R&D Systems) , in accordance with the supplier's protocols.
  • 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°C or 24°C, per the supplier's protocol.
  • HRP anti-anti-cytokine-horseradish peroxidase
  • 16-hydroxytriptolide effectively inhibited the production of IL-6 and TNF ⁇ . Production of these cytokines was suppressed to 1% of control levels in the presence of 10 "7 M 16-hydroxytriptolide. Half-maximal inhibition occurred at about 4 x 10" 9 M and 8 x 10' 9 M, respectively. As shown in Fig. 11B, 10 ⁇ 7 M 16- hydroxytriptolide also decreased the production of IL-2 to about 10% of the level present in the untreated cultures. Half-maximal inhibition of IL-2 production occurred at about 4xl0 "9 M. In contrast, 16-hydroxytriptolide did not alter IL-1 production by the cultured lymphocytes.
  • Example 7 Evaluation of Potential Cytotoxicity Potential cytotoxicity of 16- hydroxytriptolide was assessed by measurement of the compound's effect on the reduction of MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl- tetrazolium bromide) by cultured cells.
  • MTT a yellow-colored compound, is reduced by mitochondrial enzymes to form a purple crystalline reduction product (formazan) , providing an index of cellular respiration as well as a sensitive assay for cytotoxicity (Green, et al . ) .
  • Cytotoxicity was assessed in cultured human PBLs and mouse thymocytes.
  • a stock solution of MTT (Sigma, St. Louis, MO), 5 mg MTT/ml phosphate buffered saline, pH 7.4, was prepared and stored in the dark at 4°C. Following 21 hours incubation under conditions identical to those above, 25 ⁇ l of MTT solution was added to each culture well. After an additional 3 hour incubation, the experiment was terminated by addition of a solution of 10% sodium dodecyl sulfate in 0.01 N HCl. Following overnight incubation at 37°C (to solubilize the formazan crystals, the MTT reduc- tion product) , optical density was determined at
  • 16-Hydroxytriptolide showed no significant toxicity at concentrations less than 10" 7 M, the highest concentration range at which the compound produced dose-dependent inhibition of cytokine production or action. Cytotoxicity was observed at higher concentrations. At 0.5 x 10 "6 M, 16- hydroxytriptolide decreased cellular respiration by 29%, and at 2 x 10 "6 M, 16-hydroxytriptolide decreased cellular respiration by 39%.

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Abstract

L'invention se rapporte à une composition à utiliser dans des thérapies d'immunodépression, cette composition contenant un immunodépresseur, tel que la cyclosporine A, et une quantité de 16-hydroxytriptolide suffisante pour potentialiser l'effet immunodépressif du médicament immunodépresseur. Cette composition est particulièrement utile pour traiter les rejets de greffe lors de transplantations ou les maladies auto-immunes. L'invention décrit également un procédé d'immunodépression qui consiste à administrer à un sujet: (i) une quantité pharmaceutiquement efficace d'un immunodépresseur, et (ii) de la 16-hydroxytriptolide purifiée en quantité suffisante pour potentialiser l'action du médicament immunodépresseur.
PCT/US1994/004990 1993-05-06 1994-05-05 Composition de 16-hydroxytriptolide et procede d'immunotherapie WO1994026265A1 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998052933A1 (fr) * 1997-05-23 1998-11-26 Hoechst Marion Roussel, Inc. Derives triptolides convenant au traitement d'affections auto-immunes
US5849730A (en) * 1994-08-31 1998-12-15 Pfizer Inc. Process for preparing demethylrapamycins
US5972998A (en) * 1997-05-23 1999-10-26 Hoechst Marion Roussel, Inc. Triptolide derivatives useful in the treatment of autoimmune diseases
US6004999A (en) * 1997-05-23 1999-12-21 Hoechst Marion Roussel, Inc. Triptolide derivatives useful in the treatment of autoimmune diseases
US6025391A (en) * 1996-04-12 2000-02-15 Novartis Ag Enteric-coated pharmaceutical compositions of mycophenolate
US6329148B1 (en) * 1999-02-16 2001-12-11 The Board Of Trustees Of The Leland Stanford University Combined therapy of diterpenoid triepoxides and death domain receptor ligands for synergistic killing of tumor cells
EP1722806A2 (fr) * 2004-02-09 2006-11-22 Pharmagenesis, Inc. Procedes permettant d'isoler des composes a base de triptolide de tripterygium wilfordii
US7557139B2 (en) * 2000-10-02 2009-07-07 Emory University Triptolide analogs for the treatment of autoimmune and inflammatory disorders
US7662976B2 (en) 2002-05-31 2010-02-16 Pharmagenesis, Inc. Triptolide derivatives for modulation of apoptosis and immunosuppression
US7820834B2 (en) 2003-12-24 2010-10-26 Pharmagenesis, Inc. Triptolide 5,6-derivatives as immunomodulators and anticancer agents
US7863464B2 (en) 2004-03-02 2011-01-04 Pharmagenesis, Inc. Triptolide lactone ring derivatives as immunomodulators and anticancer agents
EP2583678A2 (fr) 2004-06-24 2013-04-24 Novartis Vaccines and Diagnostics, Inc. Immunopotentiateurs de petites molécules et dosages pour leur détection
US8617906B2 (en) 2004-10-13 2013-12-31 Pharmagenesis, Inc. Identification and screening of triptolide target molecules
US20200224271A1 (en) * 2017-07-14 2020-07-16 The Regents Of The University Of California Novel Methods of Predicting Transplant Rejection Risk

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 116, no. 15, 13 April 1992, Columbus, Ohio, US; abstract no. 143775f, page 72; *
MA P.C., ET AL.: "16-Hydroxytriptolide: an new active compound from Triperygium wilford H.", YAOXUE XUEBAO, vol. 26, no. 10, 1992, pages 759 - 763 *
YANG S., ET AL.: "Immunosuppression of triptolide and its effect on skin allograft survival", INT. J. IMMUNOLOG., vol. 14, no. 6, 1992, pages 963 - 969 *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5849730A (en) * 1994-08-31 1998-12-15 Pfizer Inc. Process for preparing demethylrapamycins
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
WO1998052933A1 (fr) * 1997-05-23 1998-11-26 Hoechst Marion Roussel, Inc. Derives triptolides convenant au traitement d'affections auto-immunes
US5972998A (en) * 1997-05-23 1999-10-26 Hoechst Marion Roussel, Inc. Triptolide derivatives useful in the treatment of autoimmune diseases
US6004999A (en) * 1997-05-23 1999-12-21 Hoechst Marion Roussel, Inc. Triptolide derivatives useful in the treatment of autoimmune diseases
AU741209B2 (en) * 1997-05-23 2001-11-22 Aventisub Ii Inc. Novel triptolide derivatives useful in the treatment of autoimmune diseases
US6329148B1 (en) * 1999-02-16 2001-12-11 The Board Of Trustees Of The Leland Stanford University Combined therapy of diterpenoid triepoxides and death domain receptor ligands for synergistic killing of tumor cells
US7557139B2 (en) * 2000-10-02 2009-07-07 Emory University Triptolide analogs for the treatment of autoimmune and inflammatory disorders
US8193249B2 (en) 2000-10-02 2012-06-05 Emory University Triptolide analogs for the treatment of autoimmune and inflammatory disorders
US7847109B2 (en) 2002-05-31 2010-12-07 Pharmagenesis, Inc. Triptolide derivatives for modulation of apoptosis and immunosuppression
US7662976B2 (en) 2002-05-31 2010-02-16 Pharmagenesis, Inc. Triptolide derivatives for modulation of apoptosis and immunosuppression
US7820834B2 (en) 2003-12-24 2010-10-26 Pharmagenesis, Inc. Triptolide 5,6-derivatives as immunomodulators and anticancer agents
US8048914B2 (en) 2004-02-09 2011-11-01 Pharmagenesis, Inc. Methods for isolation of triptolide compounds from Tripterygium wilfordii
EP1722806A2 (fr) * 2004-02-09 2006-11-22 Pharmagenesis, Inc. Procedes permettant d'isoler des composes a base de triptolide de tripterygium wilfordii
EP1722806A4 (fr) * 2004-02-09 2009-09-16 Pharmagenesis Inc Procedes permettant d'isoler des composes a base de triptolide de tripterygium wilfordii
US7863464B2 (en) 2004-03-02 2011-01-04 Pharmagenesis, Inc. Triptolide lactone ring derivatives as immunomodulators and anticancer agents
US8426616B2 (en) 2004-03-02 2013-04-23 Pharmagenesis, Inc. Triptolide lactone ring derivatives as immunomodulators and anticancer agents
EP2583678A2 (fr) 2004-06-24 2013-04-24 Novartis Vaccines and Diagnostics, Inc. Immunopotentiateurs de petites molécules et dosages pour leur détection
US8617906B2 (en) 2004-10-13 2013-12-31 Pharmagenesis, Inc. Identification and screening of triptolide target molecules
US20200224271A1 (en) * 2017-07-14 2020-07-16 The Regents Of The University Of California Novel Methods of Predicting Transplant Rejection Risk
US11713487B2 (en) * 2017-07-14 2023-08-01 The Regents Of The University Of California Methods of predicting transplant rejection risk

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