WO2009010021A1 - Immunostimulatory activity of trilobolide and method of preparation thereof - Google Patents

Immunostimulatory activity of trilobolide and method of preparation thereof Download PDF

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WO2009010021A1
WO2009010021A1 PCT/CZ2008/000083 CZ2008000083W WO2009010021A1 WO 2009010021 A1 WO2009010021 A1 WO 2009010021A1 CZ 2008000083 W CZ2008000083 W CZ 2008000083W WO 2009010021 A1 WO2009010021 A1 WO 2009010021A1
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trilobolide
extract
extracted
stimulation
ifn
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PCT/CZ2008/000083
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French (fr)
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Eva Kmonickova
Zdenek Zidek
Juraj Harmatha
Milos Budesinsky
Karel Vokac
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Ustav Experimentalni Mediciny Av Cr, V. V. I.
Ustav Organicke Chemie A Biochemie Av Cr, V. V. I.
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    • 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)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the invention relates to immunostimulatory activity of trilobolide, a preparation containing trilobolide and a method of preparation of trilobolide.
  • Sesquiterpene lactones are a group of natural low-molecular substances, occurring in plants and showing a wide range of activities. Trilobolide (formula I) belongs to sesquiterpene lactones.
  • Trilobolide can be obtained from roots of the plant gladich ⁇ Laser trilobum (L.) Borkh.) (Smitalova, Z.; Budes ⁇ nsky, M.; Saman, D.; Holub, M. Coll Czech Chem Commun 1986, 51, 1323-1339) or of two types of laserwort ⁇ Laserpitium archangelica WuIf. in Jacq. and Laserpitium siler L.), in which this compound is contained in substantial amounts and can be obtained rather easily by extraction and chromatographic separation, with good yields and in high purity.
  • Endoplasmic reticulum is a cell organelle, which plays the key role in maintaining calcium homeostasis in the cell.
  • ER Endoplasmic reticulum
  • the concentration gradient of Ca 2+ is 3-4 orders of magnitude higher in ER than in cytosol.
  • a signal e.g. a hormone signal
  • calcium is released from the organelle into cytosol.
  • the transport of calcium back to ER is carried out by the ATPase of sarco/endoplasmic reticulum (SERCA).
  • SERCA sarco/endoplasmic reticulum
  • SERCA 1 There are four isoforms of this enzyme, SERCA 1, SERCA 2a and 2b, SERCA 3.
  • SERCA 3 occurs in immunocompetent cells, particularly in macrophages.
  • ATP energy
  • Standard SERCA inhibitors are known: thapsigargin (TG), cyclopiazonic acid (CPA), 2,5-di-(t-butyl)-l,4-benzohydroquinone (DBHQ).
  • TG thapsigargin
  • CPA cyclopiazonic acid
  • DBHQ 2,5-di-(t-butyl)-l,4-benzohydroquinone
  • the mechanism of action of the inhibitor consists in conformational change in the enzyme and therefore in decreasing the affinity of SERCA for both Ca 2+ and ATP (Sagara, Y.; Wade, J. B.; Inesi, G. J Biol Chem 1992, 267, 1286-1992).
  • the inhibitor TG is selective for all 4 SERCA isozymes (Suplat, D.; Targos, B.; Sabala, P.; Baranska, J.; Pomorski, P.
  • CPA is a fungal toxin produced by strains Penicillium cyclopium and Aspergillus flavus. Its activity is similar to that of TG (Suplat, D.; Targos, B.; Sabala, P.; Baranska, J.; Pomorski, P.
  • the SERCA inhibitors are used experimentally for increasing the level of intracellular calcium.
  • the changes in calcium (Ca 2+ ) concentration in cytoplasm affect basically all cell functions through Ca 2+ -regulation proteins, specific kinases and phosphatases. These enzymes modulate fast processes, such as muscle contraction and secretion, or slower processes, such as cell growth and differentiation (Groenendyk, J.; Lynch, J.; Michalek, M. MoI Cells 2004, 17, 383-389).
  • the literature shows that the therapeutic potential of SERCA inhibitors is presented mainly in the field of infectious and tumor diseases. Inhibition of virus replication in relation to changes in Ca 2+ homeostasis and ER stress (e.g., rotavirus, hepatitis C virus, herpes simplex virus, cytomegalovirus etc.) was shown experimentally (Michelangeli, F.; Liprandi, F.; Chemello, M. E.; Ciarlet, M.; Ruiz, M.-C. J Virol 1995, 69, 3838-3847; Cheshenko, N.; Del Rosario, B.; Woda, C; Marcellino, D.; Satlin, L. M.; Herold, B. C.
  • ER stress e.g., rotavirus, hepatitis C virus, herpes simplex virus, cytomegalovirus etc.
  • trilobolide is an active inhibitor of Ca 2+ - ATPase with the affinity in nanomolar concentrations (Wictome, M.; Holub, M.; East, J. M.; Lee, A. G. Biochem Biophys Res Commun 1994, 199, 916-921).
  • Trilobolide inhibits SERCA in both animal cells and cells of vascular plants (Thomson, L. J.; Hall, J. L.; Williams, L. E. Plant Physiol 1994, 104, 1295-1300). So far, no therapeutical use of this lactone was proposed. Disclosure of the Invention
  • Object of the present invention is trilobolide for use for the stimulation of immune system.
  • the stimulation of immune system is the stimulation of secretion of interferon gamma (IFN- ⁇ ).
  • IFN- ⁇ interferon gamma
  • Another aspect of the invention is trilobolide for use for the treatment of disease states selected from the group comprising diseases caused by viruses, bacteria, protozoa, helminths or fungi, atopic allergies, states of decreased activity of the immune system, particularly in the glucocorticoid treatment, caused by stress situations, colorectal cancer and acute pancreatitis.
  • Immunocompetent cells are stimulated to produce the IFN- ⁇ in the presence of trilobolide.
  • This effect is caused by its ability to enter a cell, it selectively binds to the ATPase of sarco/endoplasmic reticulum (SERCA) and inhibits it irreversibly.
  • the trilobolide concentration inducing the production of IFN- ⁇ , which is applied to the cultivated cells, is in the order of magnitude of nmol/1 - ⁇ rnol/1.
  • Determination of the IFN- ⁇ concentration in the cultivation medium or in biological liquids can be carried out directly, e.g., by means of the ELISA method, on indirectly on the basis of nitrogen(II) oxide (NO) biosynthesis.
  • the activity of trilobolide can be observed in various animal species (e.g., mouse, laboratory rat, human).
  • IFN- ⁇ is a cytokine produced by immunocompetent cells, such as lymphocytes of the following groups: CD8+ T cells, ThI CD4+ T cells, NK, NK T cells and macrophages.
  • immunocompetent cells such as lymphocytes of the following groups: CD8+ T cells, ThI CD4+ T cells, NK, NK T cells and macrophages.
  • OAS 2',5'-oligoadenylate synthetase
  • PLR protein kinase R
  • Boehm U.; Klamp, T.; Groot, M.; Howard, J. C.
  • IFN- ⁇ is a typical representative of the ThI immune response and plays the key role in the process of immune system activity.
  • ThI ThI immune response
  • the Th2 immune profile is characteristic for a substantial number of infectious diseases (e.g., AIDS, tuberculosis, leishmaniasis, leprosy, etc.), in which interleukin-4 (IL-4) is of a high importance.
  • infectious diseases e.g., AIDS, tuberculosis, leishmaniasis, leprosy, etc.
  • IL-4 interleukin-4
  • From the point of view of host defence against many infectious diseases, the Th2 immune phenotype is undesirable.
  • it is desirable to increase the ThI immune response Xing, Z.; Wang, J. Curr Pharmaceut Design 2000, 6, 599-611).
  • IFN- ⁇ is considered to be a promising target in the treatment of various types of diseases and pathologic states, particularly those, in which it is necessary to reverse the ratio of Thl/Th2 immune response in favour of the ThI response, namely with regard to IFN- ⁇ production.
  • trilobolide The method of use of trilobolide is based on the known and above-mentioned mechanisms of action of IFN- ⁇ . It concerns particularly viral diseases, caused by DNA viruses, RNA viruses and retroviruses, e.g., herpes simplex type 1 and 2 (HSV-I, -2) (Gosselin, J.; Tomolu, A.; Gallo, R. C; Flamand, L. Blood 1999, 94, 4210-4219; Melkova, Z.; Esteban, M.
  • HSV-I herpes simplex type 1 and 2
  • CMV cytomegalovirus
  • VV vaccinia virus
  • HBV hepatitis B virus
  • IFN- ⁇ possesses inhibition activity also against the growth of non- viral pathogenic organisms (Murray, H. W. Am J Med 1994, 97, 459-467): protozoan parasites - Toxoplasma gondii, Leishmania donovani, L. major, L. mexicana, Trypanosoma cruzi, Plasmodium falciparum, P. vivax, P. berghei, P. chabaudi, , P. cynomolgi, Cryptosporidium parvum.
  • Entamoeba histolytica Giardia lamblia
  • helminths - Schistosoma mansoni fungi: Histoplasma capsulatum, Candida albicans,, C. parapsilosis, Cryptococcus neoformans, Coccidioides immits, Paracoccidioides brasiliensis, Pneumocystis carinii, Aspergillus fumigatus
  • bacteria Listeria monocytogenes, Legionella pneumophila, Mycobacterium tuberculosis, M. bovis, M.
  • ThI immune response In immunosuppressive states, targeted stimulation of ThI immune response is considered to be effective from the clinical point of view.
  • Increasing of the IFN- ⁇ level is suitable e.g. in the treatment of allergic diseases (e.g., atopic dermatitis), in acute pancreatitis, colorectal cancer or in a long-term treatment by glucocorticoids.
  • IFN- ⁇ The endogenous production of IFN- ⁇ is a preferable pharmacological alternative to the administration of exogenous recombinant IFN- ⁇ , the biological half-life of which is 4-8 h.
  • stimulators of IFN- ⁇ endogenous production there exist only a limited number of stimulators of IFN- ⁇ endogenous production, and in clinical practice, there is only one, which simultaneously induces further cytokines (Gupta, A. K.; Cherman, A. M.; Tyring, S. K. J Cutan Med Surg 2004, 8, 338-352). It is the 5 % cream Aldara (Laboratories 3M Sante), the active ingredient of which is imiquimod.
  • this pharmaceutical preparation is approved only for topical treatment of external genital and perianal condylomas and small external basocellular carcinoma in adults. So far, there are only a limited number of clinical experiences with this preparation. Reactions to topical application were often observed (ca. 33 % of patients) and there is lack of data about elimination of the disease after more than 24 months after finishing the treatment.
  • the benefit of this invention is the provision of a compound, which has the ability to stimulate very effectively the production of cytokines, namely IFN- ⁇ in immunocompetent cells.
  • Another aspect of the invention is the stimulation of immune system by administration of trilobolide.
  • Trilobolide stimulates the IFN- ⁇ secretion.
  • a further aspect of the invention is a method of treatment of the disease states selected from the group comprising diseases caused by viruses, bacteria, protozoan parasites, helminths or fungi, atopic allergies, states of attenuated immune system activity, particularly in connection with the glucocorticoid treatments, in stress situations, colorectal cancer and acute pancreatitis, by administering trilobolide.
  • Another aspect of the present invention is trilobolide for use in the stimulation of immune system in combination with antivirals, antibiotics and/or antifungal drugs.
  • the contemporary treatment with antibiotics is not enough effective.
  • Those skilled in the art thus recommend to perform the future treatment by means of combination of virostatics and immunostimulators, namely IFN- ⁇ , e.g., in hepatitis C (Bedossa, P.; Paradis, V. Clin Liver Dis 2003, 7, 195-210).
  • systemic mycoses e.g.
  • IFN- ⁇ can be used not only as an effective adjuvant therapy in the phase of clinical symptoms in patients with the resistance towards a standard chemotherapy, but also prophylactically. In diseases caused by protozoon (Leishmania), monotherapy by interferon- ⁇ is clinically beneficial (Sundar, S.; Murray, H. W. J Infect Dis 1995, 172, 1627-1629).
  • the combination of anti-infectious medicaments with IFN- ⁇ is beneficial also by reducing the overall time of the treatment, as shown e.g., in the hepatitis B treatment (combination of the antiviral lamivudine + IFN- ⁇ ) (Parvez, M. K.; Sehgal, D.; Sarin, S. K.; Basir, S. F.; Jameel, S. World J Gastroenterol 2006, 12, 3006-3014).
  • Object of the present invention is also a therapeutic preparation for the stimulation of immune system, containing trilobolide as an active ingredient and optionally pharmaceutically acceptable auxiliary substances.
  • the plant Laser trilobum contains trilobolide not only in the roots, from which it was isolated before, but also in other organs, particularly in seeds; this finding can be used for cultivating this plant in suitable field conditions and for collecting the seeds or whole umbels, resp., for the extraction of active ingredients.
  • the main advantage of using the seeds for the isolation of trilobolide is the fact that the plant is not destroyed during the collection of plant material, which happens when the roots are used.
  • Object of the invention is also a method of preparation of trilobolide, wherein pulverized or ground fresh or dried roots and/or seeds, optionally whole umbels, of a plant selected from the group comprising gladich (Laser trilobum (L.) Borkh.), laserwort (Laserpitium archangelica WuIf. in Jacq.) or laserwort (Laserpitium siler L, syn. Siler montanum Crantz) are gradually extracted with organic solvents and the extract containing trilobolide is determined. The solvent is subsequently evaporated from the extract containing trilobolide, and the residue is chromatographically separated by gradient elution using solvents starting from the least polar solvents and gradually supplying by more polar solvents.
  • the content of individual compounds in the thus obtained fractions is monitored after the evaporation of the solvent, e.g., by thin layer chromatography on silica gel (usually using the same solvent combinations), the fractions containing trilobolide are then combined and trilobolide is isolated.
  • the plant material i.e. roots, seeds or umbels
  • polar organic solvents such as methanol or ethanol
  • the desired fraction is extracted with ethyl acetate.
  • the plant material When the plant material is dried, it is extracted with organic solvents gradually in the order of increasing polarity, e.g., with petroleum ether, ethyl acetate, chloroform, ethanol.
  • organic solvents gradually in the order of increasing polarity, e.g., with petroleum ether, ethyl acetate, chloroform, ethanol.
  • a special process is the extraction with supercritical carbon dioxide; if needed, a small portion of polar carrier can be added, e.g., ethanol.
  • the extract containing trilobolide is determined chromatographically, e.g., by thin layer chromatography or high- performance liquid chromatography with e.g., ultra-violet or mass spectroscopy detection.
  • the chromatographic carrier for the separation of the residue of the trilobolide-containing extract is silica gel.
  • the silica gel in the column is deactivated by water, most preferably by 10 — 15 wt. % of water, referred to silica gel.
  • the chromatographic fraction of the separated residue of the extract, containing trilobolide is determined chromatographically using a standard (if the standard is available) or by evaluation of infra-red spectra or spectra obtained from high-performance liquid chromatography with mass spectroscopy detection (HPLC-MS).
  • First fraction of trilobolide can be obtained from a well worked-up chromatographic fraction (for a typical HPLC spectrum see Fig. 2) by e.g. crystallization.
  • trilobolide is separated from the crude fraction from the basic chromatographic separation on a silica gel column by another chromatography (e.g., preparative HPLC) or by crystallization.
  • the invention is based on immunostimulatory properties of trilobolide, particularly the pharmacological activity, i.e., the ability to activate the production of IFN- ⁇ .
  • the invention further comprises a facile and financially advantageous method of preparation of trilobolide from plant resources, for pharmaceutical or nutraceutical technologies.
  • Fig. 1 represents the chemical structure of trilobolide.
  • Fig. 2 represents the UV absorption of HPLC spectrum of the compounds in the fraction containing maximum trilobolide (see Example 1).
  • Analytical conditions of the chromatography column 4 x 250 mm, filled with reverse phase (Separon SGX C-18) and eluted with combination of solvents (50-100 % water in methanol) in gradient mode for 60 min at flow rate 0.6 ml/min, R t of trilobolide is 35.21 min.
  • Fig. 3 shows the effect of trilobolide on the activation of cells (peritoneal macrophages, rat) of immune system in vitro.
  • Fig. 4 shows the effect of trilobolide on the activation of cells (peritoneal macrophages, mouse) of immune system in vitro.
  • Fig. 5 shows the effect of trilobolide on the activation of cells (peritoneal macrophages, rat) of immune system.
  • Fig. 6 represents the rate of activation of the cells of immune system, based on interferon gamma (IFN- ⁇ ) secretion after the addition of trilobolide.
  • IFN- ⁇ interferon gamma
  • Fig. 7 shows the effect of trilobolide on the activation of cells (peritoneal macrophages, mouse) of immune system in vitro.
  • Fig. 8 shows the effect of trilobolide on the activation of human peripheral blood mononuclear cells (PBMC) in vitro.
  • Homonuclear 2D-COSY a 2D-ROESY spectra were used for structural determination of proton signals and heteronuclear 2D-HMQC or 2D-HMBC spectra in combination with 13 C APT spectra were used for assigning all carbon signals.
  • Mass spectra were recorded on the apparatus Waters Q-tof Microspectrometer equipped with ionization source EI or ESI (in combination with HPLC).
  • HPLC spectra were recorded on the apparatus assembled from various elements originated by the companies Waters, Spectra-Physics and LDC using columns described in the analytical conditions for HPLC recordings as shown in Fig. 2.
  • Plant material in this case roots and rhizomes, were air-dried in the shadow, ground and extracted with organic solvents.
  • a suitable type of extraction is percolation in a stoneware or a glass vessel with solvents having gradually increasing polarity (without a continuous gradient).
  • Sufficient is a process, in which the extraction with one solvent is followed by the extraction with a more polar solvent in several repetitions, until the exhaustion of the extracted substances, i.e. until zero evaporation residue (usually 3 to 5 repetitions).
  • Percolation without stirring of the material is carried out at the laboratory temperature. From the thus obtained extract, the solvent is removed by low-pressure evaporation (in a rotary vacuum evaporator). The evaporation residue of the extract is then separated by chromatography on a column filled with silica gel into several chromatographic fractions containing individual substances or groups of substances having the same polarity.
  • the material (1300 g) was first extracted five times with petroleum ether (having b.p. 50 - 80 0 C) and three times with chloroform.
  • Dried and powdered seeds (60 g) were extracted in the same apparatus as in Example 1.
  • AcOEt extract (3.8 g) was obtained, which, according to HPLC analysis (conditions as described in Example 1), contained 10 major substances, among them the fifth one was trilobolide (0.95 g). Trilobolide was then isolated from the extract by the same chromatographic fractionation and HPLC purification as described in Example 1.
  • This process reduces the use of organic solvents to the limit and makes use of the benefits of extraction with supercritical carbon dioxide in fluid state, which is performed in a specialized commercially available device for supercritical fluid extractions: SFE.
  • Dried and finely powdered seeds (4 g) were inserted into the extractor between layers of glass beads serving for dispersing the flow of the extraction fluid into the whole volume of the contents.
  • the extractor was equipped with a thermally controlled water bath. Dosage of supercritical CO 2 and ethanol was performed by high-pressure pumps. The pressure of the fluid leaving the extraction column was reduced by reduction valve to atmospheric, and individual extraction fractions were collected into glass vessels.
  • Structural and chemical identity of trilobolide is characterized by the following physico-chemical and spectroscopic data: melting point 190-192 °C; optical rotation [ ⁇ ]o 20 - 66.3° (c 0.74 in methanol). Characteristic bands in infrared spectrum 3455, 3480 cm '1 (hydroxyls), 1785 cm “1 ( ⁇ -lactone), 1725, 1250 cm “1 (acetate), 1712 (ester conjugated with double bond), 1652 cm “1 (double bond).
  • the production of nitrogen(II) oxide (NO) was measured in peritoneal macrophages, obtained from rats of the inbred line Lewis.
  • the cells were obtained by peritoneal lavage with saline solution (16 ml).
  • the cells were cultured at the density of 2 x 10 6 /ml on 96-well plates in complete RPMI -1640 culture medium (10% fetal serum albumine, 2 mmol/1 L-glutamine, 50 ⁇ g/ml gentamycin, 50 ⁇ mol/1 2- mercaptoethanol), final volume 100 ⁇ l/well, at 37 0 C, 5% CO 2 , 100% humidity, for 24 hours, without the presence (i.e., controls) or in the presence of increasing concentrations of trilobolide.
  • NO nitrogen(II) oxide
  • mice of the inbred line C57BL/6 mice of the inbred line C57BL/6.
  • the cells were obtained by peritoneal lavage with saline solution (8 ml).
  • the cells were cultured at the density of 2 x 10 6 /ml by the method described in Example 4 for 24 hours, in the absence (i.e., controls) or in the presence of increasing concentrations of trilobolide.
  • Trilobolide was administered either alone or in combination with another immunostimulatory agent, i.e. with bacterial lipopolysaccharide (LPS, 100 pg/ml).
  • LPS bacterial lipopolysaccharide
  • This experiment shows the secretion of a representative of the group of the so-called ThI cytokines, interferon-gamma (IFN- ⁇ ) in peritoneal macrophages, obtained from sewer-rats of the inbred line Lewis.
  • the cells were cultured at the density of 2 x 10 6 /rnl by the method described in Example 4 for 24 hours, without the presence (i.e., controls) • or in the presence of increasing concentrations of trilobolide.
  • the concentration of the cytokine was measured in the culture medium by immunochemical method ELISA according to the instructions for the commercially available ELISA kit for IFN- ⁇ , measured at 450 nm.
  • a substantial increase of the IFN- ⁇ production in comparison with control values occurs already in the concentration of trilobolide of 0.05 ⁇ mol/1 and with an increasing trilobolide concentration, the IFN- ⁇ production further substantially increases.
  • the results are shown in Fig. 5.
  • Example 7 Based on the results of Example 7, the secretion of interferon-gamma (IFN- ⁇ ), a representative of the group of the ThI cytokines, was measured in peritoneal macrophages obtained from the mice of the inbred line C57BL/6. The cells were cultured at the density of 2 x 10 6 /ml for 5 hours, under the same conditions as described Examples 4 and 5, in the absence (i.e., controls) or in the presence of trilobolide in the concentration of 1 ⁇ mol/1. Trilobolide was tested either alone or in combination with another reference immunostimulatory agent, i.e. with bacterial lipopolysaccharide (LPS, 100 pg/ml). Trilobolide is able to activate the production of IFN- ⁇ , the effect is highly significant. This effect is even more significant in combination with LPS. The results are shown in Fig. 7.
  • IFN- ⁇ interferon-gamma
  • PBMC human mononuclear cells
  • the cells were cultivated at the density of 2.5 x 10 6 /ml for 24 hours in the presence of 1 ⁇ mol/1 of trilobolide under conditions as described in Example 4.
  • IFN- ⁇ interferon-gamma
  • ThI cytokines a representative of the group of the so-called ThI cytokines
  • Trilobolide and its activity according to the invention can be used in pharmaceutical industry as a medicament or as a nutraceutical in human and veterinary practice.

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Abstract

The invention relates to trilobolide for use for the stimulation of immune system, particularly for the stimulation of the interferon gamma secretion, and to the use of this activity in therapy. Furthermore, it relates to a method of preparation of trilobolide from the plants gladich (Laser trilobum (L.) Borkh.), laserwort (Laserpitium archangelica Wulf. in Jacq.) or laserwort (Laserpitium siler L.).

Description

Immunostimulatory activity of trilobolide and method of preparation thereof
Technical Field
The invention relates to immunostimulatory activity of trilobolide, a preparation containing trilobolide and a method of preparation of trilobolide.
Background Art
Sesquiterpene lactones are a group of natural low-molecular substances, occurring in plants and showing a wide range of activities. Trilobolide (formula I) belongs to sesquiterpene lactones.
Figure imgf000002_0001
(I)
Trilobolide can be obtained from roots of the plant gladich {Laser trilobum (L.) Borkh.) (Smitalova, Z.; Budesϊnsky, M.; Saman, D.; Holub, M. Coll Czech Chem Commun 1986, 51, 1323-1339) or of two types of laserwort {Laserpitium archangelica WuIf. in Jacq. and Laserpitium siler L.), in which this compound is contained in substantial amounts and can be obtained rather easily by extraction and chromatographic separation, with good yields and in high purity. With regard to the fact that the collected part of the plant is the root, collecting of the plant material is difficult and for a perennial plant also destructive; furthermore, collecting is limited to unprotected regions of occurrence of the plant. For substances similar to trilobolide (e.g., thapsigargin), total synthesis was proposed (Ley, S. V.; Antonello, A.; Balskus, E. P.; Booth, D. T.; Christensen, S. B.; Cleator, E.; Gold, H.; Hδgenauer, K.; Hunger, U.; Myers, R. M.; Oliver, S. F.; Simic, O.; Smith, M. D.; Sohoel, H.; Woolford, J. A. PNAS 2004, 101, 12073-12078; Ball, M.; Andrews, S. P.; Wierschem, F.; Cleator, E.; Smith, M. D.; Ley, S. V. Org Lett 2007, 9, 663-666). It is very demanding to carry out such total syntheses, because they comprise tens of reaction steps and achieve the overall yields lower than 1 %.
In an eukaryotic cell, calcium ions play an important role, because they affect many physiological and pathophysiological mechanisms. Endoplasmic reticulum (ER) is a cell organelle, which plays the key role in maintaining calcium homeostasis in the cell. In a ,,healthy" cell, the level of free calcium ions is maintained at a low level, in the order of magnitude of 10"7 mol/1. The concentration gradient of Ca2+ is 3-4 orders of magnitude higher in ER than in cytosol. After a signal, e.g. a hormone signal, calcium is released from the organelle into cytosol. The transport of calcium back to ER is carried out by the ATPase of sarco/endoplasmic reticulum (SERCA). There are four isoforms of this enzyme, SERCA 1, SERCA 2a and 2b, SERCA 3. SERCA 3 occurs in immunocompetent cells, particularly in macrophages. During the Ca2+ transport, two Ca2+ ions are transferred from cell cytosol through ER membrane into ER lumen. This process requires energy (ATP) and comprises a conformational change (Berridge, M. J. Cell Calcium 2002, 32, 235-249; Wu, K. D.; Lee, W. S.; Wey, J.; Bungard, D.; Lytton, J. AIB J Physiol 1995, 269, C775-C784). When the Ca2+-pump (SERCA) is blocked, the level of intracellular calcium is increased by depletion of Ca2+ reservoir in ER. Simultaneously, a further transfer of Ca from extracellular space into cell cytosol occurs. That results into a long-term increase of intracellular calcium level. The dysbalance of intraluminal calcium in ER leads to ER stress. These phenomena modulate cell signalling and gene expression (Berridge, M. J. Cell Calcium 2002, 32, 235-249; Wu, K. D.; Lee, W. S.). Standard SERCA inhibitors are known: thapsigargin (TG), cyclopiazonic acid (CPA), 2,5-di-(t-butyl)-l,4-benzohydroquinone (DBHQ). The mechanism of action of the inhibitor consists in conformational change in the enzyme and therefore in decreasing the affinity of SERCA for both Ca2+ and ATP (Sagara, Y.; Wade, J. B.; Inesi, G. J Biol Chem 1992, 267, 1286-1992). The inhibitor TG is selective for all 4 SERCA isozymes (Suplat, D.; Targos, B.; Sabala, P.; Baranska, J.; Pomorski, P. Biochem Biophys Res Commun 2004, 323, 870-875) and does not show affinity for ATPases of a different type (Seidler, N. W.; Joana, L; Vegh, M.; Martonosi, A. J Biol Chem 1989, 264, 17816-17823). The SERCA isozymes inhibition is in the order of magnitude of 10~7 mol/1. CPA is a fungal toxin produced by strains Penicillium cyclopium and Aspergillus flavus. Its activity is similar to that of TG (Suplat, D.; Targos, B.; Sabala, P.; Baranska, J.; Pomorski, P. Biochem Biophys Res Commun 2004, 323, 870-875; Seidler, N. W.; Joana, L; Vegh, M.; Martonosi, A. J Biol Chem 1989, 264, 17816- 17823). DBHQ is a less active inhibitor (EC50 is 580 x 10'7 mol/1 in contrast to EC50 = 35 x 10"7 mol/1 for TG in blood platelet membranes). It is active only for the SERCA 3 isoform (Authi, K. S.; Bokkala, S.; Patel, Y.; Kakkar, V. V.; Munkonge, F. Biochem J 1993, 294, 119-126; Authi, K. S.; Bokkala, S.; Patel, Y.; Kakkar, V. V.; Munkonge, F. Biochem J 1993, 294, 119-126). The SERCA inhibitors are used experimentally for increasing the level of intracellular calcium.
The changes in calcium (Ca2+) concentration in cytoplasm affect basically all cell functions through Ca2+-regulation proteins, specific kinases and phosphatases. These enzymes modulate fast processes, such as muscle contraction and secretion, or slower processes, such as cell growth and differentiation (Groenendyk, J.; Lynch, J.; Michalek, M. MoI Cells 2004, 17, 383-389).
The literature shows that the therapeutic potential of SERCA inhibitors is presented mainly in the field of infectious and tumor diseases. Inhibition of virus replication in relation to changes in Ca2+ homeostasis and ER stress (e.g., rotavirus, hepatitis C virus, herpes simplex virus, cytomegalovirus etc.) was shown experimentally (Michelangeli, F.; Liprandi, F.; Chemello, M. E.; Ciarlet, M.; Ruiz, M.-C. J Virol 1995, 69, 3838-3847; Cheshenko, N.; Del Rosario, B.; Woda, C; Marcellino, D.; Satlin, L. M.; Herold, B. C. J Cell Biol 2003, 163, 283-293; Isler, J. A.; Maguire, T. G.; Alwine, J. C. J Virol 2005, 79, 15388-15397; Nakagawa, M.; Sakamoto, N.; Tanabe, Y.; Koyama, T.; Itsui, Y.; Takeda, Y.; Chen, C. H.; Kakinuma, S.; Oooka, S.; Maekawa, S.; Enomoto, N.; Watanabe, M. Gastroenterology 2005, 129, 1031-1041). Mycobacterial infections are commonly treated by clotrimazol (Ahmad, Z.; Sharma, S.; Khuller, G. K. FEMS Microbiol Lett 2005, 251, 19-22). This compound of imidazole type and derivatives thereof also belongs among SERCA inhibitors (Snajdrova, L.; Xu, A.; Narayanan, N. J Biol Chem 1998, 273, 28032-28039). The antitumor activity of SERCA inhibitors (TG and clotrimazol) was experimentally documented (Khalid, M. H.; Tokunaga, Y.; Caputy, A. J.; Walters, E. J Neurosurg 2005, 103, 79-86; Meira, D. D.; Marinho-Carvalho, M. M.; Teixeira, C. A.; Veiga, V. F.; Da Poian, A. T.; Holandino, C; de Freitas, M. S.; Sola-Penna, M. MoI Genet Metab 2005, 84, 354-362). The increase of intracellular Ca2+ to micromolar concentrations induces cell apoptosis. This effect is applied in the treatment of androgen-independent prostate cancer. A prodrug targeted to cancer cells is being developed (Denmeade, S. R.; Jakobsen, C. M.; Janssen, S.; Khan, S. R; Garrett, E. S.; Lilja, H.; Christensen, S. B.; Isaacs, J. T. J Nat Cancer Inst 2003, 95, 990-1000; Janssen, S.; Rosen, D. M.; Ricklis, R. M.; Dionne, C. A.; Lilja, H.; Christensen, S. B.; Isaacs, J. T.; Denmeade, S. R. Prostate 2006, 66, 358-368).
There are few data about the effect of SERCA inhibitors on immune system. Immunomodulatory activity of substances is expressed as their ability to produce cytokines and chemokines. The immunostimulatory activity of TG was documented in murine macrophages and human epithelial cells, which produced cytokines IL-6 and IL-8, resp. (Bost, K. L.; Mason, M. J. J Immunol 1995, 155, 285-296; Yu, Y.; De Waele, C; Chadee, K. Inflamm Res 2001, 50, 220-226). All three SERCA inhibitors (TG, CPA and DBHQ) activate the production of IL-4 (Onose, J.-i.; Teshima, R.; Sawada, J.-i. Immunol Lett 1998, 64, 17-22). The secretion of inflammatory cytokines TNF-α and IL- lβ is not explicit and depends on the cell type. It was shown that the secretion of IL-8 activated by TG is Ca -dependent. All three inhibitors also stimulate the production of the chemotactic protein MCP-I (Steube, K. G.; Meyer, C; Drexler, H. G. MoI Cell Biol Res. Commun. 2000, 3, 60-65). So far, no immunomudulatory medicaments based on SERCA inhibitors were prepared.
It is known that trilobolide is an active inhibitor of Ca2+- ATPase with the affinity in nanomolar concentrations (Wictome, M.; Holub, M.; East, J. M.; Lee, A. G. Biochem Biophys Res Commun 1994, 199, 916-921). Trilobolide inhibits SERCA in both animal cells and cells of vascular plants (Thomson, L. J.; Hall, J. L.; Williams, L. E. Plant Physiol 1994, 104, 1295-1300). So far, no therapeutical use of this lactone was proposed. Disclosure of the Invention
Object of the present invention is trilobolide for use for the stimulation of immune system.
It is an aspect of the invention that the stimulation of immune system is the stimulation of secretion of interferon gamma (IFN-γ).
Another aspect of the invention is trilobolide for use for the treatment of disease states selected from the group comprising diseases caused by viruses, bacteria, protozoa, helminths or fungi, atopic allergies, states of decreased activity of the immune system, particularly in the glucocorticoid treatment, caused by stress situations, colorectal cancer and acute pancreatitis.
Immunocompetent cells are stimulated to produce the IFN-γ in the presence of trilobolide. This effect is caused by its ability to enter a cell, it selectively binds to the ATPase of sarco/endoplasmic reticulum (SERCA) and inhibits it irreversibly. The trilobolide concentration inducing the production of IFN-γ, which is applied to the cultivated cells, is in the order of magnitude of nmol/1 - μrnol/1. Determination of the IFN-γ concentration in the cultivation medium or in biological liquids can be carried out directly, e.g., by means of the ELISA method, on indirectly on the basis of nitrogen(II) oxide (NO) biosynthesis. The activity of trilobolide can be observed in various animal species (e.g., mouse, laboratory rat, human).
IFN-γ is a cytokine produced by immunocompetent cells, such as lymphocytes of the following groups: CD8+ T cells, ThI CD4+ T cells, NK, NK T cells and macrophages. There are several mechanisms of its direct antiviral and other antimicrobial activities, which include particularly the stimulation of proteins of the 2',5'-oligoadenylate synthetase (OAS) family, the stimulation of protein kinase R (PKR) (Esteban, M.; Patino, C. J Interferon Cytokinee Res 2000, 20, 867-877), adenosine deaminase (Boehm, U.; Klamp, T.; Groot, M.; Howard, J. C. Annu Rev Immunol 1997, 15, 749-795), indolamin 253-dioxygenase (Bodaghi, B.; Goureau, O.; Zipeto, D.; Laurent, L.; Virelizier, J.-L.; Michelson, S. J Immunol 1999, 162, 957- 964), and also inducible nitrogen(II) oxide synthase (iNOS) (Karupiah, G.; Xie, Q.-w.; Buller, R. M. L.; Nathan, C; Duarte, C; MacMicking, J. D. Science 1993, 261, 1445- 1448).
IFN-γ is a typical representative of the ThI immune response and plays the key role in the process of immune system activity. At an insufficient ThI response, the sensitivity of the organism to infectious and tumor disease development increases. The Th2 immune profile is characteristic for a substantial number of infectious diseases (e.g., AIDS, tuberculosis, leishmaniasis, leprosy, etc.), in which interleukin-4 (IL-4) is of a high importance. From the point of view of host defence against many infectious diseases, the Th2 immune phenotype is undesirable. On the contrary, from the point of view of the treatment, it is desirable to increase the ThI immune response (Xing, Z.; Wang, J. Curr Pharmaceut Design 2000, 6, 599-611). Thus, IFN-γ is considered to be a promising target in the treatment of various types of diseases and pathologic states, particularly those, in which it is necessary to reverse the ratio of Thl/Th2 immune response in favour of the ThI response, namely with regard to IFN- γ production.
The method of use of trilobolide is based on the known and above-mentioned mechanisms of action of IFN-γ. It concerns particularly viral diseases, caused by DNA viruses, RNA viruses and retroviruses, e.g., herpes simplex type 1 and 2 (HSV-I, -2) (Gosselin, J.; Tomolu, A.; Gallo, R. C; Flamand, L. Blood 1999, 94, 4210-4219; Melkova, Z.; Esteban, M. J Immunol 1995, 155, 5711-5718), cytomegalovirus (CMV) (Bodaghi, B.; Goureau, O.; Zipeto, D.; Laurent, L.; Virelizier, J.-L.; Michelson, S. J Immunol 1999, 162, 957-964; Lucin, P.; Jonjic, S.; Messerle, M.; Polic, B.; Hengel, H.; Koszinowski, U. H. J Gen Virol 1994, 75, 101-110), Epstein-Barr virus (EBV) (Kawanishi, M. Intervirology 1995, 38, 206-213), polio virus (Komatsu, T.; Bi, Z.; Reiss, C. S. J Neuroimmunol 1996, 68, 101-108), vaccinia virus (VV) (Tanaka- Kataoka, M.; Kunikata, T.; Takayama, S.; Iwaki, K.; Ohashi, K.; Ikeda, M.; Kurimoto, M. Cytokine 1999, 11, 593-599), vesicular stomatitis virus (Komatsu, T.; Bi, Z.; Reiss, C. S. J Neuroimmunol 1996, 68, 101-108; Lohoff, M.; Marsig, E.; Rollinghoff, M. J Immunol 1990, 144, 960-963), hepatitis B virus (HBV) (Guidotti, L. G.; McClary, H.; Loudis, J. M.; Chisari, F. V. J Exp Med 2000, 191, 1247-1252), hepatitis C virus (Sharara, A. L; Perkins, D. J.; Misukonis, M. A.; Chan, S. U.; Dominitz, J. A.; Weinberg, J. B. J Exp Med 1997, 186, 1495-1502), coxsackie virus B4 (Flodstrom, M.; Horwitz, M. S.; Maday, A.; Balakrishna, D.; Rodriguez, E.; Sarvetnick, N. Virology 2001, 281, 205-215) etc.
It was found that IFN-γ possesses inhibition activity also against the growth of non- viral pathogenic organisms (Murray, H. W. Am J Med 1994, 97, 459-467): protozoan parasites - Toxoplasma gondii, Leishmania donovani, L. major, L. mexicana, Trypanosoma cruzi, Plasmodium falciparum, P. vivax, P. berghei, P. chabaudi, , P. cynomolgi, Cryptosporidium parvum. Entamoeba histolytica, Giardia lamblia; helminths - Schistosoma mansoni; fungi: Histoplasma capsulatum, Candida albicans,, C. parapsilosis, Cryptococcus neoformans, Coccidioides immits, Paracoccidioides brasiliensis, Pneumocystis carinii, Aspergillus fumigatus; bacteria: Listeria monocytogenes, Legionella pneumophila, Mycobacterium tuberculosis, M. bovis, M. intracellular, Salmonella typhimurium, Francisella tularensis, Brucela abortus, Nocardia asteroids, Chlamydia psittaci, C. trachomatis, Klebsiella pneumoniae, Rickettsia prowazekii, R. conorii, R. tsutsugamushi, Yersinia enterocolitica, Rhodococcus equi, Ehrlichia risticii, Staphylococcus aureus.
In immunosuppressive states, targeted stimulation of ThI immune response is considered to be effective from the clinical point of view. Increasing of the IFN-γ level is suitable e.g. in the treatment of allergic diseases (e.g., atopic dermatitis), in acute pancreatitis, colorectal cancer or in a long-term treatment by glucocorticoids.
Current data about the beneficial therapeutic effects of IFN-γ are based on experimental and clinical tests, in which IFN-γ itself was administered as the medicament, but not any substance (medicament) activating the endogenous synthesis of IFN-γ in the organism. The limiting factors of the therapy by interferon-γ itself are particularly: a complex manufacturing process, method of administration to patients and a high market price. Dosage recommended for the therapeutic antimicrobial effect is in the range of 25-100 μg/m2, whereas in active infections 5-7 injections per week are recommended (Murray, H. W. Am J Med 1994, 97, 459-467). The endogenous production of IFN-γ is a preferable pharmacological alternative to the administration of exogenous recombinant IFN-γ, the biological half-life of which is 4-8 h. (Grassegger, A.; Hδpfl, R. Clin Exp Dermatol 2004, 29, 584-588). However, there exist only a limited number of stimulators of IFN-γ endogenous production, and in clinical practice, there is only one, which simultaneously induces further cytokines (Gupta, A. K.; Cherman, A. M.; Tyring, S. K. J Cutan Med Surg 2004, 8, 338-352). It is the 5 % cream Aldara (Laboratories 3M Sante), the active ingredient of which is imiquimod. In the Czech Republic (and similarly in other countries), this pharmaceutical preparation is approved only for topical treatment of external genital and perianal condylomas and small external basocellular carcinoma in adults. So far, there are only a limited number of clinical experiences with this preparation. Reactions to topical application were often observed (ca. 33 % of patients) and there is lack of data about elimination of the disease after more than 24 months after finishing the treatment. The benefit of this invention is the provision of a compound, which has the ability to stimulate very effectively the production of cytokines, namely IFN-γ in immunocompetent cells.
Another aspect of the invention is the stimulation of immune system by administration of trilobolide. Trilobolide stimulates the IFN-γ secretion.
A further aspect of the invention is a method of treatment of the disease states selected from the group comprising diseases caused by viruses, bacteria, protozoan parasites, helminths or fungi, atopic allergies, states of attenuated immune system activity, particularly in connection with the glucocorticoid treatments, in stress situations, colorectal cancer and acute pancreatitis, by administering trilobolide.
Another aspect of the present invention is trilobolide for use in the stimulation of immune system in combination with antivirals, antibiotics and/or antifungal drugs. In some serious viral diseases, the contemporary treatment with antibiotics is not enough effective. Those skilled in the art thus recommend to perform the future treatment by means of combination of virostatics and immunostimulators, namely IFN-γ, e.g., in hepatitis C (Bedossa, P.; Paradis, V. Clin Liver Dis 2003, 7, 195-210). In the development of systemic mycoses (e.g. Cryptococcus neoformans, Aspergillus fumigatus) in immunosuppressed patients that did not respond to the treatment with commonly used antimycotics, the use of IFN-γ resulted very quickly in complete cure during 4-6 weeks (Summers, S. A.; Dorling, A.; Boyle, J. J.; Shaunak, S. Am J Transplant 2005, 5, 2067-2069). The combination with IFN-γ helps to decrease the resistance towards classical antivirals in hepatitis B (Parvez, M. K.; Sehgal, D.; Sarin, S. K.; Basir, S. F.; Jameel, S. World J Gastroenterol 2006, 12, 3006-3014). A similar situation concerning the resistance towards standard medicaments exists in other infectious diseases (e.g., life-threatening chronic granulomatous disease (Marciano, B. E.; Wesley, R.; De Carlo, E. S.; Anderson, V. L.; Barnhart, L. A.; Darnell, D.; Malech, H. L.; Gallin, J. L; Holland, S. M. Clin Infect Dis 2004, 39, 692-699), TBC (Suarez-Mendez, R.; Garcia-Garcia, I.; Fernandez-Olivera, N.; Valdes-Quintana, M.; Milanes-Virelles, M. T.; Carbonell, D.; Machado-Molina, D.; Valenzuela-Silva, C. M.; Lόpez-Saura, P. A. BMC Infect Dis 2004, 4, 44). IFN-γ can be used not only as an effective adjuvant therapy in the phase of clinical symptoms in patients with the resistance towards a standard chemotherapy, but also prophylactically. In diseases caused by protozoon (Leishmania), monotherapy by interferon-γ is clinically beneficial (Sundar, S.; Murray, H. W. J Infect Dis 1995, 172, 1627-1629). The combination of anti-infectious medicaments with IFN-γ is beneficial also by reducing the overall time of the treatment, as shown e.g., in the hepatitis B treatment (combination of the antiviral lamivudine + IFN-γ) (Parvez, M. K.; Sehgal, D.; Sarin, S. K.; Basir, S. F.; Jameel, S. World J Gastroenterol 2006, 12, 3006-3014).
Object of the present invention is also a therapeutic preparation for the stimulation of immune system, containing trilobolide as an active ingredient and optionally pharmaceutically acceptable auxiliary substances.
Based on the analysis of resource availability and of practical and economical demandingness of various methods of preparation of pure compounds or analytically precisely characterized preparations containing trilobolide, we have selected a method of preparation using easily available plant resources, which can be cultivated in field conditions.
We have found that the plant Laser trilobum contains trilobolide not only in the roots, from which it was isolated before, but also in other organs, particularly in seeds; this finding can be used for cultivating this plant in suitable field conditions and for collecting the seeds or whole umbels, resp., for the extraction of active ingredients. The main advantage of using the seeds for the isolation of trilobolide is the fact that the plant is not destroyed during the collection of plant material, which happens when the roots are used.
Object of the invention is also a method of preparation of trilobolide, wherein pulverized or ground fresh or dried roots and/or seeds, optionally whole umbels, of a plant selected from the group comprising gladich (Laser trilobum (L.) Borkh.), laserwort (Laserpitium archangelica WuIf. in Jacq.) or laserwort (Laserpitium siler L, syn. Siler montanum Crantz) are gradually extracted with organic solvents and the extract containing trilobolide is determined. The solvent is subsequently evaporated from the extract containing trilobolide, and the residue is chromatographically separated by gradient elution using solvents starting from the least polar solvents and gradually supplying by more polar solvents. The content of individual compounds in the thus obtained fractions is monitored after the evaporation of the solvent, e.g., by thin layer chromatography on silica gel (usually using the same solvent combinations), the fractions containing trilobolide are then combined and trilobolide is isolated.
It is an aspect of the invention that the plant material (i.e. roots, seeds or umbels), when it is fresh, is extracted with water in combination with polar organic solvents, such as methanol or ethanol, and after evaporating part of the organic solvent, the desired fraction is extracted with ethyl acetate.
When the plant material is dried, it is extracted with organic solvents gradually in the order of increasing polarity, e.g., with petroleum ether, ethyl acetate, chloroform, ethanol. A special process is the extraction with supercritical carbon dioxide; if needed, a small portion of polar carrier can be added, e.g., ethanol.
It is another aspect of the invention that the extract containing trilobolide is determined chromatographically, e.g., by thin layer chromatography or high- performance liquid chromatography with e.g., ultra-violet or mass spectroscopy detection. In a preferred embodiment of the invention, the chromatographic carrier for the separation of the residue of the trilobolide-containing extract is silica gel. Preferably, the silica gel in the column is deactivated by water, most preferably by 10 — 15 wt. % of water, referred to silica gel.
It is an aspect of the invention that the chromatographic fraction of the separated residue of the extract, containing trilobolide, is determined chromatographically using a standard (if the standard is available) or by evaluation of infra-red spectra or spectra obtained from high-performance liquid chromatography with mass spectroscopy detection (HPLC-MS). First fraction of trilobolide can be obtained from a well worked-up chromatographic fraction (for a typical HPLC spectrum see Fig. 2) by e.g. crystallization.
It is a further aspect of the invention that trilobolide is separated from the crude fraction from the basic chromatographic separation on a silica gel column by another chromatography (e.g., preparative HPLC) or by crystallization.
From mother liquors, purified on a short silica gel column, further fractions of trilobolide can be obtained. The last portion of trilobolide and its minor derivatives can be obtained from the residual mother liquors by preparative HPLC. For a practical use, the process comprising crystallization is economically advantageous. By repeated crystallization, up to 98 % purity can be achieved (determined by analytical HPLC).
The invention is based on immunostimulatory properties of trilobolide, particularly the pharmacological activity, i.e., the ability to activate the production of IFN-γ. The invention further comprises a facile and financially advantageous method of preparation of trilobolide from plant resources, for pharmaceutical or nutraceutical technologies.
Figures
Fig. 1 represents the chemical structure of trilobolide.
Fig. 2 represents the UV absorption of HPLC spectrum of the compounds in the fraction containing maximum trilobolide (see Example 1). Analytical conditions of the chromatography: column 4 x 250 mm, filled with reverse phase (Separon SGX C-18) and eluted with combination of solvents (50-100 % water in methanol) in gradient mode for 60 min at flow rate 0.6 ml/min, Rt of trilobolide is 35.21 min.
Fig. 3 shows the effect of trilobolide on the activation of cells (peritoneal macrophages, rat) of immune system in vitro.
Fig. 4 shows the effect of trilobolide on the activation of cells (peritoneal macrophages, mouse) of immune system in vitro.
Fig. 5 shows the effect of trilobolide on the activation of cells (peritoneal macrophages, rat) of immune system. Fig. 6 represents the rate of activation of the cells of immune system, based on interferon gamma (IFN-γ) secretion after the addition of trilobolide.
Fig. 7 shows the effect of trilobolide on the activation of cells (peritoneal macrophages, mouse) of immune system in vitro.
Fig. 8 shows the effect of trilobolide on the activation of human peripheral blood mononuclear cells (PBMC) in vitro.
The invention is further illustrated in the following examples, which should not be construed as further limiting the scope of the invention.
Examples
Methods
Melting points were determined on the Koffler block from the company Boetius without correction. Optical rotation was measured by the polarimeter Rudolph Research Analytical Autopol IV. Infrared spectra were recorded on the apparatus Bruker IPS-88. NMR spectra were measured by the spectrometer Varian Unity-500 (1H at 500 MHz, 13C at 125.7 MHz). Chemical shifts of proton nuclei were referred to TMS (v CDCl3) and of carbon nuclei to the solvent signal 5(CDCl3) 77.0. Homonuclear 2D-COSY a 2D-ROESY spectra were used for structural determination of proton signals and heteronuclear 2D-HMQC or 2D-HMBC spectra in combination with 13C APT spectra were used for assigning all carbon signals. Mass spectra were recorded on the apparatus Waters Q-tof Microspectrometer equipped with ionization source EI or ESI (in combination with HPLC). HPLC spectra were recorded on the apparatus assembled from various elements originated by the companies Waters, Spectra-Physics and LDC using columns described in the analytical conditions for HPLC recordings as shown in Fig. 2.
Example 1 Preparation of trilobolide
Plant material, in this case roots and rhizomes, were air-dried in the shadow, ground and extracted with organic solvents. A suitable type of extraction is percolation in a stoneware or a glass vessel with solvents having gradually increasing polarity (without a continuous gradient). Sufficient is a process, in which the extraction with one solvent is followed by the extraction with a more polar solvent in several repetitions, until the exhaustion of the extracted substances, i.e. until zero evaporation residue (usually 3 to 5 repetitions). Percolation without stirring of the material (only by several hours standing with the solvent overlaying the whole contents) is carried out at the laboratory temperature. From the thus obtained extract, the solvent is removed by low-pressure evaporation (in a rotary vacuum evaporator). The evaporation residue of the extract is then separated by chromatography on a column filled with silica gel into several chromatographic fractions containing individual substances or groups of substances having the same polarity.
In this case, the material (1300 g) was first extracted five times with petroleum ether (having b.p. 50 - 80 0C) and three times with chloroform. The obtained evaporation residue of the petroleum ether extract (50 g) was after removing crystalline laserolide (from a thickened extract) chromatographically separated on a silica gel column (silica gel deactivated with 12 % water) by elution with a mixture of petroleum ether and ethyl acetate (gradual gradient Pe - AcOEt = 1:0 to 1:1). This chromatography yielded twenty fractions (combined according to the content of identical substances detected by TLC monitoring) and from the trilobolide containing fractions, the first portion of trilobolide was isolated by crystallization (0.8 g), obtained in the purity of 98 %, followed by subsequent chromatography of mother liquors (1.5 g) yielding another fraction of trilobolide (0.6 g).
The chloroform fraction (20 g) contained according to HPLC analysis (performed on a column with reverse phase Separon SGX C- 18) another 43 % of trilobolide. It was also fractionated by column chromatography (in the same mode as above) and then purified by preparative HPLC on the same reverse phase, packed in a column 26 x 600 mm, performed in gradient mode with a combination of solvents (55-100 % water in methanol) for 360 min, at flow rate 5 ml/min. In further feedings, the percolation with petroleum ether was reduced (two repetitions only) for the removal of low-polar substances (including also several sesquiterpene lactones), so that trilobolide became the major component in the chloroform extract, from which it was advantageously obtained by crystallization.
Example 2
Preparation of trilobolide
This improved process avoids the use of an inconvenient solvent (chloroform), disadvantaged by rather high toxicity and ecological burden (chloroform); and also avoids destruction of the plant by using its seeds instead of roots and rhizoma.
Dried and powdered seeds (60 g) were extracted in the same apparatus as in Example 1. Short-term percolation (1 h) with petroleum ether was carried out to remove low- polar aliphatic (waxy and oily) substances and non-polar parts of chlorophyll. Five times repeated percolation with ethyl acetate (AcOEt) followed, until complete exhaustion of the extracted compounds. After evaporating the solvent in a vacuum evaporator, AcOEt extract (3.8 g) was obtained, which, according to HPLC analysis (conditions as described in Example 1), contained 10 major substances, among them the fifth one was trilobolide (0.95 g). Trilobolide was then isolated from the extract by the same chromatographic fractionation and HPLC purification as described in Example 1.
Example 3
Preparation of trilobolide
This process reduces the use of organic solvents to the limit and makes use of the benefits of extraction with supercritical carbon dioxide in fluid state, which is performed in a specialized commercially available device for supercritical fluid extractions: SFE. Dried and finely powdered seeds (4 g) were inserted into the extractor between layers of glass beads serving for dispersing the flow of the extraction fluid into the whole volume of the contents. The extractor was equipped with a thermally controlled water bath. Dosage of supercritical CO2 and ethanol was performed by high-pressure pumps. The pressure of the fluid leaving the extraction column was reduced by reduction valve to atmospheric, and individual extraction fractions were collected into glass vessels. The extraction was performed at a slightly increased extraction temperature (40 0C) at lower pressures (200 bar) and with gradual addition of ethanol as a modifier of extraction fluid polarity. First, the majority of low-polar ballast substances was removed by pure CO2. Trilobolide was obtained in the next fractions, most of it at the addition of 10 % ethanol to CO2. Fractions containing trilobolide were combined and from this combined extract, polar ballast substances were then removed by chromatography on a short column. From the thus purified fraction of the extract, pure trilobolide was obtained by crystallization.
Structural and chemical identity of trilobolide is characterized by the following physico-chemical and spectroscopic data: melting point 190-192 °C; optical rotation [α]o20 - 66.3° (c 0.74 in methanol). Characteristic bands in infrared spectrum 3455, 3480 cm'1 (hydroxyls), 1785 cm"1 (γ-lactone), 1725, 1250 cm"1 (acetate), 1712 (ester conjugated with double bond), 1652 cm"1 (double bond). Characteristic data of mass spectrum ESI-MS: 545 (522+23; M+Na) and fragmentation from EI-MS: m/z 462 (522-60; M-HOAc), m/z 362 (522-60-100; M-HOAc-C5-unsaturated acid), m/z 360 (522-60-102; M-HOAc-C5-saturated acid), m/z 260 (522-60-100-102; M-all three esters). Full structural information based on proton and carbon nuclear magnetic resonance (1H and 13C NMR) is summarized in Table 1).
Figure imgf000017_0001
Example 4
Effect of trilobolide on the activation of immune cells in vitro - production of NO
The production of nitrogen(II) oxide (NO) was measured in peritoneal macrophages, obtained from rats of the inbred line Lewis. The cells were obtained by peritoneal lavage with saline solution (16 ml). The cells were cultured at the density of 2 x 106/ml on 96-well plates in complete RPMI -1640 culture medium (10% fetal serum albumine, 2 mmol/1 L-glutamine, 50 μg/ml gentamycin, 50 μmol/1 2- mercaptoethanol), final volume 100 μl/well, at 37 0C, 5% CO2, 100% humidity, for 24 hours, without the presence (i.e., controls) or in the presence of increasing concentrations of trilobolide. A substantial increase of NO biosynthesis, determined on the basis of the resulting nitrite concentration (measured spectrophotometrically with Griess reagent at 540 run) in the culture medium, occurs in comparison with control cells at the concentrations of trilobolide of 0.1 - 1.0 μmol/1. The results are shown in Fig. 3.
Example 5
Effect of trilobolide on the activation of immune cells in vitro - production of NO
The production of nitrogen(II) oxide (NO) was measured in peritoneal macrophages, obtained from mice of the inbred line C57BL/6. The cells were obtained by peritoneal lavage with saline solution (8 ml). The cells were cultured at the density of 2 x 106/ml by the method described in Example 4 for 24 hours, in the absence (i.e., controls) or in the presence of increasing concentrations of trilobolide. Trilobolide was administered either alone or in combination with another immunostimulatory agent, i.e. with bacterial lipopolysaccharide (LPS, 100 pg/ml). In murine cells, a small increase of NO production occurred when trilobolide alone was administered. The NO biosynthesis increases substantially when the combination with LPS is administered, using the trilobolide concentrations of 1-5 μmol/1. The results are shown in Fig. 4. Example 6
Effect of trilobolide on the activation of immune cells in vitro — secretion of IFN-γ
This experiment shows the secretion of a representative of the group of the so-called ThI cytokines, interferon-gamma (IFN-γ) in peritoneal macrophages, obtained from sewer-rats of the inbred line Lewis. The cells were cultured at the density of 2 x 106/rnl by the method described in Example 4 for 24 hours, without the presence (i.e., controls) • or in the presence of increasing concentrations of trilobolide. The concentration of the cytokine was measured in the culture medium by immunochemical method ELISA according to the instructions for the commercially available ELISA kit for IFN-γ, measured at 450 nm. A substantial increase of the IFN- γ production in comparison with control values occurs already in the concentration of trilobolide of 0.05 μmol/1 and with an increasing trilobolide concentration, the IFN-γ production further substantially increases. The results are shown in Fig. 5.
Example 7
Rate of activation of immune cells
The time course of the activation of peritoneal cells based on interferon-gamma (IFN- γ) secretion after the addition of trilobolide in the concentration of 0.1 μmol/1. The experiment was performed in vitro using peritoneal macrophages (2 x 10 /ml), obtained from rats and cultivated by the method of Example 4. While control cells produce almost none IFN-γ, within the period of 2-5 hours a substantial secretion of this cytokine into culture medium occurs. The results are shown in Fig. 6.
Example 8
Effect of trilobolide on the activation of immune cells in vitro
Based on the results of Example 7, the secretion of interferon-gamma (IFN-γ), a representative of the group of the ThI cytokines, was measured in peritoneal macrophages obtained from the mice of the inbred line C57BL/6. The cells were cultured at the density of 2 x 106/ml for 5 hours, under the same conditions as described Examples 4 and 5, in the absence (i.e., controls) or in the presence of trilobolide in the concentration of 1 μmol/1. Trilobolide was tested either alone or in combination with another reference immunostimulatory agent, i.e. with bacterial lipopolysaccharide (LPS, 100 pg/ml). Trilobolide is able to activate the production of IFN-γ, the effect is highly significant. This effect is even more significant in combination with LPS. The results are shown in Fig. 7.
Example 9
Effect of trilobolide on the activation of human peripheral blood mononuclear cells in vitro
The effect of trilobolide on the activation of human immune system is shown on the cultivation of human mononuclear cells (PBMC). The cells were cultivated at the density of 2.5 x 106/ml for 24 hours in the presence of 1 μmol/1 of trilobolide under conditions as described in Example 4. The secretion of interferon-gamma (IFN-γ), a representative of the group of the so-called ThI cytokines, is almost zero in control, i.e. non-stimulated, cells, while the production of IFN-γ induced by trilobolide is increased by more than 300 times. The results are shown in Fig. 8.
Industrial Applicability
Trilobolide and its activity according to the invention can be used in pharmaceutical industry as a medicament or as a nutraceutical in human and veterinary practice.

Claims

1. Trilobolide for use for the stimulation of immune system.
2. Trilobolide for use according to claim 1, wherein the stimulation of immune system is the stimulation of secretion of interferon gamma (IFN-γ).
3. Trilobolide for use according to claim 2 for use for the treatment of disease states selected from the group comprising diseases caused by viruses, bacteria, protozoa, helmints or fungi, atopic allergies, states of attenuated activity of the immune system, particularly in the glucocorticoid treatment, stress situations, colorectal carcinoma and acute pancreatitis.
4. Trilobolide for use according to claim 2 or 3 in combination with antivirals, antibiotics and/or antimycotics.
5. Therapeutical preparation for the stimulation of immune system, characterized in that it contains trilobolide as an active ingredient and optionally pharmaceutically acceptable auxiliary substances.
6. A method of preparation of trilobolide, characterized in that pulverized or ground fresh or dried roots and/or seeds, optionally whole umbels, of a plant selected from the group comprising gladich (Laser trilobum (L.) Borkh.), laserwort (Laserpitium archangelica WuIf. in Jacq.) or laserwort (Laserpitium siler L. , syn. Siler montanum Crantz) are gradually extracted with organic solvents, the extract containing trilobolide is determined, from said extract, the solvent is evaporated and the residue is chromatographically separated on a column by gradient elution using solvents starting from the least polar solvents and gradually supplying by more polar solvents, wherein the content of individual compounds in the thus obtained fractions is monitored after the evaporation of the solvent, the fractions containing trilobolide are then combined and trilobolide is isolated.
7. The method according to claim 6, characterized in that when the plant material, i.e. roots, seeds or umbels, is fresh, it is extracted with water in combination with polar organic solvents, preferably methanol or ethanol, and after evaporating part of the organic solvent, the desired fraction is extracted with ethyl acetate.
8. The method according to claim 6, characterized in that when the plant material is dried, it is extracted with organic solvents gradually in the order of increasing polarity, preferably with petroleum ether, ethyl acetate, chloroform, ethanol.
9. The method according to claim 6, characterized in that the dried plant material is extracted with supercritical carbon dioxide.
10. The method according to claim 9, characterized in that the dried plant material is extracted with supercritical carbon dioxide with the addition of a polar carrier, preferably ethanol.
11. The method according to claim 6, characterized in that the extract containing trilobolide is determined chromatographically, preferably by thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC).
12. The method according to claim 6, characterized in that the chromatographic carrier for the separation of the residue of the trilobolide-containing extract is silica gel, preferably deactivated by water, most preferably by 10 — 15 wt. % of water, referred to silica gel.
13. The method according to claim 6, characterized in that the chromatographic fraction of the separated residue of the extract, containing trilobolide, is determined chromatographically using a standard or by evaluation of infra-red spectra or spectra obtained from high-performance liquid chromatography with mass spectroscopy detection (HPLC-MS).
14. The method according to claim 6, characterized in that trilobolide is isolated from the fraction obtained by the chromatographic separation of the residue of the extract, containing trilobolide, by chromatography or by crystallization.
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