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WO1999024067A1 - Preparation de mycobacterium vaccae comprenant des proteines produites dans des conditions hypothermiques et utilisation de cette preparation lors de therapies - Google Patents

Preparation de mycobacterium vaccae comprenant des proteines produites dans des conditions hypothermiques et utilisation de cette preparation lors de therapies Download PDF

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Publication number
WO1999024067A1
WO1999024067A1 PCT/GB1998/003346 GB9803346W WO9924067A1 WO 1999024067 A1 WO1999024067 A1 WO 1999024067A1 GB 9803346 W GB9803346 W GB 9803346W WO 9924067 A1 WO9924067 A1 WO 9924067A1
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WIPO (PCT)
Prior art keywords
cold
vaccae
shock
disease
proteins
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PCT/GB1998/003346
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English (en)
Inventor
John Lawson Stanford
Graham Mcintyre
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Stanford Rook Limited
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Priority to AU10430/99A priority Critical patent/AU1043099A/en
Publication of WO1999024067A1 publication Critical patent/WO1999024067A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • A61K2039/55594—Adjuvants of undefined constitution from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel methods of preparing Mycojbacterium vaccae and to preparations of M. vaccae or antigens therefrom obtainable by the method.
  • compositions for the alleviation of the symptoms of, and for the treatment or diagnosis of, arthritic disease which comprise as active ingredient the whole organism of M. vaccae . It is stated that the preparations of M. vaccae are useful for the treatment of various autoimmune diseases and especially arthritic conditions including rheumatoid arthritis, ankylosing spondylitis or Reiter's syndrome.
  • compositions comprising antigenic and immunoregulatory material derived from M. vaccae as being generally useful in the treatment of pathological conditions in which the proportion of agalactosyl IgG (i.e. IgG which lacks terminal galactose from the N-linked oligosaccharides on the heavy chains) is increased.
  • Diseases of this kind include not only the arthritic disease e.g. rheumatoid arthritis, and mycobacterial disease, e.g. tuberculosis and leprosy, mentioned in the other specifications referred to above, but also Crohn's disease.
  • agalactosyl IgG Other diseases in which this may play a part but in which an increased level of agalactosyl IgG is not easily detectable by current methods include primary biliary cirrhosis, sarcoidosis, ulcerative colitis, psoriasis, systemic lupus erythematosus (especially when accompanied by Sjogren's syndrome), multiple sclerosis, Guillain-Barre syndrome, primary diabetes mellitus, and perhaps some aspects of graft rejection.
  • Such diseases may also be described as that class of chronic inflammatory disorder which is caused by, or accompanied by, abnormally high release by macrophages of the cytokines interleukin-6 and/or tumour necrosis factor (cachectin) .
  • cytokines interleukin-6 and/or tumour necrosis factor (cachectin) .
  • the specific conditions involved are, of course, the same as those already named.
  • W091/02542 provides a method for the treatment of a pathological condition (other than mycobacterial disease, e.g. tuberculosis or leprosy, and arthritic disease, e.g. rheumatoid arthritis, mentioned in the specifications referred to above) in a patient in which the patient's IgG shows an abnormally high proportion of agalactosyl IgG which comprises administering to the patient suffering from such a condition an effective amount of a therapeutic composition comprising antigenic and immunoregulatory material derived from M. vaccae .
  • a pathological condition other than mycobacterial disease, e.g. tuberculosis or leprosy, and arthritic disease, e.g. rheumatoid arthritis, mentioned in the specifications referred to above
  • a pathological condition other than mycobacterial disease, e.g. tuberculosis or leprosy, and arthritic disease, e.g. rheum
  • W091/02542 also provides a method for the treatment of a chronic inflammatory disorder (other than an arthritic disease, e.g. rheumatoid arthritis) caused or accompanied by an abnormally high release from macrophages of interleukin-6 and/or tumour necrosis factor which comprises administering to a patient suffering from such a disorder an effective amount of the said therapeutic agent.
  • a chronic inflammatory disorder other than an arthritic disease, e.g. rheumatoid arthritis
  • W092/08488 further discloses that killed cells of M. vaccae can be used to stimulate and/or modify in a favourable way the immune response to antigens which are not endogenous to M. vaccae . It is suggested that the beneficial effect of using killed M. vaccae as an adjuvant may also be associated with the 65 kDa mycobacterial heat shock protein (hsp 65) described by Young et al . "Stress proteins are immune targets in leprosy and tuberculosis", Proc . Natl. Acad. Sci . U.S.A. 85 . (1988), pp4267-4270 in form obtained from M. bovis . The preferred autoclaved M. vaccae cells used in the invention of 092/08488 are believed to provide an effective package of adjuvant, hsp 65 and other substances.
  • hsp 65 mycobacterial heat shock protein
  • W092/08484 further describes the use of M. vaccae for the treatment of uveitis.
  • Uveitis is a condition, often observed in leprosy patients but also found in other individuals, which is difficult to treat and leads to permanent blindness.
  • W092/08484 is founded upon the surprising observation that compositions comprising antigenic and immunoregulatory material derived from M. vaccae are useful in the treatment of uveitis .
  • a method for the treatment of uveitis comprises administering to the patient suffering from such a condition an effective amount of a therapeutic composition comprising antigenic and immunoregulatory material derived from ⁇ f. vaccae .
  • W094/06466 provides the use of antigenic and/or immunoregulatory materials derived from ⁇ f. vaccae for the manufacture of a medicament useful in the therapy of AIDS and also in the therapy of HIV-positive asymptomatic patients, with or without associated tuberculosis.
  • Such material may be administered to a subject, for example a subject who is HIV positive and shows overt symptoms of AIDS with or without infection by M. vaccae, in an amount sufficient at least to arrest the progression of the symptoms.
  • the therapeutic agent conveniently, and therefore preferably, comprises dead cells of M. vaccae, most preferably cells which have been killed by autoc ⁇ aving.
  • South African patent application 95/2644 teaches that immunotherapy with M. vaccae may therefore be expected to be effective against tumours of mesodemal , endodermal and ectodermal origin, including breast and bronchial tumours, by preventing or delaying the growth or spread of such tumours.
  • the application provides the use of antigenic and/or immunoregulatory materials derived from M. vaccae for the manufacture of a medicament useful in delaying or preventing the growth and spread of tumours . Such material may be administered to a subject in an amount sufficient at least to delay or prevent the growth or spread of a tumour.
  • Raynaud's phenomenon has been defined as the occurrence of episodic attacks of well demarcated blanching or cyanosis of one or more fingers on exposure to the cold (Coffman, 1989) .
  • the toes can also be affected, as well as the nose, ears, face, chest and lips. Digits often turn red when recovering from the attack and throbbing pain may occur during this phase.
  • Cold is the most common precipitating factor, followed by pressure on the digits, and less commonly, emotional or mental factors.
  • RP is further divided into Raynaud's disease (RD) , where no associated condition is found to cause the symptoms, and Raynaud's syndrome (RS) , where other disorder with pathology which can lead to Raynaud' s-like symptoms can be found.
  • RD Raynaud's disease
  • RS Raynaud's syndrome
  • These latter occur in a host of different clinical situations including several connective tissue disorders, peripheral vascular disorder, blood abnormalities and vasculitis (Roath, 1989) .
  • Other conditions in which can induce similar symptoms include frostbite and hypothermia.
  • some medical procedures require low temperature treatments, for example during heart surgery and transplantation.
  • cold shock treatment of M. vaccae induces a cold shock response which results in the production of cold shock proteins.
  • Such cold shocked M. vaccae or preparations derived from such M. vaccae which include one or more cold shocked proteins are provided by the present invention as novel products useful in the treatment of the conditions described above.
  • Preferred conditions which may be treated include autoimmune disease, chronic inflammatory disease and conditions associated with exposure to cold including Raynaud's phenomenon, Raynaud's disease, hypothermia and frostbite.
  • treatment refers to therapy which is designed to alleviate the symptoms of a disease or condition, for example by modulation of the immune system, as well as to therapy designed to cure such diseases or conditions.
  • the present invention thus provides a composition which comprises cold-shocked ⁇ f. vaccae .
  • the composition may further comprise a pharmaceutically acceptable carrier or diluent.
  • the composition may comprise one or more ⁇ f. vaccae cold-shock proteins together with a pharmaceutically acceptable carrier or diluent.
  • the above compositions may further comprise an antigen exogenous to ⁇ f. vaccae .
  • compositions of the invention may be administered in a unit dosage form comprising material from 10 3 to 10 10 , for example from 10 7 to 10 10 ⁇ f. vaccae microorganisms.
  • compositions of the invention may be used in a method of treatment of the human or animal body, including the treatment of an autoimmune disease, chronic inflammatory disease or a condition associated with exposure to cold including Raynaud's phenomenon, Raynaud's disease, hypothermia and frostbite.
  • compositions of the invention are also useful in the study of the cold shock response, for example as antigens for western blotting of samples from patients with diseases and conditions associated with cold.
  • the presence of cold-shock antigens in such patients will allow therapies for such patients to be reviewed, and for example to be modified to include anti- mycobacterial treatment if deemed appropriate.
  • M. vaccae strains The preferred strain of ⁇ f. vaccae is SRL172, which is available for human use under several investigator IND's from the Federal Drug Administration, and CTX's from the Medicines Control Agency in the UK. GLP acute toxicology has been performed by Huntingdon Research. Phase 1 and Phase 2 safety data have been obtained in the USA, and lodged with the FDA. SRL172 is in Phase 3 trials for the immunotherapy of tuberculosis. SRL172 may be preferred for use in the present invention.
  • SRL172 is a ⁇ f. vaccae formulation derived from the strain denoted R877R which was deposited under the Budapest Convention at the National Collection of Type Cultures (NCTC) Central Public Health Laboratory, Colindale Avenue, London NW9 5HT, United Kingdom, on 13 February 1984 under the number NCTC 11659. R877R was originally isolated from mud samples from the Lango district of Central Kenya (Stanford and Paul) .
  • ⁇ f. vaccae strains may be used instead of SRL172.
  • An organism can be identified as belonging to ⁇ f. vaccae by biochemical and antigenic criteria (Bonicke et al . ) , or by molecular methods such as PCR and restriction enzyme analysis as described by Telenti et al .
  • a cold-shocked ⁇ f. vaccae is one which contains one or more proteins either within its cell or on its cell surface which is expressed at a higher level when the cell is grown at a lower-than-physiological (e.g. about 35 °C) temperature.
  • proteins include those identified in the accompanying examples, which are proteins which are visible on one- dimensional SDS gels after about 4 hours incubation at between 6 and 12 °C, and which have approximate molecular weights (as estimated by SDS-PAGE) of 32, 35, 63, 78, 112 and 158 kDa.
  • ⁇ f. vaccae cells may be grown on a suitable solid medium and cold-shocked while present on such medium.
  • a modified Sauton's liquid medium may be preferred, solidified with agar, preferably about 1% agar. After aerobic incubation, generally at 35 * C for 10 days, the organisms may be cold-shocked, harvested, optionally killed (see below) then weighed and suspended in diluent, ready for administration. Storage, if required before use, may be at 4 * C.
  • a liquid medium such as the modified Sauton's medium may be employed, for instance in a fermentor.
  • cells Before cells are harvested for use in the present invention, they will be cold-shocked. This will involve placing cells growing at about the normal temperature (e.g. about 35 °C) in a cold environment. It may also include an intermediate step of heat-shocking or cold-storage of cells first, but this is not at present preferred. We have found that different cold-shock regimes produce different patterns of cold-shock protein production in ⁇ f. vaccae and these different patterns may be valuable for the treatment of different disease conditions. Thus cold-shock may occur within a wide range of temperatures and for a wide range of times.
  • any cold shock treatment may be used where the cells are grown at less than 25°C (for example from 0-20°C, preferably from 5-20°C, more preferably from 6-12°C) for a period of time sufficient to induce cold-shock protein production.
  • the cold-shock may be effected at a range of temperatures.
  • the cells may be initially placed in an environment at about 0-5°C and the temperature raised over the course of the cold-shock treatment for example to about 15-20°C, or vice versa . Where the temperature is raised or lowered, the change may be effected linearly, stepwise, logarithmically, etc.
  • the duration of the cold-shock may be any suitable time to provide for the production of one or more cold-shock proteins. It is likely that initiation of synthesis of such proteins occurs rapidly - within a few minutes - of exposure of ⁇ f. vaccae to cold, although in accordance with the present invention it is preferred that cold- shock be administered for at least 30 minutes, an more preferably at least 1, for example at least 4 hours. There is no upper limit as such on the length of exposure to cold since maintenance of a low temperature will provide for maintenance of expression of one or more cold-shock genes. However since some genes may be expressed at higher levels in an initial response to the change in temperature, it is preferred that the cold-shock is carried out for between from 1 to 10 hours, for example from 2 to 6 hours .
  • the Mycobacterium vaccae material used in compositions of the invention may be or include dead cells of ⁇ f. vaccae .
  • Such cells may be killed, for instance using irradiation, e.g. from 60 Cobalt at a dose of 2.5 megarads, chemically, or by any other means, although autoclaving is preferred, e.g. at 69kPa for 10 minutes at 115 * C-125 * C.
  • Autoclaving may yield a more effective preparation than irradiation. Where cells are killed this is desirably immediately following cold-shock, or if this is not possible, soon enough afterwards such that one or more cold-shock proteins are present in the cell.
  • compositions of the invention may include ⁇ f. vaccae cold-shock proteins which have been obtained or are obtainable from cold-shocked ⁇ f. vaccae . There are two preferred ways in which this may be achieved.
  • ⁇ f. vaccae which has been cold-shocked may be fractionated to obtain a protein fraction comprising one or more heat shocked proteins.
  • a protein fraction comprising one or more heat shocked proteins.
  • the cells may be osmotically shocked and the proteins and nucleic acid separated.
  • the proteins may then be purified and separated by size using techniques such as column chromatography and suitable size fractions retained. For example, we have found two cold-shock proteins with estimated molecular weights of about 35 and 63 kDa respectively. A protein fraction spanning this range of sizes may be recovered to include these proteins .
  • the genes encoding one or more cold-shock proteins may be isolated and placed under the control of a promoter functional in a mycobacterium which promoter is able to express constituitively the gene at 35oc or can be induced at 35°c to express the gene.
  • Genes from ⁇ f. vaccae may be cloned by conventional techniques including recovering cold-shocked proteins, analysing their sequence and making gene probes based upon said protein sequences or by using sequences of other bacterial cold-shock proteins as probes under conditions of medium to high stringency on a cDNA or genomic library from ⁇ f. vaccae .
  • the cold-shock gene cspA of E it is known the cold-shock gene cspA of E.
  • coli is 63% homologous to a gene sequence identified in ⁇ f. tuberculosis and this sequence from M. tuberculosis could be used as a probe for a corresponding gene in ⁇ f. vaccae.
  • antibodies against cold shock proteins may be made and used as probes on expression library made from ⁇ f. vaccae cDNA obtained from a cold-shocked
  • the cold-shock gene operably linked to a promoter can be introduced into ⁇ f. vaccae in the form of an independently replicating plasmid or it may be integrated into the genome of ⁇ f. vaccae, for example by homologous recombination with the corresponding wild type gene. In the latter circumstance this will lead to replacement of the wild-type gene with a copy under the control of the exogenous constitutive or inducible promoter.
  • Suitable promoters functional in mycobacteria are known as such in the art .
  • Abou Zeid describes the expression of a recombinant antigen from ⁇ f. tuberculosis in ⁇ f. vaccae using a shuttle vector and such a vector may also be used for the expression of cold shock antigens.
  • ⁇ f. vaccae cells may be cultured at normal physiological temperatures and still produce cold-shock proteins.
  • modified ⁇ f. vaccae may be used in the present invention and former further aspect of it.
  • cold-shock proteins from ⁇ f. vaccae may be expressed recombinantly in other bacterial or eukaryotic expression systems in accordance with established techniques in the art and these proteins recovered and used to formulate compositions of the invention.
  • the recovered proteins may be used alone or mixed with non cold-shocked ⁇ f. vaccae in order to provide a composition of the present invention.
  • the pharmaceutically acceptable diluent may be unbuffered saline, pyrogen-free .
  • the diluent is borate- buffered, preferably containing a surfactant such as Tween 80 ® .
  • a suitable borate buffer is: Na 2 B 4 0 7 10H 2 O - 3.63 g, H 3 B0 3 - 5.25 g, NaCl - 6.19 g, Tween 80 ® 0.0005%, distilled water to 1 litre. These diluents are pharmaceutically acceptable.
  • the ⁇ f. vaccae material is administered free or substantially free from non- ⁇ f. vaccae antigenic or immunoregulatory material.
  • the medicament or composition to be administered may include, or may consist essentially of, ⁇ f. vaccae antigenic and/or immunoregulatory material, such as dead cells, an extract or derivative thereof, and a pharmaceutically acceptable diluent .
  • Administration is preferably in a "therapeutally effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom.
  • the actual amount administered, and rate and time- course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, eg decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.
  • a single dosage (where dead cells are to be administered) will generally contain from 10 7 to 10 10 killed ⁇ f. vaccae microorganisms. Patients may be administered a single dose of 10 8 to 10 9 killed ⁇ f. vaccae, though the dose may be repeated if need be, for instance at intervals from 2 weeks to 6 months.
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • compositions according to the present invention may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which is preferably by injection, e.g. cutaneous, subcutaneous or intra-dermal .
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • Suitable diluents which are pharmaceutically acceptable and may be preferred, have been discussed already above.
  • Oral administration may be used, in which case the pharmaceutical composition may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol , propylene glycol or polyethylene glycol may be included. The invention is illustrated by the following non-limiting examples .
  • Mycobacterium vaccae was grown on Suaton's medium and incubated at 35oC.
  • Sauton's medium is: Asparagine 6.0g, Glycerol 30.0ml, Citric acid 2.0g, di-Potassium hydrogen phosphate (K 2 HP0 4 ) 1.5g, Magnesium sulphate (MgS0 4 ) 0.25g, Ferric ammonium citrate 0.05g, Agar 10. Og and Distilled water 1 litre.
  • the pH of the medium was adjusted to 6.2 using approximately 1.0ml of concentrated ammonia solution.
  • the medium was autoclaved at 15lbs for 15 minutes. When it had cooled to 50- 54"C, 25ml of 40% glucose solution (sterilized by filtration using 0.2 ⁇ m filter) was added.
  • Bacterial cells were grown at 35 'C, collected and resuspended in phosphate-buffered saline (PBS), pH6.8 at 35 * C. 0.5ml portions were cold-shocked for 1, 2 and 4 hours at different temperatures, namely 10°C, 15°C, 20°C and 25 °C.
  • PBS phosphate-buffered saline
  • Protein synthesis in mycobacteria during cold-shock was examined by adding L- [ 35 S] methionine (190 ⁇ Ci ml, ICN) during the last hour of cold-shock treatment. Radioactive labelling was terminated with lOmM-L-methionine. The cells were collected and washed with PBS containing ImM-L-methionine followed by PBS alone. Cells were lysed with an equal amount of glass beads, and proteins were solubilized by boiling the sample for 5-10 minutes in an SDS sample buffer. Proteins were separated by vertical one-dimensional SDS-PAGE using a 10% ExcelGel ® SDS gradient 8-18 precase gel (Pharmarcia Biotech) , together with a standard marker mixture (Sigma) .
  • L- [ 35 S] methionine 190 ⁇ Ci ml, ICN
  • Radioactive labelling was terminated with lOmM-L-methionine.
  • the cells were collected and washed with PBS containing ImM
  • Gels were stained with either Coomassie blue stain and then silver stain or only with silver stain. After silver staining, the gels were soaked in a fluorographic reagent, Amplify (Amersham life sciences) .
  • Example 1 The experiments of Example 1 were repeated with cold shock treatment of cells at 8 * C and 12 * C for 1, 4, 8, 16, 20, 24, 33 and 48 hours, and the samples of cell run out on a silver stained gel as above.
  • Example 2 The experiment of Example 2 was repeated at 6 * C. Two bands were clearly visible in the silver stained gel as they were more deeply stained than the rest . When the gel was compared to the control at 35'C, band 1 appeared to be also present in the control but band 2 was not. A standard curve of the molecular weight markers run on this gel was plotted and it was estimated that the size of protein 1 is 32kDa and 2 is 112kDa .
  • the first two samples were plasma from London pulmonary tuberculosis patients with high antibodies to Hsp65 and 70.
  • Samples three to six were plasma from London healthy controls which also had high antibodies to Hsp65 and 70.
  • Samples seven to eleven were sera from South African pulmonary tuberculosis patients whose antibodies to heat-shock proteins were not tested.
  • the different serum samples were diluted 100 times in PTB and 4ml was used for incubation with the blot under standard conditions. Each sample on the membrane was cut into 0.5mm strips which were placed in the wells with sera and incubated for two hours at room temperature on a shaker. The strips were then washed three times for ten minutes in PTB at room temperature on a shaker. The secondary antibody, anti-human IgG-peroxidase (Sigma) at a dilution of 1:1000, was then added and then incubated and washed as for the sera. Several bands were detected from the Western blots. From the eleven primary antibody samples used, six plasma and five sera, only two detected proteins in both the cold-shock treatments but not in the control .

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Abstract

Cette invention concerne une composition qui comprend les éléments suivants: des M. vaccae produites dans des conditions hypothermiques ou des protéines produites dans des conditions hypothermiques et issues de M. vaccae; et un excipient ou un diluant acceptables sur le plan pharmaceutique. Ces compositions sont utilisées dans un procédé permettant de traiter le corps d'êtres humains ou d'animaux qui souffrent notamment de troubles associés à une exposition au froid, tel que le phénomène de Raynaud, la maladie de Raynaud, l'hypothermie et les engelures.
PCT/GB1998/003346 1997-11-07 1998-11-09 Preparation de mycobacterium vaccae comprenant des proteines produites dans des conditions hypothermiques et utilisation de cette preparation lors de therapies WO1999024067A1 (fr)

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AU10430/99A AU1043099A (en) 1997-11-07 1998-11-09 (micobacterium vaccae) preparation comprising cold-shock proteins and its use intherapy

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GB9723630.1 1997-11-07
GBGB9723630.1A GB9723630D0 (en) 1997-11-07 1997-11-07 M.vaccae preparation and its use

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7393540B2 (en) 2001-07-04 2008-07-01 Health Protection Agency Mycobacterial antigens expressed during latency
US8017753B2 (en) 2001-06-22 2011-09-13 Health Protection Agency Mycobacterial antigens expressed under low oxygen tension

Citations (1)

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WO1991002542A1 (fr) * 1989-08-25 1991-03-07 University College London Traitement d'etats inflammatoires chroniques

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US8017753B2 (en) 2001-06-22 2011-09-13 Health Protection Agency Mycobacterial antigens expressed under low oxygen tension
US8404826B2 (en) 2001-06-22 2013-03-26 Health Protection Agency Mycobacterial antigens expressed under low oxygen tension
US7393540B2 (en) 2001-07-04 2008-07-01 Health Protection Agency Mycobacterial antigens expressed during latency
EP2196473A1 (fr) 2001-07-04 2010-06-16 Health Protection Agency Antigènes mycobacteriens exprimes pendant la phase de latence
US8003776B2 (en) 2001-07-04 2011-08-23 Health Protection Agency Mycobacterial antigens expressed during latency
US8399650B2 (en) 2001-07-04 2013-03-19 Health Protection Agency Mycobacterial antigens expressed during latency

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