Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
  • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
  • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
  • C07H13/06—Fatty acids
• • 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
  • A61P31/04—Antibacterial agents
  • A61P31/06—Antibacterial agents for tuberculosis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
  • G01N33/5695—Mycobacteria
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)

Definitions

• the present invention relates to new sulfoglycolipid antigens, their process of preparation and their use in the treatment or the prophylaxis of tuberculosis.
• tuberculosis is the estimated cause of 3 million deaths.
• the causative agent of this disease is a bacterium, Mycobacterium tuberculosis, by which one of every three people is infected worldwide. It is transmitted through the air by sneezes or coughs of infected persons.
• the bacterium may be harboured in an inactive state by infected persons who will never develop the disease. However, under certain conditions, such as age or depression of the immune system, the bacterium may become active and cause the onset of tuberculosis.
• mycobacterial envelope accounts for an important part of the virulence of bacteria of the Mycobacterium genus. Indeed, up to 40% of mycobacteria dry weight is constituted by lipids. Among those lipids, some of them seem restricted to Mycobacterium tuberculosis, such as sulfoglycolipids for instance (Vergne I. and Daffe M. Frontiers in Bioscience (1998) 3:865-876).
• This family of glycolipids is typified by a sulfate substituent on position 2' of a trehalose unit (i.e. ⁇ -D-glucopyranosyl-CI- ⁇ - ⁇ '-D-glucopyranoside) (A).
• the fatty acids substituents notably comprise palmitic acid, stearic acid, phthioceranoic acid and hydroxyphthioceranoic acid.
• the latter two fatty acids are characteristic of sulfoglycolipids.
• the most abundant sulfoglycolipid is SL-I (Goren et al. Biochemistry (1976) 15:2728-2735) (B).
• the vaccine used for the prophylaxis of tuberculosis consists of a live attenuated bacterium of the Mycobacterium bovis species. It is named BCG, Bacillus of Calmette and Guerin, after the two scientists who first devised it, at the beginning of the 20 th century.
• BCG Bacillus of Calmette and Guerin
• BCG protective efficacy in adults range from 0% to 80% according to varying studies, besides, it fails to protect against pulmonary tuberculosis, the most prevalent disease form in adults.
• antibiotic resistant strains of M. tuberculosis have been found in over 35 countries. Hence, it is a subject of the present invention to provide a more effective way of treating and/or preventing tuberculosis.
• Further immunogenic glycolipids extracted from Mycobacterium tuberculosis are disclosed in WO2004/092192.
• these natural products are obtainable in limited amount; additionally, they are obtained from a pathogenic strain and their preparation may be hazardous and requires specific safety installations.
• the invention notably provides new synthetic sulfoglycolipid antigens, which have been shown to stimulate CD 1 -restricted T lymphocytes in vitro.
• the invention relates to compounds of the following general formula (I):
• R 2 ', R 3 ', identical or different, are independently chosen from H, SO 3 H or SO 3 VM + , provided that at least one of R 2 ', R 3 ' is SO 3 H or SO 3 VM + ; Preferably, R 2' is SO 3 H or SO 3 VM + and R 3' is H.
• - M + is the cation of a metal, such as Na + , K + .
• R 2 , R 3 are independently chosen from: a) fatty acyl groups o
• X is an unsaturated linear or ramified hydrocarbon chain optionally substituted with one or more substituents; where at least one of R 2 , R 3 is b); and its enantiomers, diastereoisomers, mixtures thereof and pharmaceutically acceptable salts or esters.
• X is of formula (b-1):
• - Y is a saturated or unsaturated, preferably saturated, linear or ramified hydrocarbon chain optionally substituted with one or more substituents.
• Y is a saturated linear alkyl chain optionally substituted with one to ten, more preferably one to four alkyl groups, preferably methyl groups.
• the methyl groups are located at C-1 , C-3, C-7 or C-9 positions of the Y chain.
• the substituted carbon atoms exhibit the (S) configuration.
• R 1 , R j identical or different, are independently chosen from H, Alkyl; preferably, R' is Methyl and R j is H.
• R 1 and R J are such that the configuration may be (E) or (Z).
• the b) chain is of formula (b-2):
• r is an integer chosen from 1 , 2 or 3; preferably, 1 ;
• I is an integer chosen from 0 to 10; preferably, 1 , 2 or 3; q is an integer chosen from 0 to 50; preferably, 5 to 50; more preferably 10 to 20; provided that I + q >1 ; each T', identical or different, is independently chosen from alkyl groups; preferably, each T' is methyl.
• the carbon atom to which T' is attached is asymmetric.
• said carbon atom exhibits a (S) configuration.
• T 2 is an alkyl group; preferably a Methyl.
• Fatty acyl groups are derived from fatty acid groups which esterify the hydroxyl groups in position 2 or 3 of the 2'- or 3'-sulfated trehalose unit.
• Fatty acid groups are aliphatic carboxylic acids which can be linear or ramified, saturated or unsaturated, unsubstituted or substituted by groups such as hydroxyl, or ketone.
• they contain from 5 to 50, preferably 15 to 50 carbon atoms, and more particularly may be selected from the group comprising:
• n is an integer from 2 to 10
• fatty acyl groups are selected from the group comprising palmitic acyl and stearic acyl.
• the invention more particularly relates to compounds of formula I, wherein R 2 represents a fatty acyl group, preferably, a palmitic acyl group or a stearic acyl group and R 3 represents a (b-2) chain, namely compounds of following formula (II):
• R 2 , R 3 , r, p, I, q, T 2 , T 1 are defined as above, or the corresponding salt thereof, and its enantiomers, diastereoisomers, mixtures thereof and pharmaceutically acceptable salts or esters.
• the invention more specifically relates to compounds of following formulae:
• pharmaceutically acceptable salts refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof.
• the pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
• such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, tartaric, citric, glutamic, benzoic, salicylic, toluenesulfonic, oxalic, and the like.
• the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
• such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.
• non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, the disclosure of which is hereby incorporated by reference.
• the invention relates to a composition comprising at least two different compounds of formula I such as defined above.
• the invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising at least one compound as defined above, or a composition as defined above, in association with a pharmaceutically acceptable vehicle.
• a pharmaceutically acceptable carrier comprises in particular liposomes.
• composition may also comprise vaccine adjuvants.
• vaccine adjuvants for use in human individuals or animals are well known to the man skilled in the art, a list of such salts can be found for instance in "A compendium of vaccine adjuvants and excipients" 2 nd edition, Vogel et a).
• Particular vaccine adjuvants notably comprise aluminum salts or M59, for example.
• the invention particularly relates to a pharmaceutical composition as defined above, characterized in that it is presented in a form intended for administration by oral or injectable route.
• the invention more particularly relates to a pharmaceutical composition as defined above, characterized in that it comprises one or more other products useful for the treatment or the prophylaxis of tuberculosis, such as BCG or mycobacterial proteins.
• BCG stands for Bacillus of Calmette and Guerin, the different strains of BCG currently used for vaccination are notably described in Behr M.A. et al. Science (1999) 284:1520-1523.
• mycobacterial proteins refers to proteins, or fragments thereof, which are encoded by the genome of bacteria of the Mycobacterium genus and notably by the genome of Mycobacterium tuberculosis, such proteins may be advantageously recombinant. According to a preferred embodiment said mycobacterial proteins are antigens of M. tuberculosis.
• kits for the treatment or the prophylaxis of tuberculosis notably comprise immunomodulators, such as cytokines, DNA fragments encoding M. tuberculosis antigens, live M. tuberculosis deletion mutants, such as mutants in which virulence genes have been deleted, or live recombinant BCG, such as BCG expressing antigens of M. tuberculosis.
• immunomodulators such as cytokines, DNA fragments encoding M. tuberculosis antigens, live M. tuberculosis deletion mutants, such as mutants in which virulence genes have been deleted, or live recombinant BCG, such as BCG expressing antigens of M. tuberculosis.
• tuberculosis refers to the disease caused in humans by the bacterium Mycobacterium tuberculosis, but also to the corresponding disease in animals.
• the invention relates to products comprising:
• the invention also relates to the use of at least one compound as defined above, or of an above mentioned composition, for the preparation of a medicament, notably a vaccine, intended for the treatment or the prophylaxis of tuberculosis.
• the vaccine may comprise a vaccine adjuvant such as described above.
• the invention relates to the use of at least one compound as defined above, or of a composition according as defined above, as an immune reaction activator, and more particularly an inflammatory reaction activator.
• an immune reaction activator is meant a compound which has the ability to activate components or processes of the immune reaction, in vitro or in vivo, in particular cells of the immune system, such as T lymphocytes, B lymphocytes, antigen presenting cells (APCs), such as dendritic cells or macrophages, monocytes or granulocytes.
• inflammatory reaction activator is meant a compound which has the ability to activate components or processes of the inflammatory reaction, in vitro or in vivo, such as diapedesis, capillary permeabilization, macrophage activation or fever onset for instance.
• the invention also relates to the use of at least one compound as defined above, or of a composition as defined above, to induce the activation of T lymphocytes, notably CD 1 -restricted T lymphocytes.
• T lymphocytes notably CD 1 -restricted T lymphocytes.
• the activation can proceed in vitro or in vivo.
• T lymphocytes can be assessed by several methods, such as measuring cell multiplication or cytokine production such as IFN- ⁇ (interferon- ⁇ ).
• IL-2 interleukine-2
• IL-4 interleukine-4
• TNF- ⁇ tumor necrosis factor ⁇
• CD1 -restricted T lymphocytes are T lymphocytes which are activated by antigens presented by CD1 molecules.
• the invention also relates to the use of at least one compound as defined above, or of a composition as defined above, to induce the production of IFN- ⁇ ,
• TNF- ⁇ , IL-4 or granulysin This production induction can be done in vitro or in vivo.
• the production of IFN-Y, TNF- ⁇ , IL-4 or granulysin can be measured for instance by immunoassays, such as ELISA (enzyme linked immunosorbent assay) or EIA (enzyme immunoassay).
• the invention also relates to the use of at least one compound as defined above, or of an above mentioned composition, for the preparation of a composition for the diagnosis of the infection by Mycobacterium tuberculosis.
• Tuberculosis diagnosis may prove to be difficult as the current test (tuberculin or so-called "PPD” test) can not differentiate those PPD + individuals who received BCG from those PPD + patients actually infected with latent tuberculosis.
• the compounds of the invention are highly specific to Mycobacterium tuberculosis species. They may be used to discriminate PPD + individuals into those vaccinated with BCG and those actually infected with tuberculosis.
• the present invention thus provides a method for diagnosing infection by Mycobacterium tuberculosis comprising the following steps:
• R 2 ', R 3 ', identical or different, are independently chosen from H, SO 3 H or SO 3 VM + , provided that at least one of R 2' , R 3' is SO 3 H or SO 3 VM + ;
• R 2' is SO 3 H or SO 3 VM + and R 3' is H.
• - M + is the cation of a metal, such as Na + , K + .
• R 2 , R 3 are independently chosen from: a) fatty acyl groups
• X is an unsaturated linear or ramified hydrocarbon chain optionally substituted with one or more substituents; and its enantiomers, diastereoisomers, mixtures thereof and pharmaceutically acceptable salts or esters,
• T lymphocytes e.g. where IFN- ⁇ release increased after administering a compound of the invention are infected with Mycobacterium tuberculosis.
• the present invention also provides a kit for diagnosing tuberculosis comprising:
• - dendritic cells - means for detecting T lymphocytes activation, e.g. release of IFN- ⁇ .
• the sulfoglycolipid activation of T cells is detected by measuring the IFN- ⁇ released in the supernatant 48 hr after stimulation with dendritic cells and sulfoglycolipids, using any human IFN- ⁇ ELISA detection kit (for example, purchased from BD Pharmingen).
• the present invention also relates to the use of the compounds of the invention to assess the efficacy of the vaccines prepared with recombinant Mycobacterium tuberculosis.
• Vaccines have been developed using recombinant living bacteria obtained from Mycobacterium tuberculosis or Mycobacterium bovis BCG, which may e.g. overexpress a protein. It is highly desirable to assess whether such vaccines are effective and protect the patient administered therewith. Assessment is made possible by carrying out the diagnosis method of the invention.
• the present invention also relates to specific ligands to the compounds of formula (X).
• a “specific ligand” relates to any compound which can be prepared or elicited to specifically bind to a compound according to the invention.
• Specific ligands notably encompass antibodies and aptamers.
• the term "antibodies” relates to complete antibodies, in particular monoclonal antibodies, but also to antibody fragments which retain the ability to specifically bind to the compounds of the invention, such as the Fab, Fab', F(ab') 2 , Fv or scFv fragments.
• Antibody preparation is well known to the man skilled in the art. Usually, a preparation of a compound according to the invention is administered to an animal, such as a mouse, a rat, a rabbit, or a goat, by various routes, such as the intramuscular, intravascular or intraperitoneal route. Additional administration can be performed after the first administration in order to increase the production of antibodies. Antibodies are then obtained from blood drawn from the animals, or from blood derivatives such as serum or plasma, and optionally purified, for instance using affinity chromatography. Monoclonal antibodies can notably be prepared as described by Koller & Milstein (1975) Nature 256:495-499.
• “Aptamers” may be of the peptide type or of the nucleic (e.g. desoxyribonucleic or ribonucleic) type. Nucleic aptamers can notably prepared using the well known SELEX method or as described by Ellington & Szostak (1990) Nature 346:818-22. Peptide aptamers can be obtained as described by
• General formula (X) includes formula (I) as disclosed in WO 2004/092192 incorporated herein by reference as well as compounds of formula (I) and (II) of the present invention.
• the compounds of formula (X) are those of formulae (I) and (II) and their preferred embodiments as disclosed above.
• the present invention also relates to the process of preparation of a compound of formula (I) as defined above.
• the compounds and process of the present invention may be prepared in a number of ways well known to those skilled in the art.
• the compounds can be synthesized, for example, by application or adaptation the methods described below, or variations thereon as appreciated by the skilled artisan.
• the appropriate modifications and substitutions are readily apparent and well known or readily obtainable from the scientific literature to those skilled in the art.
• reagents and starting materials are commercially available, or readily synthesized by well-known techniques by one of ordinary skilled in the arts.
• suitable solvents include: hydrocarbons, which may be aromatic, aliphatic or cycloaliphatic hydrocarbons, such as hexane, cyclohexane, benzene, toluene and xylene; amides, especially fatty acid amides, such as dimethylformamide; and ethers, such as diethyl ether and tetrahydrofuran.
• hydrocarbons which may be aromatic, aliphatic or cycloaliphatic hydrocarbons, such as hexane, cyclohexane, benzene, toluene and xylene
• amides especially fatty acid amides, such as dimethylformamide
• ethers such as diethyl ether and tetrahydrofuran.
• the reactions can take place over a wide range of temperatures. In general, we find it convenient to carry out the reaction at a temperature of from about 0 0 C to about 150 0 C (more preferably from about room temperature to about 100°C).
• the time required for the reaction may also vary widely, depending on many factors, notably the reaction temperature and the nature of the reagents. However, provided that the reaction is effected under the preferred conditions outlined above, a period of from about 3 hours to about 20 hours will usually suffice.
• the compound thus prepared may be recovered from the reaction mixture by conventional means.
• the compounds may be recovered by distilling off the solvent from the reaction mixture or, if necessary after distilling off the solvent from the reaction mixture, pouring the residue into water followed by extraction with a water-immiscible organic solvent and distilling off the solvent from the extract.
• the product can, if desired, be further purified by various well techniques, such as recrystallization, reprecipitation or the various chromatography techniques, notably column chromatography or preparative thin layer chromatography.
• the present invention provides a process of preparation of a compound of formula (I) as defined above comprising the steps of:
• each of P 4 , P 5 , P 4 ', P 5 ' identical or different represents H or a OH- protecting group or P 4 and P 5 and/or P 4 ' and P 5 ' form together a cyclic OH-protecting group.
• One or two sulfonating and acylating reactions may be required.
• the sulfonating and acylating steps may be carried out in any order, successively or alternatively.
• One or more protecting and/or deprotecting steps may be required before and/or after the acylating and/or sulfonating steps, as appropriate.
• Such protecting groups include acetals or ketal, such as
• the process of the invention comprises the further step of hydrolyzing said protecting groups. This reaction is usually carried out in acid conditions.
• Said sulfonation reaction is generally carried out with any suitable sulfonating agent, such as the pyridine-sulfur trioxide complex or trimethylamine-sulfur trioxide complex.
• any suitable sulfonating agent such as the pyridine-sulfur trioxide complex or trimethylamine-sulfur trioxide complex.
• the two required acylations may be carried out in one or two steps.
• the acylation step(s) is/are usually conducted with coupling agents such as DMAP and/or DCC.
• the process of the invention may also comprise the final step of isolating the desired compound.
• the 2', 3' bi-sulfonated compound (7 ⁇ ) may be obtained by using excess of the sulfonating reagent.
• the 3'-sulfonated compound (T_) is obtained as a byproduct of the sulfonating reaction; usually, the selectivity of the sulfonating reaction is about 5/1.
• Compounds 7, T_ and T ⁇ are separated by chromatography.
• the compound of formula (IV) is generally commercially available.
• R 3 represents the b) chain as defined above
• the compounds of formula (IV) may be prepared by application or adaptation of the following illustrative scheme:
• R represents the corresponding optionally substituted unsaturated linear or
• steps are carried out up to G which then comprises an additional methyl group with the required (S) stereochemistry.
• G is then reduced into reduced A' leading to desired B', then C which in turn is saponified to lead to the desired compound of formula (IV).
• the process may be carried out by applying or adapting the experimental conditions and/or starting products given in the examples.
• Figures 1 and 2 represent the amount of IFN- ⁇ (in pg/mL, vertical axis) produced by T cell clone Z4B27 in response to stimulation (horizontal axis) by the compounds of the invention comparative compounds.
• the dibenzylidene derivative 7, T_ or 7 ⁇ was treated with a solution of chloroform/methanol/1.7 % H 2 SO 4 (60/40/8) for 2 days at room temperature.
• the reaction mixture was made neutral with a solution of NaHCO 3 . This deprotection afforded quantitatively the corresponding diacyiated sulfoglycolipid 8, 8_[ or 8".
• the conjugated ester C (1 eq.) was hydrogenolyzed in ethyl acetate (0.4 M) using 10% palladium on carbon as catalyst.
• the saturated ester D was obtained in 77 % as a 1/1 enantiomeric (diastereoisomeric) mixture on the C-2 position .
• Ester D (1 eq.) was heated overnight at 110 0 C in a solution of potassium hydroxyde (12 eq.) in water/ethanol 2/3 (0.2 M). This reaction afforded quantitavely the corresponding acid E as a racemic (1/1 diastereoisomeric) mixture on the C-2 position.
• Acid E (1 eq.) was heated under reflux with oxalyl chloride (10 eq.) for 1 h. Excess reagent was removed under reduced pressure. Dry dichloromethane (0.4 M) and DMAP (1.2 eq.) followed by (R)-2-phenylglycinol (1.1 eq.) were added to the crude acyl chloride and the reaction mixture wax stirred at room temperature overnight. A flash chromatography permitted the separation of the diastereoisomers. The 2S diastereisomer F was the less polar compound and was isolated in 36 % yield.
• Acid G (1 eq.) was dissolved in a solution of BH 3 in THF (1 M, 1.7 eq.) and this mixture was stirred overnight at room temperature. Ethanol was added, then 80 % aqueous acetic acid and the mixture was made neutral with NaHCO 3 . The alcohol was obtained quantitatively in pure form.
• lFN- ⁇ release is measured after stimulation with the purified sulfoglycolipids.
• 2 x 10 5 PBMC per/well are incubated for 4 days in the presence of GM-CSF (500 U/mL) and IL- 4 (5 ng/mL).
• GM-CSF 500 U/mL
• IL- 4 5 ng/mL
• Autologous effector T-cells are incubated in 10% human serum during this time.
• the sulfoglycolipids (10 ⁇ g/mL) are added to the irradiated, CD1 -expressing antigen-presenting cells.
• effector cells are added (2 x 10 5 /well) and IFN- ⁇ release is measured by ELISA in the supernatants after 18 hours.
• the IFN- ⁇ ELlSA is performed in 96-well immunosorbent plates, which are coated with an IFN- ⁇ capture antibody (2 ⁇ g/mL) overnight. Non-specific binding sites are blocked with PBS containing 1 % bovine serum albumin. The supernatants are diluted 1 :1 and added in a final volume of 100 ⁇ l. Plates are incubated at room temperature for 2 hours and removed by thorough washing (3-4 times). Finally, a biotinylated anti-IFN- ⁇ antibody is added for 1 hour (2 ⁇ g/mL). For detection of immunoreactive IFN- ⁇ horseradish-peroxidase is added for 30 min. Finally, a chromogenic substrate (TMB, Endogen, MA, USA) is added. After 20 min.
• TMB Endogen, MA, USA
• PPD + donors positive to the tuberculin test
• PPD ' donors negative to the tuberculin test
• the response to the sulfoglycolipids is measured by assessing the IFN- ⁇ production with the ELISA test (15 pg/mL) in each patient in each group. The responses in each group are compared.
• Example 4 Antigen presentation assay
• CD1 + APC were preincubated at 5x10 4 cells/well for 2 h at 37 0 C with sonicated antigen (1-10 ⁇ g/mL) before addition of T cells (5x10 4 /well in triplicate). After 36 h released TNF- ⁇ and IFN- ⁇ were measured using sandwich ELISA kits (Instrumentation Laboratory). Data are expressed as mean ng/mL or pg/mL ⁇ SD of triplicates. All experiments were repeated at least 2 times.
• Results are illustrated in Figure 1 for compound e (cpd e) of the invention.

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