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WO1993005011A1 - Nouveaux immunosuppresseurs - Google Patents

Nouveaux immunosuppresseurs Download PDF

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Publication number
WO1993005011A1
WO1993005011A1 PCT/EP1992/001995 EP9201995W WO9305011A1 WO 1993005011 A1 WO1993005011 A1 WO 1993005011A1 EP 9201995 W EP9201995 W EP 9201995W WO 9305011 A1 WO9305011 A1 WO 9305011A1
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WO
WIPO (PCT)
Prior art keywords
ala
thr
lys
cha
amino acid
Prior art date
Application number
PCT/EP1992/001995
Other languages
English (en)
Inventor
Sylvain Cottens
Hans Fliri
Edouard Lier
Beat Weidmann
Original Assignee
Sandoz Ltd.
Cytel Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB919118498A external-priority patent/GB9118498D0/en
Priority claimed from GB919118499A external-priority patent/GB9118499D0/en
Priority claimed from GB929202059A external-priority patent/GB9202059D0/en
Application filed by Sandoz Ltd., Cytel Corporation filed Critical Sandoz Ltd.
Publication of WO1993005011A1 publication Critical patent/WO1993005011A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/22—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated the carbon skeleton being further substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/30—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and unsaturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/16—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and unsaturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0207—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)4-C(=0), e.g. 'isosters', replacing two amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link

Definitions

  • This invention relates to certain novel peptides which
  • the body's own (“self”) cells do not trigger an immune response. It is believed that one way in which the immune response is mediated is by selective presentation of antigens to T-cells by identifying molecules, the production of which is mediated by a gene sequence known as the major histocompatibility complex ("MHC").
  • MHC major histocompatibility complex
  • the nature of these MHC molecules may vary depending on the type and location of the cell as well as on the type of antigen presented.
  • MHC major histocompatibility complex
  • self antigens are presented, there is ordinarily no population of T cells recognizing such antigens, and no immune response occurs. In some cases, however, presentation of self antigens can lead to an inappropriate, autoimmune, response.
  • One object of this invention is to provide blocker compounds which inactivate the MHC molecules associated with various autoimmune diseases.
  • the MHC blockers of this invention have primarily been evaluated in connection with rheumatoid arthritis, which has been associated with the MHC alleles HLA-DR1, HLA-DR4w4, and/or HLA-DR4wl4.
  • autoimmune diseases which may be treated, prevented or relieved using the peptides of this invention include ankylosing spo ⁇ dylitis, Hashimoto's disease, multiple sclerosis, Sjogren syndrome, scleroderma, polymyositis, dermatomyositis, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile diabetes, myasthenia gravis, bullous pemphigoid, pemphigus,
  • autoimmune hemolytic anemia r pernicious anemia idiopathic thrombocytopenic purpura
  • Grave's disease Addison's disease, graft rejections and the like, as well as autoimmune diseases of animals.
  • rheumatiod arthritis include corticosteroids, anti-malarial drugs, gold salts, and nonsteroidal anti-inflammatory drugs, have proved, in many cases, to be ineffective in the treatment of autoimmune diseases or to have serious side effects or both.
  • Conventional immuno- suppressants which work systemically to suppress the entire immune system have the disadvantage of increasing the risk of cancers and opportunistic infections, as well as other side effects. There is thus a clear and long felt need for immunosuppressants which are relatively nontoxic and which can be used to treat the specific autoimmune response associated with particular autoimmune diseases.
  • the MHC blocker peptides of this invention meet that need.
  • Peptides which bind to MHC were known in the art prior to this invention.
  • immunogenic peptides such as influenza hemaglutinin (HA), ragweed protein and tetanus toxin have been extensively studied.
  • HA hemaglutinin
  • ragweed protein ragweed protein
  • tetanus toxin tetanus toxin
  • peptides generally only bind well to a single type of MHC receptor, such as DR1.
  • the challenge is therefore to develop peptides which bind to the desired MHC receptors competitively with the antigens to be blocked, but which do not themselves stimulate T cell proliferation.
  • the blocker peptides should be relatively stable in vivo. This is generally a problem with peptides, which tend to have poor bioavailability due to lack of stability against proteases under physiologic conditions and rapid excretion by the kidneys. Moreover, due to the fact that new MHC molecules are constantly forming on the surface of antigen presenting cells, it is particularly important for an MHC blocker to be available over a substantial period of time so as to be able to bind to the newly formed MHC molecules.
  • novel unnatural residues at specified points in the MHC blocker peptide will enhance the bioavailability and stability of the peptide and will inhibit T-cell activation by masking or blocking selected MHC molecules.
  • novel peptides are shown in vivo to be useful in the propylaxis and treatment of disease, particularly autoimmune disease.
  • the unnatural residues of the invention may be novel amino acids, halo-olefin dipeptide isosteres, or cyclic tripeptides, all as more fully described herein. Incorporation of these residues at selected points in the molecule appears to alter the configuration or presentation of the non-MHC binding portion of peptide without significantly changing the configuration or presentation of the side chains which are critical to MHC binding, thereby permitting or even enhancing MHC binding, and at the same time interfering with or eliminating recognition of the peptide by T-cells or by peptidases.
  • peptides of the invention which have a strong affinity for MHC molecules associated with particular autoimmune diseases are also useful in a variety of diagnostic assays to determine the presence of MHC molecules associated with autoimmune disease in the early stages of the disease, before the outward symptoms of the disease become apparent. Such early diagnosis aids greatly in the treatment and management of autoimmune disease. Peptides of the invention can also be used in competitive screening assays to measure MHC binding to other molecules of interest.
  • the present invention thus provides stable, soluble, relatively nontoxic compounds which are antagonistic to MHC molecules associated with autoimmune diseases and which are not immunogenic.
  • the invention provides novel peptides incorporating novel, unnatural residues, which peptides show a strong affinity for HLA-DR molecules, especially DR1, DR4w4 and DR4w14, the molecules associated with rheumatoid arthritis.
  • novel peptides have proved stable under physiologic conditions and are relatively nontoxic in vivo. The development of these novel compounds
  • the invention provides novel residues which are useful in other applications of peptide chemistry in that they confer unexpectedly useful stability and resorption properties to peptides in which they are incorporated.
  • novel amino acids of the invention are as follows:
  • AA-I is an O-substituted serine of formula I
  • nonnatural amino acid O-triethyleneglycol serine monomethylether (the compound of formula I in which m is zero, n is three, and R a is methyl) is not novel per se, it may be utilized in place of AA-I in the MHC blocker peptides of the invention, and MHC blocker peptides incorporating this amino acid are novel.
  • AA-II is N-[6-(2,4-dimethoxypyrimidyl)] alanine of formula II:
  • AA -III is 0-(2,3-dihydroxypropyl) serine of formula III:
  • halo-olefin dipeptide isosteres of this invention may, in principle, be incorporated into any peptide without altering the geometry of the peptide or the chirality of the amino acid residues.
  • the advantages of and processes for making these peptide isosteres are thus not limited to MHC blocking peptides, but could be utilized to enhance the stability, binding activity and bioavailability of a wide variety of biologically active peptides in addition to the ones exemplified herein.
  • novel halo-olefin peptide isosteres comprise residues of two or more amino acids (other than isosteres of diglycine and phenylalanyl glycine) linked by a halo-olefin group as shown in Formula IV:
  • W is a halogen, preferably fluorine or chlorine
  • Z represents an amino acid residue, a peptide, a protective group, or hydrogen
  • Z' represents an amino acid residue in protected or unprotected form, a peptide, in protected or unprotected form, a protective group, or hydroxyl;
  • Y and Y' represent side chains of ⁇ -amino acid residues, (e.g., where the halo-peptide isostere is an isostere of phenylalanyl alanine, for example, Y is C 6 H 5 -CH 2 - and Y' is CH 3 -); and
  • the configuration of the olefin bond is preferably trans (E).
  • Compounds of formula IV may be in free form or in the form of their acid or base addition salts, or be in protected form at the N-terminus, the C-terminus or at both.
  • residues have chiral centers and halo-olefin isosteres of peptides having more than two amino acid residues are entirely novel.
  • phenylalanine-glycine are known, such isosteres have not previously been used in formulating MHC blocker peptides, therefore MHC blocker peptides incorporating these isosteres are novel.
  • the process for making these halo-olefin isosteres, disclosed herein, is novel and has the significant advantage that chirality of the amino acid residues is controlled, which is not possible using previously known methods.
  • cylic tripeptides of the invention in free acid form, are of formula V:
  • A is a linear C 2-4 alkylene group, which may be interrupted by an oxygen atom
  • B is the side chain of an oamino acid, preferably of phenylalanine (i.e., benzylmethyl) or alanine (i.e., methyl).
  • A is the group -(CH 2 ) 3 - and B is methyl
  • the peptide of formula V is the tripeptide Lys-Ala-Glu in which a further peptide bond has been formed between the ⁇ -amino group of the lysine residue and the Y-carboxy group of the glutamic acid residue.
  • the compound of formula V may be in free form or in the form of its acid or base addition salts, or be in protected form at the N-terminus, the C-terminus or at both.
  • novel MHC antagonist compounds described herein are small peptides having eight to seventeen residues of amino acids, and comprising one or more of the novel residues of Formulas I, II, III, IV, or V, arranged from the N-terminus to the C-terminus according to Formula VI:
  • R 1 is a series of from zero to three amino acid residues
  • P is a hydrophobic L-amino acid residue or, where NX and R 1 are not present, may be a hydrophobic carboxylic acid residue;
  • R 2 is a series of three to five L-amino acid residues
  • T is a an amino acid residue selected from the group containing
  • R3 is a series of two to five amino acid residues
  • CX is a residue of an amino acid, an amino acid amide, or an amino alcohol.
  • the preferred MHC blocker peptides of the invention are those where NX, R 1 , P, R 2 , T, R 3 , and CX of Formula VI are, independently, as follows:
  • NX represents an N-terminal protective group, a residue of a D-amino acid, a residue of an amino acid in protected form, or is not present.
  • An N-terminal protective group is defined as a group which tends to protect peptides from degradation in vivo, including such groups as alkyls, cycloalkyls, aryls, arylalkyls, acyls, polyethylene glycols and polyethylene glycol carboxylic acids, pyroglutamic acid, succinyl, methoxy succinyl, benzoyl phenylacetyl, 2-, 3- or 4- pyridylalkanoyl, aroyl, alkanoyl (including acetyl and cycloalkanoyl), arylalkanoyl (including pyridyls), arylaminocarbonyl, alkylaminocarbonyl, cycloaminocarbonyl, alkyloxycarbonyl (e.
  • NX is preferably butyloxycarbonyl, fluorenylmethoxycarbonyl, or a residue of D-proline, D-tyrosine, D-alanine, polyethylene glycol carboxylic acid, O-triethyleneglycol serine monomethylether, AA-I, or
  • B 1 is a series of from zero to three amino acid residues, preferably L-amino acid residues. Where R 1 is present, it preferably contains residues of one or more of the following amino acids:
  • alanine, lysine, O-triethyleneglycol serine monomethylether AA-I, AA-II, or AA-III.
  • P is a hydrophobic L-amino acid residue selected from the group containing L-phenylalanine and hydrogenated analogues thereof
  • TMSA trimethylsilylalanine
  • P is a residue of a carboxylic acid
  • NX and R 1 are preferably not present.
  • R 2 is a series of any three to five L-amino acid residues.
  • R2 preferably contains four amino acids selected from the group containing glycine, L-threonine, L-alanine, L-lysine, amino- isobutyric acid, O-triethyleneglycol serine monomethylether, AA-I, AA-III, or contains the cyclic tripeptide residue of Formula V and one other L-amino acid.
  • the tripeptide is preferably adjacent to T, and the residue in R2 adjacent to P is preferably an L-amino acid, most preferably L-valine.
  • the cyclic tripeptide may also span R 1 -P-R 2 , e.g., as -Lys-Phe-Glu-, or -Orn-Phe-Glu-, and where this is the case, R 2 may contain an additional two to three residues, e.g., -Ala-Lys- or -Ala-Ala-Lys-.
  • T is a residue of L-threonine, L-serine, L-proline, or
  • L-hydroxyproline preferably L-threonine or L-trans-4-hydroxyproline, most preferably L-threonine.
  • R3 is a series of two to five amino acid residues, preferably L-amino acid residues, and most preferably residues of the L-forms of one or more of the following amino acids: alanine, leucine, lysine, phenylalanine, O-triethyleneglycol serine monomethylether, AA-I, or AA-III.
  • R2 comprises the cyclic tripeptide of Formula V
  • R3 is preferably Leu-Lys-Ala.
  • CX is a residue of an amino acid, an amino acid amide, or an amino alcohol, preferably L-alaninol, L-threoninol,
  • One or more amide bond may be replaced with halo-olefin groups at any position in the peptide.
  • the Phe or Cha portion of the isostere is P and the Ala portion is the first residue of R 2 .
  • peptide isostere refers to a moiety that i) conformationally and functionally serves as a substitute for two or more amino acid residues in a peptide of the present invention, permitting the interaction of the peptide with its MHC receptor, and ii) differs from a peptide in that one or more amide bonds is replaced with a bond or entity that conformationally and functionally serves as a substitute for an amide bond, such as the halo-olefin group described herein.
  • Peptide isosteres and peptides incorporating peptide isosteres may be synthesized and screened for effect on binding in a variety of assays, e.g., in a competition assay with an unsubstituted parent peptide.
  • assays e.g., in a competition assay with an unsubstituted parent peptide.
  • amino acid residues is considered to include the amino acid residue analogues in peptide isosteres, as veil as conventional amino acid residues linked by amide bonds.
  • peptide as used herein is broadly defined to include (i) such peptide isosteres, and (ii) peptides which are modified at the N- or C-terminals as described herein, as well as peptides comprising amino acid residues linked by amide bonds.
  • the peptides of the invention may be synthesized from natural or unnatural amino acids, using conventional methods of peptide synthesis or on an inert polymeric support peptide synthesizer, and purified using reversed-phase high-performance liquid chromatography (HPLC). The purity of the peptides may be confirmed by amino acid sequence and/or composition analysis using conventional means.
  • the N-terminal of the peptide may be capped by conventional means with the capping groups disclosed above.
  • the C-terminal of the peptide may be reduced to the amino alcohol or may be
  • the affinity of the novel peptides for the HLA-DR molecules and the stability of the peptides under physiological conditions may be assessed in a variety of ways, which are known to one skilled in the art, generally by labelling the peptide with a radionuclide, enzyme, fluorescer, or other labeller, and determining the specific binding activity to the MHC molecule in question. Stability may be measured in vitro as a half-life in minutes under physiologic conditions (e.g., in human serum at 37° C).
  • Binding to MHC The procedure used to determine relative specific activities such as those given in the Examples below is a time-resolved fluorescence assay which measures the relative binding activity of the novel peptides to human MHC HLA-DRl, -DR4w4, and -DR4w14, using essentially the following methods:
  • EBV-transformed homozygous cell lines are used as a source of DR molecules.
  • Cell lines are routinely monitored for DR expression by FACS analysis. Their DR types are confirmed by serological typing and RFLP analysis, using conventional methods.
  • Cell lines used are maintained in vitro by culture in RMPI 1640 medium, supplemented with 10% heat inactivated fetal calf serum or horse serum. Large quantities of cells are grown in 1 liter roller bottles. Cells are then lysed at a concentration of 10 8 cells/ml in PBS pH 7.2, containing 1% NP40, 5 mM sodium thoranadate, 25 mM iodoacetamide, and
  • DR molecules are purified using the monoclonal antibody LB3.1, covalently coupled to CNBr-Sepharose CL-4B.
  • Epstein-Barr virus transformed human B cell lines, homozygous at the DR locus, are used as a source of DR molecules.
  • Aliquots of cell lysates equivalent to approximately 5 grams of cells are passed sequentially through the following columns: Sepharose CL-4B (10 ml), protein A-Sepharose (5 ml), LB3.1-protein A-Sepharose (15 ml), using a flow rate of 30 ml/h.
  • the columns are washed with 10 column volumes of PBS, 1% n-octylglucoside, 0.05% NP40 (5 ml/hr); 2 column volumes of PBS and IX octylglucoside.
  • the DR is eluted with 0.05 M diethylamine in 0.15 M NaCl containing 1% octylglucoside and 0.05% NP40 (pH 10.5), immediately neutralized with
  • protease inhibitors may be, for example, as follows: 1 mM PMSF, 1.3 mM ortho-phenanthroline, 73 ⁇ M pepstatin A, 8 mM EDTA, 6 mM N-ethyl maleimide, and 200 uM N ⁇ -p-tosyl -L-lysine chloromethyl ketone
  • TLCK The final detergent concentration in the incubation mixture is 0.05% NP-40.
  • One or more of the peptides screened may be hydrophobic and require DMSO to maintain solubility and reduce peptide adsorption to surfaces. In these instances peptide stock solutions are prepared in neat DMSO, and the final DMSO concentration in the incubation mixture is adjusted to 5%. Control experiments may be performed to demonstrate that the IC 50% values obtained with or without DMSO are similar.
  • the DR-peptide complexes are separated from free peptide by use of a monoclonal anti-DR antibody coupled to microtiter plates.
  • the amount of peptide bound to the MHC is determined in the case of a biotin labelled assay by use of europium-labelled streptodine and time-resolved fluorescence, or by measurement of radiation in the case of a radio-labelled assay.
  • LK-2 An EBV positive, DR1+ homozygous typing cell line, LK-2, is washed 3X with Hank's Balanced Salt Solution (HBSS), suspended to 5 x 10 6 /ml in HBSS, and fixed with 0.5% p-formaldehyde (w/v) at room temperature for 20 minutes.
  • HBSS Hank's Balanced Salt Solution
  • the fixed LG-2 cells are washed IX with HBSS, washed 2X with RPMI media supplemented with L-glutamine, non-essential amino acids, sodium pyruvate, antibiotics, and 10% human sera type AB (Complete Media, CM), resuspended at 10 6 /ml in CM, and then plated as 100 ⁇ l into individual wells of a 96 well microtiter dish.
  • the fixed LG-2 cells are then pulsed simultaneously for 2 hours at 37°C with 50 ⁇ l of test peptide and 50 ⁇ l of stimulatory peptide which have been dissolved in CM.
  • the amount of stimulatory peptide added to each test well is constant and is calculated so as to yield 60-80% of the maximal T cell proliferative response.
  • the potential inhibitor peptide is evaluated in duplicate at several concentrations
  • the stimulatory peptide concentration usually 3X, 10X, 50X, and 250X.
  • the plates are centrifuged, and the media carefully aspirated and replaced with fresh medium. The plates are washed ' in this manner three times in order to ensure complete removal of unbound peptide.
  • 50 ⁇ l of CM remains in the well.
  • increasing concentrations of stimulatory peptide are assayed in the absence of inhibitory peptide in order to obtain dose-response curves for the stimulatory peptide and to ensure that the amount of stimulatory peptide used in test wells is truly limiting.
  • Antigen-specific T cells are washed 2X in CM, suspended to 2 x 10 5 /ml in CM, and plated as 150 ⁇ l into the wells containing the peptide-pulsed LG-2 cells. The cultures are incubated for three days at 37°C, and pulsed with 0.1 ⁇ Ci/well of 3 H-thymidine during the last 16 hours of culture. At the termination of the cultures the cells are harvested onto glass fiber and the amount of 3 H-thymidine, which has been incorporated by the responder T cell, determined using liquid scintillation counting.
  • each peptide is determined relative to a reference peptide of known activity. For each test peptide the amount of inhibition is plotted as a function of the inhibitor concentration, and this graph is used to determine a 50% inhibitory dose. The inhibitory activity is then expressed as the ratio of the 50% inhibitory dose of the standard peptide (determined in a parallel experiment) to that of the test peptide. These data are presented in the last three columns of Table 1. This inhibitory value constitutes an additional means by which the MHC binding peptides can be compared.
  • lymph nodes draining the injection site are removed and 4 x 10 5 cells are cultured in wells of microtiter plates in HL-1 medium supplemented with 2 mM L-glutamine, 50 ⁇ M 2-ME and 50 ⁇ M/ml gentamicin with the indicated antigen concentrations. Cultures are set up in triplicate from each mouse and from pooled lymph node cells, cultures are incubated for three days in a humidified atmosphere of 5% CO 2 in air and were pulsed 10 hours before
  • the half life of the peptides is determined using a 25% human serum assay.
  • the reaction medium is prepared by mixing 1 part human serum with 3 parts cell culture medium (RPMI) , vortexing the mixture, filtration through a 0.45 ⁇ filter, and incubation at 37°C for 20 min.
  • the test peptide (0.5mg) is dissolved in 50 ⁇ l DMSO, and 10 ⁇ l of this solution is diluted with 1.5ml of the reaction medium and left to stand at 37°C. Samples of 100 ⁇ l of the mixture are taken at intervals and added to 200 ⁇ l of ice-cold 6% aqueous trichloroacetic acid left to stand in ice for 15 min and finally centrifuged for 2 min.
  • the drug can be delivered parenterally, orally, nasally, topically, by aerosol, or
  • the drug is administered parenterally, in a sterile solution, preferably an aqueous solution such as water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid, or the like.
  • a sterile solution preferably an aqueous solution such as water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid, or the like.
  • the composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like.
  • the concentration of peptide in the solution may vary widely and will depend on the desired fluid volume, viscosity, etc., in accordance with the particular mode of administration selected.
  • the peptides of the invention may be combined with one another or with other chemotheraputic agents for increased efficacy.
  • the final solution should be sterile, sterilization being accomplished by well known conventional means, especially sterile filtration.
  • the solution may be packaged for use as is or may be lypholized for subsequent mixture with a sterile solution prior to administration.
  • a typical pharmaceutical composition for intravenous infusion could contain on the order of 5% peptide by volume in sterile Ringer's solution.
  • nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, or other pharmaceutically acceptable solid carrier.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active
  • oral administration is not generally suitable for peptides having only amide bonds, but may be preferred for certain peptide isoteres, e.g., the halo-olefin peptide isosteres of this invention.
  • the peptides of the invention are preferably supplied in finely divided form along with a surfactant and a propellant. Typical percentages of peptides are 0.01%-20% by weight, preferably 1%-10%.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Appropriate aerosol carriers will be well known to one skilled in the art.
  • compositions containing the peptides of the invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already suffering from an autoimmune disease, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications, this amount being defined as a therapeutically effective dose.
  • the therapeutically effective dose will depend on the severity and nature of the disease and on the weight and general state of the patient, but generally range from about 0.1 mg to about 2000 mg of peptide per day for a 70 kg patient, with dosages of from about 0.5 mg to about 1000 mg of peptide per day being more commonly used.
  • compositions containing the peptides of the invention are administered to a patient susceptible to or otherwise at risk of a particular autoimmune disease to enhance the patient's own immunoregulatory capabilities, in an amount defined top be a prophylactically effective dose.
  • the prophylactically effective dose again depends on the nature of the disease and .the patient's weight and general health, but generally ranges from about 0.1 mg to about 500 mg per 70 kilogram patient, more commonly from about 0.5 mg to about 200 mg per 70 kg of body weight.
  • compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • pharmaceutical formulations should provide a quantity of the peptide of the invention sufficient to effectively treat the patient.
  • the peptides may also find use as diagnostic reagents.
  • a peptide may be used to determine the susceptibility of a particular individual to a treatment regimen which employs the peptide or related peptides, and thus may be helpful in modifying an existing treatment protocol or in determining a prognosis for an affected individual.
  • cells bearing the selected MHC antigen e.g., leukocytes, will be obtained from an individual, incubated in vivo with the peptide(s), an immunostimulatory peptide, and a collection of T cells from the same individual.
  • proliferation of T cells can then be assayed in the presence or absence of the immunomodulatory peptide or peptide mixture.
  • Peptides of the invention may be synthesized by any conventional method of peptide synthesis. Solid state synthesis is preferred, and suitable methods may be found in any standard textbook on peptide synthesis, e.g., as described by Atherton & Shephard, "Solid Phase Peptide Synthesis, a Practical Approach", IRL Press, 1989.
  • the resin is washed three times with DMF and methanol. A ninhydrin test establishes that the coupling is complete. The resin is then pondered with DMF, deprotected with 50% piperidine in DMF and the next Fmoc-amino acid or isostere in DMF added. This procedure is repeated until the desired sequence had been assembled.
  • the peptide is cleaved from the resin by treatment with 2.0ml trifluoroacetic acid and 0.1ml water for 90 minutes at room
  • the peptide is precipitated by adding 30ml diethyl ether and centrifuged. A further 30ml diethyl ether is added and this procedure repeated four times. The peptide is stirred with 30ml ethyl acetate / diethyl ether (1.5:1),
  • the peptide is purified using HPLC (250 x 21.4mm Dynamax column RP-18, 12 ⁇ M Spherical 300A, gradient 90% acetronitrile / 10% water / 0.1% trifluoroacetic acid increasing to 25% acetontrile / 75% water / 0.1% trifluoroacetic acid), the fractions collected (UV detector 200nm) and lyophillized.
  • HPLC 250 x 21.4mm Dynamax column RP-18, 12 ⁇ M Spherical 300A, gradient 90% acetronitrile / 10% water / 0.1% trifluoroacetic acid increasing to 25% acetontrile / 75% water / 0.1% trifluoroacetic acid
  • the peptide is then converted to its acetate by dissolving in 10% aqueous acetic acid and passing through a Bio Rad A G4-X4 column.
  • the fractions are collected, lyophillised, and analysed by standard procedures.
  • the peptide may be protected and deprotected by standard means.
  • the N-terminus may be protected by standard caps or may be a carboxylic acid rather than an amino acid, as described elsewhere herein.
  • C-terminus modifications they may be performed after the peptide is synthesized, or, alternatively, the initial starting resin may be varied.
  • the above starting resin yields alanylamide at the C-terminus.
  • the above synthesis is performed starting with 187mg.
  • threoninol-p-benzyloxybenzylalcohol resin (Kilo-labor) instead of the 4-(2',4'-dimethoxyphenyl-Fmoc-amino- methyl)-phenoxy resin.
  • AA-I may be synthesized by reacting a compound of formula VII with a compound of formula VIII CH 2 -OH
  • the hydrochloride obtained above is dissolved in a mixture of 160ml water and 100ml dioxane containing 16g sodium hydroxide, the solution cooled to 0-5°C, and slowly treated with llg FMOC-chloride.
  • the reaction mixture is allowed to warm to room temperature over 3hr, acidified with cone, hydrochloric acid, and extracted with ethyl acetate.
  • the crude product is purified by chromatography using as mobile phase 6:4 ethyl acetate/hexane acidified by shaking with 5% cone, hydrochloric acid. The yield of pure product is 5.6g.
  • AA-II may be produced by reacting 2-methoxy-4,6-dichloro- pyrimidine of formula IX with alanine t-butyl ester of formula X
  • the carboxy group of AA-II may be linked to an amino acid benzyl ester, preferably phenylalanine benzyl ester, then the benzyl group may be removed to permit linkage to the remaining peptide chain.
  • the process is substantially as follows: a) N-[6-(2-methoxy-4-chloropyrimidyl)]alanine t-butyl ester
  • step b) in the form of the DCHA salt, (5g, 12.25 mmol) is dissolved in 250 ml dichloromethane. To this solution is added at 0°C L-phenylalanine benzyl ester in free base form (3.3g, 13 mmol) dissolved in 50 ml dichloromethane, together with BOP-Cl (3.4g, 13.5 mmol), and the mixture is stirred at room temperature overnight. The precipitate of BOP DCHA salt is filtered off, and the filtrate is diluted with ethyl acetate and washed first with potassium
  • step c) The product of step c) (4.5g, 9.7 mmol) is hydrogenated in 200 ml ethanol at room temperature in the presence of 0.5g of a 10% Pd/C catalyst. After one hour no further hydrogen is taken up. The reaction mixture is filtered through Hyflo/talc and evaporated to give 3g of an oily residue. Chromatographic separation on 440g
  • Kieselgel (0.040 - 0.063 mm) using as eluent a mixture of
  • AA-III may be prepared by the following reaction scheme:
  • This reaction may be carried substantially as follows : a) BOC-(O-allyl)serine (Compound of formula XIII)
  • step (a) (19 g, 0.077 mol) is dissolved in 400 ml toluene, and warmed to 90°. N,N-dimethylformamide dibenzyl acetal (55ml, 0.19 mol) is added dropwise over 45 min. to give a dark yellow solution, which is stirred for 1 hr at 90°, then evaporated to give a brown oil which is then purified by chromatography on kieselgel to give the title product as a yellow liquid (20g) . [ ⁇ ] D 20 - -5.34° (1% in CH 2 Cl 2 ) c) BOC-(O-2,3-epoxypropyl)serine benzoate (Compound of Formula XV)
  • step (b) The product of step (b) (18g, 53.7 mmol) is dissolved in 200 ml methylene chloride and m-chloroperbenzoic acid (15.4g, 80 mmol) is added. The resulting colourless solution is stirred overnight under argon, to give a white suspension which is filtered, evaporated, and the residue taken up in ether. The ether solution is washed with sodium bicarbonate solution then with brine, then dried and
  • step (d) The product of step (d) (8.0 g, 21 mmol) is dissolved in 50 ml of a 1:4 mixture of trifluoroacetic acid/methylene chloride and stirred 5 hr at room temperature. Evaporation and washing with toluene gives the title product as a yellow oil (7.5 g).
  • the product may be deprotected, reprotected and coupled by standard procedures.
  • reaction scheme illustrates, by way of example, synthesis routes for chloro- and fluoro-olefin dialanine isosteres in protected form, and subsequent incorporation at various points on an MHC blocker peptide:
  • Analogous processes may be used to produce different distereoisomers, different amino acid residue combinations, and different MHC blocker peptides within the scope of the invention. Isosteres of other dipeptides may be synthesized and incorporated into peptides by analogous procedures.
  • step b) 1.379g of the product of step b) is dissolved in 6ml methylene chloride, cooled to 0°C and 98mg l,8-diazabicyclo[5.4.0]undec-7- en(l,5-5) added. This solution is added over ten minutes to a solution of 0.681g trichloroacetonitrile in 2ml methylene chloride at the same temperature. After stirring for 45 minutes at 0°C the solvent Isevaporated, the residue dissolved in diethyl ether and chromatographed through silca gel using 10% diethyl ether / hexane to elute the product. This is dissolved in 25ml o-xylene and heated to reflux (ca.
  • the mixture is stirred at -78°C for one hour and then at 0°C for two hours when the boronic complex is decomposed by treatment with 35ml phosphate buffer (pH 7), 120ml methanol, and 35ml 30% hydrogen peroxide for thirty minutes.
  • the aqueous phase is extracted with methylene chloride, the organic layer washed with ice cold 1N sodium sulphite followed by water, dried over Na 2 SO 4 , and evaporated to give a colourless oil. This is chromatographed over silica gel using 10 to 50% diethyl ether in hexane to elute the product, which is then crystallised from diethylether / hexane.
  • 6.561g of the product of step a) is dissolved in 30ml methylene chloride, cooled to 0°C and 0.43ml 1,8-Diazabicyclo[5.4.0]undec-7- en(l,5-5) added. This mixture is added over ten minutes to a solution of 3.08g trichloroacetonitrile in 10ml methylene chloride at the same temperature. After stirring for 45 minutes at 0°C, the solvent is evaporated, the residue dissolved in diethyl ether and rapidly filtered through 2x7cm silca gel with methylene chloride / hexane to elute the product.
  • step c) The product of step c) (5.601g) is dissolved in 170ml tetrahydrofuran, cooled to 0°C, and treated with a solution of 985mg lithium hydroxide in 56ml water for five minutes followed by 5.32ml 30% hydrogen peroxide. After sirring the cloudy solution for 45 minutes at 0°C, the hydrogen peroxide is decomposed with a solution of 5.91g sodium sulphite in 25ml water, the mixture extracted with toluene followed by diethyl ether, the aqueous layers are acidifed with IN HCl and the product extracted with ethyl acetate.
  • step c) The product of step c) (2.15g) is dissolved in 40ml ethanol, cooled to 0°C and stirred overnight with 33ml 6N NaOH under argon. The solvent is evaporated, the residue dissolved in 40ml water, the pH brought to 5 with 12N HCl and a solution of 2.52g 9-Fluorenylmethyl- succinimidyl carbonate in 50ml dioxan added. The mixture is stirred overnight at 0°C under argon, acidified with 2N HCl, and the organic product extracted with ethyl acetate. After washing to pH 7 the organic phase is dried (Na 2 SO 4 ), evaporated, and chromatographed over silica gel. The required product is eluted with 10% ethyl acetate in toluene and triturated with hexane to give a foam.
  • a solution of 50ml dibutylboryl-trifluoromethanesulphonate (1M in methylene chloride) is added to a mixture of 11.6g of (4R)-3-(1- Oxopropyl)-4-phenylmethyl-2-oxazolidinone in 75ml methylene chloride and 10.2ml N-ethyldiisopropylamine at 0°C over thirty minutes. After sixty minutes at this temperature, the orange solution is cooled to -78°C and a mixture of 55ml.
  • diethyl aluminium chloride (1.8M in toluene) in 50ml methylene chloride and 8.81g (Z)-2-Fluoro-but-2-enal at -78°C is added during ten minutes. This is stirred at -78°C for one hour when 150ml phosphate buffer (pH 7) followed by 200ml diethyl ether is added. The aqueous phase is extracted with diethyl ether, the organic layer evaporated and the residue dissolved in 200ml methanol. This is treated with 50ml 30% hydrogen peroxide diluted with 200ml water for one hour, diluted with 200ml water evaporated to dryness at room temperature.
  • This intermediate is dissolved in 28ml o-xylene and added to 500ml refluxing o-xylene and the mixture refluxed for approximately three hours until the starting material has disappeared (TLC, absorption at 1660cm -1 ). The solvent is evaporated, and the residue dissolved in ethyl acetate and chromatographed over silica gel (2 x 8cm.). The required product is eluted with ethyl acetate and
  • step c) The product of step c) (1.787g) is dissolved in 40ml ethanol and stirred overnight under argon with 29.2ml 6N NaOH. The solvent is evaporated, the residue dissolved in 40ml water, the pH brought to 9 and a solution of 1.97g 9-Fluorenylmethyl-succinimidyl carbonate in 50ml dioxan added. The mixture is stirred overnight at room
  • This intermediate is dissolved in 15ml o-xylene and added to 300ml refluxing o-xylene and then refluxed for approximately three hours until the starting material has disappeared (TLC, absorption at 1660cm -1 ). The solvent is evaporated, the residue dissolved in diethyl ether / hexane and chromatographed over silica gel. The required product is eluted with diethyl ether to give an oil.
  • Butyl lithium in hexan (1.6M) is added during 70 minutes to a solution of 62g (4S)-4-phenylmethyl-2-oxazolidinone in 1.51 tetrahydrofuran at -78°C until the colour changes from bright yellow to dark yellow, the mixture stirred for 30 minutes at this
  • Boc-Lys(Z)-Ala-Glu(0Bzl)-0Me (0.7 g, 1 mmol), prepared in fully protected form by standard peptide synthesis, is dissolved in 30 ml of methanol and hydrogenated over 10 mg Pd/C for two hours. The catalyst is removed by filtration and the solvent evaporated in vacuo to obt0in ca. 0.4 g of the title compound.
  • Boc-Lys-Ala-Glu-OMe (3.1 g, 6.7 mmol) suspended in 2.5 1 of dichloromethane is treated with triethylamine (3.6 ml, 26 mmol) and benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophos- phate (5.8 g, 14 mmol). The mixture is stirred for 48 hours at 25° C. The solvent is removed in vacuo and the residue triturated twice with 200 ml of diethyl ether. The solid is filtered and chromatographed over silica gel eluting with ethyl acetate/methanol (9:1) to obtain ca. 2.7 g of the title compound.
  • Cyclic(3-1)-Boc-Lys-Ala-Glu (1,2 g, 2.8 mmol) is dissolved in 10 ml of dichloromethane/trifluoroacetic acid (1:1). After two hours at 20° C, the solvents are removed under reduced pressure. The residue is dissolved in 20 ml of 10% sodium carbonate and added to a solution of FM0C-ONSU (0.95 mg, 2.8 mmol) in 40 ml of tetrahydrofuran. The reaction mixture is stirred overnight at 20° C. The pH is adjusted to 6.9 with citric acid and the organic solvent is removed under reduced pressure. The precipitate is filtered , washed with water and triturated twice with ethyl acetate to obtain ca. 1.2 g of the title compound. MS :(FAB) 551 (MI+H).
  • Boc-Lys(Z)-Ala-Glu(0Bzl)-0Me (1g), prepared in fully protected form by standard peptide synthesis, is suspended in 40 ml of methanol and hydrogenated by 4 bars over 100 mg Pd/C for two hours at r.t.. The catalyst is removed by filtration and the solvent evaporated in vacuo to obtain ca. 100% of the title compound.
  • Boc-Lys-Phe-Glu-OMe (470 mg, 0.877 mmol) suspended in 40 ml of dichloromethane and triethylamine (360 mg, 3.56 mmol) is treated with a solution of benzotriazol-1-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate (780 mg) in 180 ml dichloromethane in 5 portions until 1 hour by r.t.. The mixture in vacuo and the residue is chromatographed over 70 g silicagel eluting with ethyl
  • Example 13 Peptides incorporating novel cyclic tripeptides
  • Example 13 Cyclic(5-3)-Boc-Phe-Ala-Lys-Ala-Glu Cyclic(3-l)-Boc-Lys-Ala-Glu-0Me (1.2 g, 2.7 mmol) is stirred in 12 ml of dichloromethane/trifluoroacetic acid (1:1) at 20° C for three hours. The solvents are evaporated and the residue is
  • N,N-dicyclohexylcarbodiimide (0.87 g, 4.2 mmol) and Boc-Phe -Ala-OH (1.1 g, 3.1 mmol).
  • the reaction mixture is stirred overnight at 20° C and filtered.
  • the solid is triturated 3x with methanol (5 ml), washed with dichloromethane and dried to obtain ca. 1.1 g of
  • Cyclic (3-1)-Boc-Lys-Phe-Glu-0Me (2.85 mmol) is dissolved in a few volume of ether abs. and stirred with a satured solution (20 ml) of HCl in ether for 2 hours (controled by TLC). The mixture is evaporated in vacuo and the residue is removed and evaporated with 50 ml ether. The final residue is dried over potassium hydroxide in vacuo for 2 hours.
  • Cyclic (4-2)-Boc-Pro-Orn-Glu-OMe (340 mg) is dissolved in 15 ml of tetrahydrofuran and treated with 2N sodium hydroxide (0.5 ml,
  • Example 10(d) The product of Example 10(d) is used in a standard solid-phase peptide synthesis as described, e.g., in Example 1, supra.
  • the title compound in which -Orn- is ornithine (i.e., the 7-5 cyclic tripeptide is a residue of formula V where A is -CH 2 -CH 2 -) may be produced by solid phase
  • the compound of example 12 is used in a standard solid-phase peptide synthesis as described in example 11 and the structure
  • AA-III or the halo-olefins of formula IV may be produced by
  • a variety of novel blocker peptides can be synthesised using the methods described above. Table I lists examples such peptides. Table II shows their MHC binding affinity relative to native peptides with known MHC binding activity. Table III shows the ability of the novel blockers to inhibits MHC restricted antigen dependent T-cell proliferation.
  • BOC is butyloxycarbonyl
  • PEG is heptaethyleneglycol carboxylic acid monomethyl ether
  • TES is O-triethyleneglycol serine monomethyl ether
  • CHP is cyclohexylpropanoic acid
  • Aib is L-aminoisobutyric acid
  • TMSA is trimethylsilyl alanine
  • Cha is cyclohexylalanine
  • I a is the L-form of the amino acid of formula I where m is zero, n is two and R a is methyl,
  • I b is the L-form of the amino acid of formula I where m is one, n is two and R a is isopropyl,
  • III is the L-form of the amino acid of formula III
  • OH at C-terminus signifies that the carboxy has been reduced to an alcohol.
  • H signifies an unmodified N-terminus
  • (7-5) indicates a that the side chains of the seventh and fifth residues, counting from the N-terminus, are linked; (4-2) indicates linkage of the fourth and second residues.
  • Peptide A has the sequence:
  • Binding affinity for DR4w14 is relative to native peptide
  • Peptide B 1. K D of peptide B is approximately 67 nM.
  • Peptide B has the sequence: Tyr-Ala-Arg-Phe-Gln-Ser-Gln-Thr-Thr-Leu-Lys-Gln-Lys-Thr. Results are given where available for both biotin-labelled and radio-labelled ( 125 I) assays, as described above.

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Abstract

Des peptides nouveaux qui comprennent un nouvel acide aminé, des isostères dipeptides halo-oléfiniques, et/ou des tripeptides cycliques, sont utiles pour le diagnostic, la prophylaxie, le traitement et le suivi de maladies auto-immunes.
PCT/EP1992/001995 1991-08-29 1992-08-28 Nouveaux immunosuppresseurs WO1993005011A1 (fr)

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EP0610743A1 (fr) * 1993-02-09 1994-08-17 Bayer Corporation Nouveaux aminométhylène-peptides immunosuppressifs
WO1996040753A1 (fr) * 1995-06-07 1996-12-19 G.D. Searle & Co. Derives d'acide pipecolique obtenus a partir d'amides de threoline et de proline utiles dans le traitement de l'arthrite rhumatoide
EP0735893A4 (fr) * 1993-09-14 1998-02-04 Cytel Corp Alteration de la reponse immunitaire a l'aide de peptides se liant a des alleles pan dr
WO1998025951A1 (fr) * 1996-12-12 1998-06-18 Zeneca Limited Inhibiteurs peptides se liant a des proteines cmh de classe ii
US5965532A (en) * 1996-06-28 1999-10-12 Trustees Of Tufts College Multivalent compounds for crosslinking receptors and uses thereof
US6087336A (en) * 1996-02-23 2000-07-11 Zeneca Limited Peptide derivatives useful in treating autoimmune diseases
US6100234A (en) * 1997-05-07 2000-08-08 Tufts University Treatment of HIV
US6162828A (en) * 1995-03-31 2000-12-19 Takeda Chemical Industries, Ltd. Cysteine protease inhibitor
US6207644B1 (en) 1996-10-19 2001-03-27 Zeneca Limited Peptide analogues containing a 7-membered lactam ring
US6258597B1 (en) 1997-09-29 2001-07-10 Point Therapeutics, Inc. Stimulation of hematopoietic cells in vitro
US6300314B1 (en) 1998-05-04 2001-10-09 Point Therapeutics, Inc. Hematopoietic stimulation
US6355617B1 (en) 1996-11-27 2002-03-12 Syngenta Limited Peptide derivatives
US6355614B1 (en) 1998-06-05 2002-03-12 Point Therapeutics Cyclic boroproline compounds
US6413935B1 (en) 1993-09-14 2002-07-02 Epimmune Inc. Induction of immune response against desired determinants
US6541453B2 (en) 1996-06-07 2003-04-01 Syngenta Limited Peptide derivatives
US6692753B2 (en) 1997-05-07 2004-02-17 Trustees Of Tufts College Potentiation of the immune response
US6825169B1 (en) 1991-10-22 2004-11-30 Trustees Of Tufts College Inhibitors of dipeptidyl-aminopeptidase type IV
US6890904B1 (en) 1999-05-25 2005-05-10 Point Therapeutics, Inc. Anti-tumor agents
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US7230074B2 (en) 1991-10-22 2007-06-12 Trustees Of Tufts College Inhibitors of dipeptidyl-aminopeptidase type IV
US6825169B1 (en) 1991-10-22 2004-11-30 Trustees Of Tufts College Inhibitors of dipeptidyl-aminopeptidase type IV
EP0610743A1 (fr) * 1993-02-09 1994-08-17 Bayer Corporation Nouveaux aminométhylène-peptides immunosuppressifs
EP0735893A4 (fr) * 1993-09-14 1998-02-04 Cytel Corp Alteration de la reponse immunitaire a l'aide de peptides se liant a des alleles pan dr
US7202351B1 (en) 1993-09-14 2007-04-10 Pharmexa Inc. Alteration of immune response using pan DR-binding peptides
US6413935B1 (en) 1993-09-14 2002-07-02 Epimmune Inc. Induction of immune response against desired determinants
US6162828A (en) * 1995-03-31 2000-12-19 Takeda Chemical Industries, Ltd. Cysteine protease inhibitor
WO1996040753A1 (fr) * 1995-06-07 1996-12-19 G.D. Searle & Co. Derives d'acide pipecolique obtenus a partir d'amides de threoline et de proline utiles dans le traitement de l'arthrite rhumatoide
US6087336A (en) * 1996-02-23 2000-07-11 Zeneca Limited Peptide derivatives useful in treating autoimmune diseases
US6541453B2 (en) 1996-06-07 2003-04-01 Syngenta Limited Peptide derivatives
US5965532A (en) * 1996-06-28 1999-10-12 Trustees Of Tufts College Multivalent compounds for crosslinking receptors and uses thereof
US6875737B1 (en) 1996-06-28 2005-04-05 Trustees Of Tufts College Multivalent compounds for crosslinking receptors and uses thereof
US6207644B1 (en) 1996-10-19 2001-03-27 Zeneca Limited Peptide analogues containing a 7-membered lactam ring
US6355617B1 (en) 1996-11-27 2002-03-12 Syngenta Limited Peptide derivatives
US6184207B1 (en) * 1996-12-12 2001-02-06 Zeneca Limited Inhibitors of peptide binding to MHC class II proteins
WO1998025951A1 (fr) * 1996-12-12 1998-06-18 Zeneca Limited Inhibiteurs peptides se liant a des proteines cmh de classe ii
US6503882B2 (en) 1997-05-07 2003-01-07 Trustees Of Tufts College Treatment of HIV
US6692753B2 (en) 1997-05-07 2004-02-17 Trustees Of Tufts College Potentiation of the immune response
US6100234A (en) * 1997-05-07 2000-08-08 Tufts University Treatment of HIV
US6258597B1 (en) 1997-09-29 2001-07-10 Point Therapeutics, Inc. Stimulation of hematopoietic cells in vitro
US7276371B2 (en) 1997-09-29 2007-10-02 Point Therapeutics, Inc. Stimulation of hematopoietic cells in vitro
US6703238B2 (en) 1997-09-29 2004-03-09 Point Therapeutics, Inc. Methods for expanding antigen-specific T cells
US6770628B2 (en) 1998-05-04 2004-08-03 Point Therapeutics, Inc. Hematopoietic stimulation
US7067489B2 (en) 1998-05-04 2006-06-27 Point Therapeutics, Inc. Hematopoietic stimulation
US6300314B1 (en) 1998-05-04 2001-10-09 Point Therapeutics, Inc. Hematopoietic stimulation
US6355614B1 (en) 1998-06-05 2002-03-12 Point Therapeutics Cyclic boroproline compounds
US6949514B2 (en) 1999-05-25 2005-09-27 Point Therapeutics, Inc. Anti-tumor agents
US6890904B1 (en) 1999-05-25 2005-05-10 Point Therapeutics, Inc. Anti-tumor agents
US7259138B2 (en) 1999-05-25 2007-08-21 Point Therapeutics, Inc. Anti-tumor agents
US7282484B2 (en) 1999-05-25 2007-10-16 Point Therapeutics, Inc. Anti-tumor agents
RU2334760C2 (ru) * 2002-03-22 2008-09-27 Гпц Биотек Аг Иммуносупрессоры

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