Classifications

• • 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
  • A61K39/35—Allergens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/415—1,2-Diazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants

Definitions

• the invention relates generally to methods for modulating the immune system of mammals and more particularly to methods of modulating the immune system using compositions comprising histamine derivatives.
• Histamine is a small molecule that has been shown to have a significant role in the immune response process in mammals. However, its ubiquitous effects on many cells that have receptors for histamine limits its possible immunotherapeutic use. Histamine derivatives that are tissue directed or effect specific would significantly aid in determining the role of histamine in immune modulation and produce valuable immunotherapeutics.
• Histamine can substantially modulate models of immune responses in mammals, particularly models of delayed hypersensitivity and T and B cell functions. Histamine is synthesized during different phases of response to antigen and is able directly or indirectly to effect further responses to antigen. It is possible that the concentration of histamine in tissue during inflammation and immune response can modify the fanction of a number of lymphoid cells. Although these effects may be substantial, the direct effect on single cell types in a mixture of cells cannot be determined unless the agonists are effect or cell specific. Ubiquitous effects of agonists on all cells that have receptors for histamine would limit any
• Histamine is an autacoid as are catecholamines, prostaglandins and some peptides. e.g., bradykinin and probably lymphokines.
• Autacoids differ from hormones in that they are made at their local sites of action and they can be made in a variety of tissues. Autacoids play an important role in mediating inflammation. During inflammation, certain events may occur which include: protein denaturation. lowering of local pH, release of "new peptides" and lysosomal enzymes, and the like.
• Autacoids affect natural suppressor cells, T cell subsets and B cells during various stages of immunity. Receptors for autacoids are non-randomly distributed
• Precursor B cells do not appear to have histamine and catecholamine receptors, while B cells committed to produce antibodies do.
• T suppressor (T s cells modulate the
• T helper T h
• T cytolitic T c
• Mitogens alter responsiveness of these cells to histamine.
• Some lymphocytes that respond to histamine have both H 1 and H 2 receptors on them while others have only H 2 receptors. In some lymphocytes the H 2 receptors seem to modify the responses to H 1 agonism, in others there is no such interplay.
• biologic response is inhibitory (e.g., reduced release of antibody from B cells: inhibition of lymphokine release or lysis of target cells by T effector cells and inhibition of release of histamine from mast cells); in others the response enhances immune function (e.g., enhanced suppression by natural suppressor and T s cells or T helper (T h ) cell proliferation.
• inhibitory e.g., reduced release of antibody from B cells: inhibition of lymphokine release or lysis of target cells by T effector cells and inhibition of release of histamine from mast cells
• the response enhances immune function (e.g., enhanced suppression by natural suppressor and T s cells or T helper (T h ) cell proliferation.
• autacoids seem to be enhancing selected early events in immune response (e.g., enhanced suppressor function) while inhibiting later phases of phenotypic
• manifestations e.g., release of lymphokines or antibodies of immunity.
• the natural suppressor cells appear briefly during the early maturation of lymphoid tissue but can be induced in adults by total lymphoid irradiation.
• the cells have the unique feature of inhibiting the antigen specific cytolytic arm of alloreactive immune response but leave the antigen-specific suppressive arm intact.
• alloreactions in the regulatory milieu of natural suppressor (NS) cells generate large numbers of antigen-specific suppressor cells that in turn maintain tolerance in vivo.
• the natural suppressor cells may play an important role in preventing the development of host versus graft and graft versus host diseases in allogenic bone marrow chimeras, and in immune tolerances in the neonatal and total lymphoid irradiated (TLI) mice.
• Histamine activates human T s cells and enhances the suppressive ability of murine NS cells in vitro. See, Khan et al., J. Immunol. (1985) 134:4100 and Sansoni et al., J. Clin. Invest. (1985) 75:650. After pretreatment of both human T s cells
• the present invention provides methods for using histamine derivatives as immunomodulators and in immunotherapeutics.
• One embodiment of the present invention provides methods for inhibiting at least a portion of an antigen specific antibody response by the immune system of a mammal comprising administering to the mammal an effective amount of a composition comprising at least one histamine derivative having binding specificity for at least one histamine receptor and in conjunction therewith,optionally administering a predetermined antigen or immunogenic portion thereof.
• the invention also provides methods of treating sensitivity to a particular antigen in an individual by administering to the individual a therapeutically effective amount of a composition comprising at least one histamine derivative having binding specificity for at least one histamine receptor, and a pharmaceutically acceptable carrier or diluent.
• the histamine derivative is administered in conjunction with a predetermined antigen or an immunogenic portion thereof to which the individual is sensitive.
• the histamine derivative is administered to the individual in conjunction with a peptide having T- cell stimulating activity derived from a predetermined antigen such as a protein allergen or autoantigen to which the individual may be sensitive.
• the histamine derivative is administered to an individual in conjunction with both a predetermined antigen or immunogenic portion thereof to which an individual may sensitive, and a peptide having T-cell stimulating activity derived from the same predetermined antigen.
• Fig. 1a-c is a graphic representation depicting the results of an ELISA assay showing anti Fel d I IgG antibody response in three groups of 6 mice which had been treated with either saline (PBS) (Fig. 1a) or 50 mg/kg Compound 1 (Fig. 1b) or 100 mg/kg
• Compound 1 (Fig. 1c) one day before treatment with the Fel d I antigen (day-1) or 2 days after Fel d I antigen treatment (day+2).
• Fig. 2a-c is a graphic representation depicting the results of an ELISA assay showing IgE of anti-Fel d I antibody response in three groups of 6 mice which had been treated with either saline (PBS) (Fig. 2a) or 50 mg/kg Compound 1 (Fig.2b) or 100 mg/kg
• Compound 1 (Fig. 2c) one day before treatment with the Fel d I antigen (day-1) or 2 days after Fel d I antigen treatment (day+2).
• Fig. 3a-b is a graphic representation depicting the results of an ELISA assay showing the IgG anti Fel d I antibody response in 2 groups of 6 mice which had been treated with either saline (PBS) (Fig. 3a) or 50 mg/kg Compound 1 (Fig.3b) one day before treatment with the Fel d I antigen (day-1) or 2 days after Fel d I antigen treatment (day+2).
• PBS saline
• Compound 1 Fig.3b
• Fig.4a-b is a graphic representation depicting the results of an ELISA assay showing the IgE an ⁇ -Fel d I antibody response in 2 groups of 6 mice which had been treated with either saline (PBS) (Fig. 4a) or 50 mg/kg Compound 1 (Fig.4b) one day before treatment with the Fel d I antigen (day-1) or 2 days after Fel d I antigen treatment (day+2).
• PBS saline
• Compound 1 Fig.4b
• Fig. 5a-d is a graphic representation depicting the results of an ELISA assay showing the IgG anti Fel dl antibody response in 4 groups of 6 mice which had been treated with either saline (PBS) (Fig. 5a) or 50 mg/kg Compound 1 (Fig. 5b) or 5 mg/kg Compound 1 (Fig. 5c) or 0.5 mg/kg Compound 1 (Fig. 5d) one day before treatment with the Fel d I antigen (day-1) or 2 days after Fel d I antigen treatment (day+2).
• PBS saline
• Fig. 6a-d is a graphic representation depicting the results of an ELISA assay showing the IgE anti Fel d I antibody response in 4 groups of 6 mice which had been treated with either saline (PBS) (Fig. 6a) or 50 mg/kg Compound 1 (Fig. 6b) or 5 mg/kg Compound 1 (Fig. 6c) or 0.5 mg/kg Compound 1 (Fig. 6d) one day before treatment with the Fel d I antigen (day-1) or 2 days after Fel d I antigen treatment (day+2).
• PBS saline
• Fig. 6b 50 mg/kg Compound 1
• Fig. 6c Compound 1
• Fig. 6d 0.5 mg/kg Compound 1
• Fig. 7a-b is a graphic representation depicting the results of an ELISA assay showing the IgG anti Fel d I antibody response in 2 groups of 6 pre-primed mice which had been treated with either saline (PBS) (Fig. 7a) or 50 mg/kg Compound 1 (Fig. 7b) one day before 2° (secondary boost of antigen) treatment with the Fel d I antigen (day-1) or 2 days after 2° Fel d I antigen treatment (day+2).
• PBS saline
• Compound 1 Fig. 7b
• Fig. 8a-b is a graphic representation depicting the results of an ELISA assay showing the IgE anti Fel d I antibody response in 2 groups of 6 pre-primed mice which had been treated with either saline (PBS) (Fig. 8a) or 50 mg/kg Compound 1 (Fig. 8b) one day before 2° treatment with the Fel d I antigen (day-1) or 2 days after 2° Fel d I antigen treatment (day+2).
• PBS saline
• Compound 1 Fig. 8b
• Fig. 9a-d is a graphic representation depicting the results of an ELISA assay showing the IgG anti Fel d I antibody response in 4 groups of 6 pre-primed mice which had been treated with either saline (PBS) (Fig. 9a) or 50 mg/kg Compound 1 (Fig. 9b) or 75 mg/kg Compound 1 (Fig. 9c) or 100 mg/kg Compound 1 (Fig. 9d) one day before 2° treatment with the Fel d I antigen (day-1) or 2 days after 2° Fel d I antigen treatment (day+2).
• PBS saline
• Fig. 9b 50 mg/kg Compound 1
• Fig. 9c 75 mg/kg Compound 1
• Fig. 9d 100 mg/kg Compound 1 (Fig. 9d) one day before 2° treatment with the Fel d I antigen (day-1) or 2 days after 2° Fel d I antigen treatment (day+2).
• Fig. 10a-d is a graphic representation depicting the results of an ELISA assay showing the IgE anti Fel d I antibody response in 4 groups of 6 pre-primed mice which had been treated with either saline (PBS) (Fig. 10a) or 50 mg/kg Compound 1 (Fig. 10b) or 75 mg/kg Compound 1 (Fig. 10c) or 100 mg/kg Compound 1 (Fig. 10d) one day before 2° treatment with the Fel d I antigen (day-1) or 2 days after 2° Fel d I antigen treatment (day+2).
• Fig. 11. is a graphic representation of the results of a total IgG assay performed on the four groups of 6 mice discussed in Fig. 9 to determine whether there was any change in IgG in mice that received 100 mg/kg, 75 mg/kg, or 50 mg/kg dosages of Compound 1 compared to the saline control group.
• Fig. 12a-c is a graphic representation depicting the results of an ELISA assay showing the IgG anti Fel d I antibody response in three groups of 6 mice which had been treated with either saline (PBS) (Fig. 12a) or 50 mg/kg Compound 1 (Fig. 12b) or 100 mg/kg Compound 1 (Fig. 12c) one day before treatment with the ovalbumin antigen (day-1) or 2 days after Ovalbumin antigen treatment (day+2).
• PBS saline
• Fig. 12b 50 mg/kg Compound 1
• Fig. 12c 100 mg/kg Compound 1
• Fig. 13a-b is a graphic representation of lie results of an ELISA assay showing the IgG anti Fel d I antibody response in a total of six groups of 6 mice which had been treated with 10 mg/kg of Compound 1 for (day-4)(day+4) days subcutaneously (s.c.) (Fig. 13a) or intraperitoneally (i.p.) (Fig. 13b) after treatment with the Fel d I antigen on day 0.
• Fig. 14 is a graphic representation of the results of an ELBA assay showing the IgG anti Fel dl antibody response in the saline control group of six mice (Group 7) in the experiment referred to in Fig. 13, the control group had been treated with 10 mg/kg of Fel d I antigen on day 0.
• Fig. 15 is a graphic representation of an ELISA assay showing the IgG anti h-Mb antibody response in mice which had been treated subcutaneously (sc) with 35 mg/Kg Compound 1 or Compound 3 or saline (PBS) control on day-1 and day 2, and 100 ug of h-Mb in complete Freunds Adjuvant (CFA) on day 0 and 100 ug of h-Mb in Incomplete Freund's Adjuvant (IFA) on day 21. Mice were bled on day 33 and sera was assayed for h-Mb specific IgG. The mean antibody binding from 5 mice is shown.
• Fig. 16 is a graphic representation of an ELISA assay showing the IgG anti h-Mb antibody response in mice which had been treated subcutaneously (sc) with 35 mg/Kg Compound 1 or Compound 3 or saline (PBS) control on day-1 and day 2, and 100 ug of h-Mb in complete Freunds Adjuvant (CFA) on day 0 and 100 ug of h-Mb in
• IFA Incomplete Freund's Adjuvant
• Fig. 17 is a graphic representation of an ELISA assay showing the IgG anti h-Mb antibody response in mice which had been treated subcutaneously (sc) with 35 mg/Kg Compound 1 or Compound 3 or saline (PBS) control on day-1 and day 2, and 100 ug of h-Mb in complete Freunds Adjuvant (CFA) on day 0 and 100 ug of h-Mb in Incomplete Freund's Adjuvant (IFA) on day 21.
• Mice were bled on day 33 and sera was assayed for h-Mb specific IgG2b. The mean antibody binding from 5 mice is shown.
• Fig. 18 is a graphic representation of an assay showing the effect of Compound 1 and Compound 3 on h-Mb specific T cell proliferation
• 3 mice were given either 35 mg/kg or PBS (control on day -2 and day -1 intravenously), the mice were primed with 100ug h-Mb/CF A subcutaneously on day 0. Lymph nodes were pooled and harvested on day 7, proliferation of lymph node T cells is shown.
• Fig. 19 is a graphic representation showing the effect of Compound 1 only.
• Fig. 20 is a graphic representation showing the effect of Compound 1 only, Compound 3 only, Compound 1 and Compound 3 together, or saline (PBS) on days 90 and 91 of life.
• the incidence of diabetes was measured by serum glucose levels.
• Fig. 20 is a graphic representation showing the effect of Compound 1 only,
• Compound 1 only.
• Compound 3 only, Compound 1 and Compound 3 together, or saline (PBS) on day 76 of life at each data point shown.
• the incidence of diabetes was measured by serum glucose levels.
• Fig. 21 is a graphic representation of the effect of Compound 1 or Compound 3 on the IgM response of mice which were treated subcutaneously with 35 mg/Kg
• the present invention provides methods for inhibiting at least a portion of an antigen specific antibody response by the immune system of a mammal comprising administering to the mammal an effective amount of a composition comprising at least one histamine derivative having binding specificity for at least one histamine receptor.
• histamine derivatives include histamines which are modified at the histamine side chain primary amine by derivatiz ⁇ ng with an aliphatic, e.g., alkyl, or aralkyl (aryl group bound to an aliphatic chain) where the aliphatic chain may be branched or unbranched of variable length, which may include oxocarbonyl.
• modified histamine agonists may be further modified by linkage to carrier molecules such as amino acids, polypeptides, proteins, or derivatives thereof.
• the histamine derivatives and pharmacologically acceptable salts thereof are formed by derivatizing the primary amine in histamine to produce a variable length side chain having 0 to 1 branch of from 1 to 3 carbons, preferably methyl, particularly alpha to the amino group: 0 to 2 non-oxo-carbonyl groups: 0 to 4 heteroatoms, other than the non-oxo-carbonyl oxygen: 0 to 1 aryl or substituted aryl group, preferably the substituent being methyl or trifluoromethyl located para to the histamine linking chain: and 0 to 1 covalentiy bonded amino acid, polypeptide. protein, or derivative thereof.
• histamine derivatives which are administered in the method of the invention have the formula:
• His-NH intends the histaminyl residue, with the NH being the side chain amino (2-(4'-imidazolinyl)ethylamino): n indicates the number of methylene groups in the chain and is usually 0 to
• X is a carbonyl, a methylene, or alkylidene, i.e., -CHR-, where R is an alkyl chain of from 1 to 3 carbons, preferably methyl: Y is a terminal group, either a methyl or amide, i.e., -CONHZ.
• Z is hydrogen or preferably Z is an organic group, thereby producing an N-substituted amide, where the N-substituent is an alkyl group, particularly a straight chain, i.e., - (CH 2 ) m -CH 3 , wherein m is usually from 0 to 10, more usually 2 to 6 and preferably 2 to 5: an aryl or substituted aryl group, i.e.,
• phi is phenylene, particularly para-phenylene
• D is hydrogen, methyl or heteroatom-substituted methyl, preferably halomethyl, more particularly
• A is a physiologically acceptable counterion such as acetate, chloride, sulfate, phosphate, and the like, preferably chloride: and b indicates the number of additional protons and counterions found in the salt (e.g., the number of basic amines available for neutralization) and is usually 0 to 2, preferably from 1 to 2, with the proviso that when Y is an amino acid, polypeptide, protein, or derivative thereof, b may be greater than 2 to neutralize partially or totally any additional charge introduced by Y.
• b indicates the number of additional protons and counterions found in the salt (e.g., the number of basic amines available for neutralization) and is usually 0 to 2, preferably from 1 to 2, with the proviso that when Y is an amino acid, polypeptide, protein, or derivative thereof, b may be greater than 2 to neutralize partially or totally any additional charge introduced by Y.
• N is other than 4.
• Histamine derivatives of particular interest include compounds of the formulas:
• His-NH is the histaminyl residue, with the NH being the side chain amino:
• X' is CO, CH 2 or CHCH 3 ;
• phi is phenylene, particularly para-phenylene:
• E is any naturally occurring (especially genetically encoded) amino acid residue side chain: i.e., E is H (in which case the amino acid is glycine) or a side chain of an amino acid bonded to the alpha-carbon of glycine (in which case the amino acid is an amino acid other than glycine):
• G is OH, NH 2 or NHCH 3 ;
• n' is an integer of from 2 to 5, usually 3 to 5:
• n" is an integer of form 2 to 3;
• p' is an integer of from 1 to 8.
• histamine derivatives of interest come within the structure:
• the histamine derivatives may be synthesized by various methods according to procedures well known in the art. A discussion of the synthesis of the above- described histamine derivatives can be found in U.S. Pat. No. 4,996,221 incorporated herein by reference.
• the acylated derivatives may be prepared from histamine and the appropriate carboxylic acid via the mixed anhydride, carbodiimide or aryl halide method.
• Unbranched alkylated derivatives may be synthesized either by a displacement reaction using a halide or pseudohalide compound, e.g., bromo, chloro, tosyl, etc.
• alkylated derivatives may be prepared by reductive amination of histamine with the appropriate methyl ketone derivative or by halide or pseudohalide displacement (using conditions that favor displacement over elimination). Although these are possible synthesis routes, other methods well known in the art are contemplated as also producing compounds of the subject invention.
• the histamine derivatives may be purified by conventional purification techniques, such as crystallization, or by chromatographic techniques, such as column chromatography, high performance liquid chromatography, preparative thin-layer chromotography, or the like.
• the subject invention includes derivatives of histamine wherein histamine is connected by a linking group to an amino acid of poly(amino acid) molecule thereby defining a conjugate.
• the histamine derivative may be linked to a carrier such as polypeptides, proteins, glycoproteins or derivatives thereof (all included within the name poly(amino acid).
• the conjugates may serve a variety of functions, changing the physiological character of the histamine derivative, acting as immunogens, providing for cell specific binding and the like.
• the nature of the histamine derivative may be modified to lesser or greater degrees by adding additional functionalities, substituting groups or the like.
• a group may be substituted for another group, e.g., methyl or trifluoromethyl with carboxyl.
• immunogens substitution at histamine or intermediate the ends of the histamine derivative may be desirable.
• the conjugates may be bonded through a wide variety of functionalities to form amide, methyleneamine, thioether, disulfide, sulfonamide, azo, amidine, etc.
• the particular functionality chosen will depend upon the purpose of the conjugate, ease of synthesis, stability of the linking functionality, affect of the linking group on the physical, chemical like.
• T is an amino acid derivative or poly(amino acid)
• d is the number of histamine derivatives per T, usually being on the average in the range of 1 to 50, more usually 1 to 20, and frequently 1 to 10:
• W is a bond or linking group of at least one atom other than hydrogen and may be methylene, e.g., by reductive amination of a periodate cleaved sugar or other aldehyde, non-oxo-carbonyl, thio, alkylene-non-oxo-carbonyl alkylene, alkylenethia, aryllene-non-oxo-carbonyl. arylazo, etc., the particular linking group not being critical except as indicated herein:
• T is an amino acid or poly(amino acid) of from about 2 to 2000, usually about 2 to 1000, amino acid residues, which may also include sugars or lipids, and may be a carrier for antibody formation, e.g., bovine serum albumin, keyhole limpet hemocyanin, ⁇ -globin, etc., a poly(amino acid) usually of at least about 100 amino acids, or for site specific binding, may be a hormone, lymphokine, growth factor, or the like.
• amino acid residues which may also include sugars or lipids, and may be a carrier for antibody formation, e.g., bovine serum albumin, keyhole limpet hemocyanin, ⁇ -globin, etc.
• a poly(amino acid) usually of at least about 100 amino acids, or for site specific binding, may be a hormone, lymphokine, growth factor, or the like.
• the linking group may provide for linkage which is resistant or susceptible to hydrolytic cleavage under physiological conditions.
• the functionalities bonded to the histamine derivative and carrier are selected so as to complement one another in such a way as to allow the formation of a suitable chemical bond between the two.
• the carrier contains an amine functional groups, e.g.,lysine or p-aminophenylalanine side chains
• the functionality of the histamine derivative may be a carboxylic acid, a sulfonic acid, etc.
• the number of histamine derivatives per carrier may be one, or any number greater than one.
• the number of histamine derivatives per carrier molecule is dependent upon the number of appropriate functional groups in the carrier and the stoichiometry used during the coupling reaction.
• Synthesis routes are well known in the art.
• One method would involve the preparation of appropriate histamine derivatives where the extended amine side chain or other location on histamine has a suitable functional group.
• One or more functionalized histamine derivatives are then, in turn, coupled to appropriate side chains of the carrier.
• a method of synthesis may involve the initial modification of the carrier by coupling the derivative group moiety containing a further functional group reactive with histamine directly to the carrier side chain.
• the resulting carrier derivative is then coupled directly to the histamine, for example, by a reductive amination reaction to produce the conjugate.
• the above histamine derivatives have been found to be useful in methods of inhibiting at least a portion of an antigen specific antibody response by the immune system of a mammal.
• administration of an effective amount of at least one histamine derivative having binding specificity for at least one histamine receptor has been found to inhibit the production of IgG and/or IgE antibodies. It appears that the production of IgM antibodies are not inhibited.
• the inhibition is dependent on the dose of the histamine derivative, can endure for several months, can be prolonged by repeated dosing of the histamine derivative, and moreover, can reverse an already established response to antigen.
• Administration of at least one histamine derivative of the present invention results in inhibition of an antigen specific antibody response by the immune system of a mammal of at least about 30% inhibition of the production of IgE antibodies and/or at least about 60% inhibition of the production of IgG antibodies, and more preferably up to about 100% inhibition of the production of IgG antibodies.
• both the production of IgE antibodies and the production of IgG antibodies is substantially inhibited (i.e., IgE production is inhibited by at least about 30% and IgG production is inhibited by at least about 60%).
• composition comprising at least one histamine derivative is administered to a mammal varies widely in accordance with methods well known in the art
• composition is preferably administered with a histamine derivative
• physiologically suitable or pharmaceutically acceptable carrier may be any physiologically acceptable buffer as is known in the art and includes but is not limited to phosphate buffered saline (PBS). Suitable methods of administration include but are not limited to: orally, parenterally, by injection subcutaneously or the like. A preferred route of administration is subcutaneously.
• Pharmaceutically effective concentrations and dosages of compositions comprising at least one histamine derivative will vary widely, depending upon the potpose, host and particular derivative employed. Concentrations may vary from less than 10 - 1 M, and preferably less than or equal to 10 -3 M. Suitable single pharmaceutically effective dosages of such compositions range from about 0.5mg/kg body weight to ab
• mg/kg is from about 1 mg/kg to 50mg/kg. preferably from about 1 to 20 mg/kg. more preferably from about 1 to 10 mg/kg.
• Suitable pharmaceutically effective daily total dosages range from about 1 mg/kg to 100 mg/kg.
• the present invention also provides methods for inhibiting at least a portion of an antigen specific antibody response to a predetermined antigen, such as a protein allergen or autoantigen, by the immune system of a mammal sensitive to the predetermined antigen (i.e., the mammal has been producing antibodies to the predetermined antigen).
• a predetermined antigen such as a protein allergen or autoantigen
• an effective amount of a composition comprising at least one histamine derivative is administered to a mammal sensitive to the predetermined antigen or immunogenic portion thereof (i.e., a portion of an antigen capable of eliciting an immune response).
• Administration of at least one histamine derivative results in at least partial inhibition of further production of antibodies to said predetermined antigen by the immune system of the mammal.
• the predetermined antigen or an immunogenic portion thereof is administered to a mammal sensitive to the predetermined antigen or immunogenic portion thereof, in conjunction with a histamine derivative of the present invention to specifically inhibit the antigen specific antibody response to the predetermined antigen or immunogenic portion thereof (i.e., further production of antibodies specifically reactive to the predetermined antigen or immunogenic portion thereof).
• predetermined antigen or immunogenic portion thereof is at least partially inhibited).
• the predetermined antigen or immunogenic portion can be administered to the mammal simultaneously or sequentially with a composition comprising at least one histamine derivative.
• antigens include but are not limited to protein allergens, autoantigens or at least an immunogenic portion of either antigen capable of eliciting an immune response.
• Another embodiment of the present invention provides methods for treating sensitivity to an antigen, such as a protein allergen or autoantigen.
• an antigen such as a protein allergen or autoantigen.
• a peptide having T cell stimulating activity and derived from a protein allergen or other antigen is administered to an individual sensitive to the protein allergen or autoantigen from which the peptide is derived in conjunction with a histamine derivative of the present invention.
• T cell stimulating activity is defined herein as induction of T cell proliferation, lymphokine secretion and/or T cell anergy/tolerization.
• Such T cell stimulating activity can be tested by culturing T cells obtained from an individual sensitive to a protein allergen or other antigen with a peptide derived from the protein allergen or other antigen and determining the presence of proliferation by the T cells in response to the peptide.
• Peptides useful in methods of the treating sensitivity to a protein allergen or other antigen have T cell stimulating activity and preferably have human T cell stimulating activity and accordingly comprise at least one T cell epitope of a protein allergen or other protein antigen.
• Preferred peptides comprise at least two T cell epitopes (e.g., the peptide comprises at least approximately eight amino acid residues, and preferably at least fifteen amino acid residues).
• Peptides derived from protein allergens preferably do not bind immunoglobulin E (IgE) or bind IgE to a
• T cell epitopes are believed to be involved in initiation and perpetuation of the immune response to a protein allergen or other protein antigen which is responsible respectively for the clinical symptoms of allergy or other diseases. These T cell epitopes are thought to trigger early events at the level of the T helper cell by binding to an appropriate HLA molecule on the surface of an antigen presenting cell and stimulating the relevant T cell subpopulation. These events lead to T cell
• T cell epitope is the basic element or smallest unit of recognition by a T cell receptor, where the epitope comprises amino acids essential to receptor recognition and may be contiguous and/or non-contiguous in the amino acid sequence of the protein. Amino acid sequences which mimic those of the T cell epitopes and which modify the allergic response to protein allergens are within the scope of this invention.
• Administration of these peptides may tolerize or anergize appropriate T cell subpopulations such that they become unresponsive to the protein allergen or other antigen and do not participate in stimulating an immune response upon such exposure.
• administration of a peptide comprising at least one T cell epitope of a protein allergen may modify the lymphokine secretion profile as compared with exposure to the naturally-occurring protein allergen or portion thereof (e.g., result in a decrease of IL-4 and/or an increase in IL-2).
• T cell subpopulations which normally participate in the response to the allergen such that these T cells are drawn away from the site(s) of normal exposure to the allergen (e.g., nasal mucosa, skin, and lung) towards the site(s) of therapeutic administration of the peptide.
• This redistribution of T cell subpopulations may ameliorate or reduce the ability of an individual's immune system to stimulate the usual immune response at the site of normal exposure to the allergen, resulting in a diminution in allergic symptoms.
• peptides When peptides are derived from protein allergens, they can comprise at least one. and preferably at least two T cell epitopes of a protein allergen such as a protein allergen selected from the group consisting of: a protein allergen of the genus
• Dermatophagoides a protein allergen of the genus Felis; a protein allergen of the genus Ambrosia; a protein allergen of the genus Lolium; a protein allergen of the genus Cryptomeria: a protein allergen of the genus Alternaria: a protein allergen of the genus Alder; a protein allergen of the genus Betula; a protein allergen of the genus Quercus; a protein allergen of the genus Olea; a protein allergen of the genus Artemisia; a protein allergen of the genus Plantago; a protein allergen of the genus Parietaria; a protein allergen of the genus Canine; a protein allergen of the genus Blattella; a protein allergen of the genus Apis; and a protein allergen of the genus Periplaneta.
• Preferred peptides are derived from protein allergens selected from the group consisting of: Der p I; Der p II; Der f I; Der f II; Amb a 1.1; Amh a I.2; Amb a I.3: Amb a I.4: Amb a II: Lol p I: Lol p IX: Cry j I: Cry j II; and Fel d I.
• Peptides useful in methods of the invention can be derived from protein antigens other than protein allergens where enhancement or depression of an antigen specific immune response is desired.
• peptides having human T cell stimulating activity of a known autoantigen involved in the pathogenesis of an autoimmune disease or peptides comprising at least one T cell epitope of a known autoantigen can be administered to decrease the antibody response to the autoantigen, to interfere with efficacy and/or decrease immune complex related side effects.
• peptides derived from the autoantigen having human T cell stimulating activity could be defined by standard T cell biology techniques, or if desired, precise T cell epitopes can be defined by fine mapping techniques and a peptide comprising at least one T cell epitope produced.
• Peptides which stimulate T cells and do not have undesired properties of the autoantigen e.g., do not bind autoantibodies in a substantial percentage of individuals sensitive to the autoantigen
• Autoantigens useful in methods of the present invention include insulin, glutamic acid decarboxylase (64K), PM-1 and carboxypeptidase in diabetes; myelin basic protein in multiple sclerosis; rh factor in erythroblastosis fetalis; acetylcholine receptors in myasthenia gravis; thyroid receptors in Graves' Disease; basement membrane proteins in Good Pasture's syndrome; and thyroid proteins in thyroiditis.
• the present invention also provides methods of treating sensitivity in an individual to an antigen, such as a protein allergen or autoantigen whereby a therapeutically effective amount of a therapeutic composition comprising a histamine derivative and a pharmaceutically acceptable carrier or diluent is administered to an individual sensitive to the antigen.
• an antigen such as a protein allergen or autoantigen
• a therapeutically effective amount of a therapeutic composition comprising a histamine derivative and a pharmaceutically acceptable carrier or diluent is administered to an individual sensitive to the antigen.
• administration of such a therapeutic composition to an individual results in inhibition of at least a portion of an antigen specific antibody response for up to 150 days after
• composition is administered to an individual at least once a year and preferably up to four times a year.
• the antigen, or an immunogenic portion thereof is optionally administered to the individual to specifically inhibit the antigen specific immune response by the individual to the antigen or immunogenic portion thereof.
• a peptide having T cell stimulating activity and derived from the antigen can be administered to the individual to desensitize the individual to the antigen.
• the peptide comprises at least one T cell epitope of the antigen.
• Such peptides are administered to individuals sensitive to an allergen or other protein antigen from which the peptide is derived, at dosages and for lengths of time effective to reduce sensitivity of the individual to the allergen or other antigen.
• Peptides having T cell stimulating activity can be administered in the form of a therapeutic composition including a physiologically acceptable vehicle.
• the peptide can be administered in combination with an appropriate diluent, a carrier, and/or an adjuvant, where appropriate.
• Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
• Pharmaceutically acceptable carriers include polyethylene glycol (Wie et al., International Archiyes of Allergy and Applied Immunology 64: 84-99 (1981)) and liposomes (Strejan et al., Journal of Neuroimmunology 7: 27 (1984)).
• Pharmaceutically acceptable adjuvants include alum.
• Such compositions will generally be administered by injection, oral administration (e.g., as in the form of a circule), inhalation, transdermal application or rectal administration.
• Fel d I protein was decolorized by loading it on a Phenyl-Sepharose column (Pharmacia) with 4N NaCl, then eluted with 2M and 1M NaCl. Decolorized Fel d I protein was recovered by dialysing the 2M and 1M salt elutes against distilled water and lyophilized.
• An alternative way to decolonization was to pass the house dust extract through a Sephacryl 200 column (Pharmacia) before the affinity purification.
• mice Incubate at room temperature for 30 minutes. Split up the sample into eppendorf tubes and spin in an eppifuge (15,000rpm) for 10 minutes. Pour supernatant into tubes so that it can be tested for the presence of antigen to be certain that it bound. Resuspend the pellet in desired volume of PBS in order to inject mice. (See:
• Coat Costar EIA plates (#3590) with 1 ⁇ g/ml Fel d I in PBS 50 ⁇ l/well overnight at 4o C. Wash plates with 1 x PBS (repeat 3 times). Block with 1% BSA (essentially globulin free) in PBS-Tween (PBS-T), 100 ⁇ l/well, 1 hour at room temperature. Add test serum diluted in (PBS-T), 100 ⁇ l/well, for 1 hour at room temperature. Wash plates with 1 X PBS-T 3 times. Add Goat anti-mouse IgG 1/5000 diluted in PBS-T, 100 ⁇ l/well, for 1 hour at room temperature. Wash plates with 1 X PBS-T 3 times.
• mice were given two treatments of Compound 1 intraperitoneally one day before antigen treatment (day -1) and two days following antigen treatment (day+2).
• the doses of Compound 1 were eimer 100mg/kg, 50 mg/kg, or saline control in 100 ⁇ l PBS.
• the three groups of mice were injected with 10 ⁇ g Fel d I Alum (obtained as described above) intraperitoneally.
• mice The priming and bleeding schedule for these three groups of mice are as follows:
• mice After the first immunization described above the mice were bled 21 days later for a 1° response. The mice were boosted and then 14 days later the mice were bled for a 2° response and bled again in another 14 days without an intervening antigen injection for a 2°B response. This boost schedule (i.e., 2o, 2oB, 3o, 3oB) was repeated until the mice were bled for the 5oB. Each time the mice were boosted they receive a day-1 & day+2 dose of Compound 1 and on day 0 they are injected with 10 ⁇ g Fel d I Alum. All the sera was tested as described above in an Elisa for IgG & IgE antibody response to Fel d I.
• mice There was some toxicity seen in the mice that received 100mg/kg Compound 1 on day-1 and day+2. In that group of mice 2 out of 6 mice died.
• mice immunized with 100 mg/kg of Compound 1 had no IgG or IgE response to Fel d I compared to the saline control mice where there was a significant IgG response and an average IgE response to Fel d.
• the mice that received 50 mg/kg of Compound 1 had low IgG & IgE response to Fel d I compared to the saline control mice.
• mice were given either 50 mg/kg in 100 ⁇ l PBS of Compound 1 or saline one day before antigen immunization and two days after antigen immunization in the first priming only.
• the immunization and bleeding schedule was the same as priming #34, however the drug was not given in subsequent boost only 10 ⁇ g Fel d I alum on day 0.
• mice immunized with 50 mg/kg of Compound 1 in the 1o had very good IgG response to Fel d I compared to the saline control group. There appears to have been a delay in the response to Fel d I response in the Compound 1 treated group. The IgE specific Fel d I response did not appear to differ significantly between the two groups (Fig. 4).
• a lesser dose of Compound 1 was administered three groups of mice.
• the groups of mice were given two treatments of Compound 1 intraperitoneally one day before antigen treatment (day-1) & two days following antigen treatment (day+2).
• the doses of Compound 1 were either 50 mg/kg, 5 mg/kg, 0.5 mg/kg in 100 ⁇ l of PBS, or a saline control.
• the four groups of mice were injected with 10 ⁇ g Fel d I alum intraperitoneally. The immunization and bleeding schedule was followed as described in priming #34.
• Fig. 5 shows that mice given 50 mg/kg of Compound 1 had a decreased IgG specific Fel d I response compared to the saline control. In the groups that received 5 mg/kg or 0.5 mg/kg of Compound 1 there was not a significant difference in IgG specific Fel d I response compared to the saline control. As shown in Fig. 6, the IgE response to Fel d I appeared to be low in all the groups.
• mice were pre-primed with 10 ⁇ g Fel d I Alum and then bled 21 days after immunization.
• the two groups of mice were either given 50 mg/kg of Compound 1 or saline intraperitoneally one day before antigen immunization (day-1) and two days following antigen immunization (day+2).
• the antigen immunization was given on day 0 witii 10 ⁇ g Fel d I alum intraperitoneally.
• the two weeks later the mice were bled and then in another 2 weeks (without boosting) they were bled again.
• the two groups of mice were then boosted only with 10 ⁇ g Fel d I alum in each of the scheduled immunizations as described in priming #34.
• Figs.7 and 8 demonstrate that mice that received drug only in the 2o boost have a much lower IgG & IgE specific Fel d I response than the mice that received saline in the 2o only. It appears as though the mice did not have a high response to Fel d I before giving them a dose of Compound 1. Thus, it is unclear whether there was a decrease in the Fel d I specific response or whether there was a decrease and delayed IgG Fel d I response, as shown in priming #37. TABLE 4
• mice immunized with 100 mg/kg of Compound 1 have no IgG or IgE response to Fel d I compared to the saline control mice where there is good IgG response and average IgE response to Fel d I.
• the mice that received 75 mg/kg and 50 mg/kg of Compound 1 have low IgG response to Fel d I compared to the saline control mice.
• a total IgG assay (Fig. 11) was performed to determine whether there was a different-amount of IgG in the drug treated groups compared to the saline control groups. There was no change in total IgG in the mice that received 100 mg/kg, 75 mg/kg, and 50 mg/kg compared to die saline control group in the early bleed.
• Fel d I has a mitogenic contaminant which causes an increase in immunoglobulin levels. The mitogenic effect appears to be decreased by the histamine congener.
• mice were given two treatments of Compound 1 intraperitoneally one day before antigen treatment (day-1) and two days following antigen treatment (day+2).
• the doses of Compound 1 were either 100 mg/kg, 50 mg/kg, in 100 ⁇ l of PBS or saline control.
• day 0 the three groups of mice were injected with 50 ⁇ g ovalbumin alum intraperitoneally.
• mice immunized with 100 mg/kg of Compound 1 have low IgG responses to ovalbumin compared to the saline control mice where there is a good IgG response to ovalbumin. It appears that mice which received 50 mg/kg of Compound 1 have a similar IgG response to ovalbumin as the saline control group indicating no decrease in antibody response. The IgE response to ovalbumin appears to be similar in all three groups (Fig. 12). TABLE 7
• Figs. 13 and 14 show that administration of 10 mg/kg Compound 1 for 9 days s.c. (a total of 90 mg/kg) shows a decrease in antibody response to Fel d I compared to the saline control and to the group that was given 10 mg/kg Compound 1 for 9 days i.p.
• Native Fel d I protein was purified from an extract of house dust as described by Chapman et al. Briefly, house dust (from vacuum containers used in homes with multiple cats) was extracted with PBS, then lyophilized and redissolved in water. The extract was applied to a column coupled with anti-Fel d I monoclonal antibody (hybridomas 6F9 and 1G4 were both provided by M. Chapman). The Fel d I was eluted from the column with 100 mM glycine pH 2.5 and was neutralized. Direct Binding ELISA
• Fel d I was coated onto Immulon 2 (Dynatech, Chantilly, VA) 96-well plates by incubation of 50 ⁇ l/well of 2 ⁇ g/ml Fel d I in PBS overnight at 4°C. The wells were incubated with 0.5% gelatin in PBS at 37°C for one hour.
• H-Mb ELISA is carried out similarly, using isotype specific polyclonal reagents.
• Antigen bound IgE was detected similarly, but using biotinylated EM95-1, a rat monoclonaTantibody specific for mouse IgE. Biotinylated goat anti-rat IgG (Dirkegaard and Perry) was used as an added signal amplification step in the IgE ELISA.
• Antigen bound IgM was detected similarly, but using anti-mouse IgM.
• the inguinal, paraaortic, and popliteal lymph nodes were removed from the animals seven days after antigen challenge.
• the cells from these organs were suspended by being forced through a stainless steel mesh with a glass pestal.
• the cells were washed two times in RPMI 1640 with 1% FCS before being cultured. All cells were cultured at 37°C in 5% CO 2 in RPMI-1640 with 10% FCS (#F4884,
• helper T cell subsets to augment different antibody isotypes.
• Murine TH 1 cells appear to stimulate the production of IgG2 while TH2 cells stimulate the production of IgG1 and IgE.
• histamine congeners can specifically effect different populations of helper T cell functions. Two different histamine congeners (Compound 1 and
• Compound 3 have been compared for their ability to effect the antibody response to
• Horse myoglobin H-Mb.
• the formula for Compound 1 was given previously in
• the IgM response specific for H-Mb was assayed on day 7 following an antigen priming with a day 0 and day 2 drug treatment (Fig. 21). There was no detectable effect of 35 mg/Kg histamine congener on the antigen specific IgM made in response to the H-Mb priming.
• mice were treated with 35 mg/Kg histamine congener on day (-1) and day 2. These mice received H-Mb on day 0 and day 21. Sera (day 33) from these mice were assayed for H-Mb specific IgG.
• Fig. 15 demonstrates the ability of Compound 1 and Compound 3 to inhibit the production of H-Mb specific IgG. The same bleeds were assayed for H-Mb specific IgG2a (Fig. 16) and IgG2b
• Compound 1 does not effect the H-Mb specific IgG2a or IgG2b.
• the decrease in IgG found after Compound 1 treatment may reflect an effect on IgGl.
• the ability of Compound 1 to decrease IgG and IgE responses to Fel d I as discussed in Examples 1-6 suggests that its target may be the TH2 pathway.
• IDM Insulin-dependent diabetes mellitus
• Type I Diabetes Mellitus is an autoimmune disease and involves lymphocyte dependent inflammatory destruction of the insulin-producing beta cells in pancreatic islets of Langerhans. T lymphocytes have been implicated in the destruction of the pancreatic cells and autoantibody production is associated with the development of insulitis, the inflammatory lesion of IDDM.
• a strain of mouse non obese diabetic mice -- NOD mice
• NOD mice non obese diabetic mice
• NOD mice non obese diabetic mice
• Many experiments using immunosuppressive drugs that showed efficacy in NOD mice predicted the value of immunosupprcssives in people susceptible to type I diabetes.
• the imunosuppressives available for the treatment of Type I disease in susceptible patients are efficacious but only during the time they are given. Those agents, with the possible exception of anti-CD4 monoclonal antibody, are too toxic to give continuously for prolonged periods.
• Compound 3 could influence the course of the disease because Compound 3 is capable of inhibiting T Cell proliferation to antigen. It should thus be able to limit a T cell-mediated cytoltic event.
• hyperglycemia was most pronounced in the groups treated with Compound 3 or 1 plus 3. It is quite likely that the apparent effects of Compound 1 plus 3 is caused by the additive H2 effects of the two drugs.
• Fig. 20 shows the absence of any disease in the group treated with compounds 1 plus 3 up to day 140.
• Examples 1-11 show that histamine congeners are potent immunosuppressants and each has a different potential mechanism of
• Compound 1 suppresses T cell dependent IgE, IgG 1 (but not IgM, IgG2a or IgG2b) antibody responses. In various antigenic responses,
• Compound 3 supresses IgG1, IgG2a and IgG2b but not IgM responses. The effects of Compound 3 on IgE will be tested. Only Compound 3 appears to directly suppress T cell proliferation to specific antigen at the doses tested. The suppression of antibody production is transferable and also can be seen after the response to the antigen is established.
• H1 and H2 receptor effects may be needed and cooperative in the effects on the NOD mouse or the apparent cooperation may simply be additive H2 effects contributed separately by the two drugs; dose dependence and blocking effects of H1 or H2 blockers will be studied to determine the pharmacologic mechanism by which the experiments work; the cellular mechanism by which the autacoids work are not established byt the selectivity of the effects on isotypes and actions of T cell proliferation suggest the target for Compound 3 could be a TH 1 cell.

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