AU3591793A - Methods for using histamine derivatives as immunomodulators and in immunotherapeutics - Google Patents

Description

METHODS FOR USING HISTAMINE DERIVATIVES AS

IMMUNOMODULATORS AND IN IMMUNOTHERAPEUTICS

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

With the increased level of understanding concerning the immune response process in mammals, there is a growing awareness that certain molecules play a significant role in immune modulation. Unfortunately, these molecules are generally nonspecific as to their effects on single cell types in a mixture of cells. A critical need exists for agonists that are effect or cell specific.

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

immunotherapeutic use of histamine. See Khan, et al., Clin Immunol. Rev. (1985) 4:1 Melmon, et al., Am. J. Med (1981) 71:100: and Roclin et al., Cell Immunol.

(1978) 37:162. 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.

Such events create a setting in which the immune system should not overreact to the new products. Yet, despite the ability of inflammation to generate likely

immunogens, the inflammatory process usually is not accompanied or followed by grossly abnormal immune responses. Autacoids appear to somehow modulate this response.

Autacoids affect natural suppressor cells, T cell subsets and B cells during various stages of immunity. Receptors for autacoids are non-randomly distributed

(in number and affinity for agonist) on cells that carry out immune functions.

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

CAMP responses of T helper (T_h) and T cytolitic (T_c) cells to histamine. Mitogens alter responsiveness of these cells to histamine. Some lymphocytes that respond to histamine have both H₁ and H₂ receptors on them while others have only H₂ receptors. In some lymphocytes the H₂ receptors seem to modify the responses to H₁ agonism, in others there is no such interplay. In some cells 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.

The 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 appearance of naturally occurring suppressor cells in the spleens of neonatal or irradiated mice may have a key role in induction of immune tolerance. See Strober et al., Ann. Rev. Immunol. (1984) 2:219: Hertel-Wulff et al, J. Immunol. (1984) 133:2791: Okada et al., J. Expt. Med. (1982) 156:522: and Okada et al., J. Immunol. (1982) 129:1892. These cells are related to NK cells in terms of their surface phenotype but differ in function. 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. In this way, 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. Thus, 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

(Leu2., 9.3) with histamine, both phytohemagglutinin-induced T_n cell proliferation and pokeweed mitogen-induced B cell differentiation were inhibited. The effects were mediated via H₂ receptors. The enhancement of natural suppressor function is via H₁ receptors. Natural suppressor cells can be propagated and clones in long-term tissue culture and cause nonspecific suppression in both in vitro and in vivo models of mixed leukocyte reactions.

Therefore, methods using histamine derivatives that have little or no systemic effects in immune modulation and immunotherapeutics would be advantageous. SUMMARY OF THE INVENTION

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. In one variation of this embodiment, the histamine derivative is administered in conjunction with a predetermined antigen or an immunogenic portion thereof to which the individual is sensitive. In another variation, 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. In yet another variation, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

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).

Fig.4a-b is a graphic representation depicting the results of an ELISA assay showing the IgE anά-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).

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).

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).

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).

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).

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).

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).

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

Incomplete Freund's Adjuvant (IFA) on day 21. Mice were bled on day 33 and sera was assayed for h-Mb specific IgG2a. The mean antibody binding from 5 mice is shown.

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.

Compound 3 only, or Compound 1 and Compound 3 together on the incidence of diabetes in NOD mice, 10 mice were treated subcutaneously with 35 mg/Kg

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 3 only, or Compound 1 and Compound 3 together on the incidence of diabetes in NOD mice, 10 mice were treated subcutaneously with 35 mg/Kg

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

Compound 1, Compound 3 or PBS (Control) on day 0 and day 2 and 100 ug of h- Mb/CFA on day 0, the mice were bled on day 7 and sera was assayed for h-Mb specific IgM. The mean antibody binding from 5 mice is show. DETAILED DESCRIPTION OF INVENTION

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. As used herein, histamine derivatives include histamines which are modified at the histamine side chain primary amine by derivatizϊng 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. e.g., keto, non-oxo-carbonyl groups, e.g., carboxamide or heteroatoms. These modified histamine agonists may be further modified by linkage to carrier molecules such as amino acids, polypeptides, proteins, or derivatives thereof.

Generally, 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.

Specifically, the histamine derivatives which are administered in the method of the invention have the formula:

His-NH-(X)-(CH₂)_n-(Y)(HA)_b

wherein:

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

10, more usually 2 to 6, and preferably 2 to 5:

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. wherein 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., - (CH₂)_m-CH₃, 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-D

where phi is phenylene, particularly para-phenylene, D is hydrogen, methyl or heteroatom-substituted methyl, preferably halomethyl, more particularly

trifluoromethyl, and the D group is para to the chain: or an amino acid, polypeptide, protein, or derivative thereof:

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.

Further, when Y is methyl and either X is carbonyl or R is methyl. N is other than 4.

Histamine derivatives of particular interest include compounds of the formulas:

His-NH-X'-(CH2)_n'-phi-D

His-NH-X'-(CH2)n'-CO(NHCH(E)CO)_p'G

His-NH-X'-(CH2)_n'-CONHZ'

His-NH-X'-(CH2)_n"-CH₃ wherein:

His-NH is the histaminyl residue, with the NH being the side chain amino: X' is CO, CH₂ or CHCH₃;

phi is phenylene, particularly para-phenylene:

D is methyl or trifluoromethyl:

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, NH₂ or NHCH₃;

Z' with the nitrogen to which it is attached is a poly(amino acid): 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.

More specifically, individual histamine derivatives of interest come within the structure:

His-NH-Q(HA)_b

wherein A and b are as defined above and Q is defined as:

-CO-(CH₂)₃-CO-NH-phi-CH₃ -CH₂-(CH₂)₄-CH₃ -CH₂-(CH₂)₃-CO-NH-phi-CH₃ -CO-(CH₂)₂-CO-NH-phi-CH₃

-CHCH₃-(CH₂)₂-CO-NH-phi-CH₃

-CHCH₃-(CH₂)₁-CO-NH-phi-CF₃

-CHCH₃-(CH₂)₂-CO-NH-phi-CF₃

-CHCH₃-(CH₂)₃-CO-NH-phi-CH₃ -CHCH_3-(CH₂)₄-CO-NH-phi-CH₃

-CHCH₃(CH₂)₅-CO-NH-phi-CH₃

-CHCH₃-(CH₂)₃-CO-NH-phi-CF₃

-CHCH₃-(CH₂)₄-CO-NH-phi-CF₃

-CHCH₃-(CH₂)₄-CO-NH-(CH₂)₃-CH₃ -CHCH₃-(CH₂)₃-CO(BOC)_{0- 1}-N(H)_1-0-Phe-Gly-NHCH₃ where BOC is the t-butyloxycarbonyl blocking group.

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. or, preferably, by reductive amination of histamine with an aldehyde in the presence of sodium cyanoborohydride or similar agent. Branched, 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.

It is understood that 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. Depending on the purpose of the conjugate, the nature of the histamine derivative may be modified to lesser or greater degrees by adding additional functionalities, substituting groups or the like. Particularly for the production of antibodies from immunogens, a group may be substituted for another group, e.g., methyl or trifluoromethyl with carboxyl. Also, in the case of

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.

For the most part, the conjugates of this invention will have the following formulas:

[(His-NH-(X)-(CH₂)_n(Y))^w]_d-T

wherein all of the symbols have been defined previously except that a hydrogen, methyl or trifluoromethyl group may be replaced by W, which is a bond or linking group to T,

wherein T is an amino acid derivative or poly(amino acid), and 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, β-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. Thus, if 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. Alternatively, 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. In particular, 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. Preferably, 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%).

The manner in which a composition comprising at least one histamine derivative is administered to a mammal varies widely in accordance with methods well known in the art The composition is preferably administered with a

physiologically suitable or pharmaceutically acceptable carrier. The 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 puipose, 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. A preferred range 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). According to this embodiment 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.

In one variation of the above described embodiment, 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 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. As used herein, 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. According to the method, 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

substantially lesser extent than the protein allergen from which the peptide is derived binds IgE.

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

proliferation, lymphokine secretion, local inflammatory reactions, recruitment of additional immune cells to the site, and activation of the B cell cascade leading to production of antibodies. One isotype of these antibodies, IgE, is fundamentally important to the development of allergic symptoms and its production is influenced early in the cascade of events, at the level of the T helper cell, by the nature of the lymphokines secreted. A 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. In addition, 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). Furthermore, exposure to the peptide may influence 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.

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. For example, 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. In order to preserve the T cell reactivity of the autoantigen, 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) are selected for use in methods of treatment as immunotherapeutics. 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. In accordance with the invention, 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

administration. Such composition is administered to an individual at least once a year and preferably up to four times a year.

Subsequently, or simultaneously therewith, 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. In addition, 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. Preferably, 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. For example, 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.

The invention is further illustrated by the following non-limiting examples.

EXAMPLE 1 Monoclonal affinity purification of Fel d I

A house dust sample collected from several homes with cats was used to isolate and purify Fel d I. Monoclonal antibodies 1G9 or 6F9 were coupled to Sepharose 4B and used for the purification according to a published protocol.

Chapman, M.D., et al., J. Immunology, 140 (3):812-818 (1988). The purified 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.

Alum Precipitation of Affinity Purified Fel d 1

In a 50 ml conical tube, add 10 ml of 10% aluminum potassium sulfate (Banco, Fort Worth, TX). While vortexing, add 22.8 ml of 0.25 N NaOH, dropwise. Incubate at room temperature for 10 minutes. Centifuge at 1000g for 10 minutes.

Remove and discard the supernatant Add 50 ml of distilled water to the pellet and resuspend the Al(OH)₃. Centifuge at 1000g for 10 minutes. One milligram of Al(OH)₃ will bind approximately 50-200μg of Fel d I protein. 200 μg/ml of Fel d I protein is combined with 1 ml of Al(OH)₃. Therefore, 1 mg of Fel d I protein diluted in 5 mis of phosphate buffered saline (PBS) is combined with 5 mis Al(OH)₃.

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:

Antibodies: A Laboratory Manual (1988) by Ed Harlow and Dave Lane, page 99.)

Anti-Feld IIgG Elisa

Coat Costar EIA plates (#3590) with 1 μg/ml Fel d I in PBS 50 μl/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 μl/well, 1 hour at room temperature. Add test serum diluted in (PBS-T), 100μl/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 μl/well, for 1 hour at room temperature. Wash plates with 1 X PBS-T 3 times. Add Streptavidϊn-HRP 1/10,000 diluted in PBS-T, 100 μl/well, for 30 minutes at room temperature. Wash plates with 1 X PBS-T 3 times. Develop using TMB Substrate from KPL. Stop the reaction with 1M Phosphoric Acid. Read on Elisa Reader at OD 450 nm.

Anti-Fel d I IgE Elisa

Coat Coming Elisa plates with 10 mg/ml Fel d I in PBS 50 μl/well overnight at 4° C. Wash plates with 1 X PBS 3 times. Block plates with 0.05% gelatin in PBS 100/μl/well for 1 hour at 37º C. Wash plates with PBS-T 3 times. Add 100 μl test serum diluted in PBS-T and incubate for 1 hour at 37° C. Wash with PBS-T 3 times. Add 100 μl of Em95 Biotin labelled 1/2000 diluted in PBS-T to each well and incubate for 1 hour at room temperature. Wash with PBS-T 3 times. Add 100 μl of Goat anti-Rat IgE (H&L) Biotin labelled 1/5000 diluted in PBS-T to each well and incubate for 1 hour at room temperature. Wash with PBS-T 3 times. Add 100 μl Streptavidin-HRP 1/10,000 diluted in PBS-T to each well and incubate for 1/2 hour at room temperature. Wash with PBS-T 3 times. Develop using TMB Peroxidase Substrate from KPL. Stop reaction with 1 M phosphoric acid. Read on Elisa Reader at OD 450 nm.

Priming Experiment #34

This experiment analyzed the effect of two different doses of a histamine derivative (also known as a congener) having the following formula

His-NH-CH₃-(CH₂)₄-CO-NH-phi-CF₃ (Compound 1) on the in vivo immune response to Fel d I. We chose to do this experiment with Balb/c mice (average weight is 25 grams) due to their good IgG response to Fel d I.

Groups of 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 μl PBS. On Day 0, the three groups of mice were injected with 10 μg Fel d I Alum (obtained as described above) intraperitoneally.

The priming and bleeding schedule for these three groups of mice are as follows:

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 μg Fel d I Alum. All the sera was tested as described above in an Elisa for IgG & IgE antibody response to Fel d I.

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.

As shown in Figs. 1 and 2, 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.

EXAMPLE 2

Priming #37

The results from priming #34 suggested that a decrease in Fel d I specific antibody in the mice resulted upon treatment with Compound 1 in each boost with antigen. In this experiment a single dose of Compound 1 in the initial injection with antigen was administered. Groups of mice were given either 50 mg/kg in 100 μl 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 μg Fel d I alum on day 0.

As shown in Fig. 3, 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).

TABLE 2

EXAMPLE 3

Priming #38

In this experiment 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 μl of PBS, or a saline control. On day 0, the four groups of mice were injected with 10 μg 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.

TABLE 3

EXAMPLE 4

Priming #40

In an attempt to inhibit the Fel d I response of groups of already primed mice, two groups of mice were pre-primed with 10 μg Fel d I Alum and then bled 21 days after immunization. In the next boost (2º) 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 μg Fel d I alum intraperitoneally. 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 μg 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

EXAMPLE 5

Priming #41

Priming #34 was repeated, with the addition of a group of mice which received 75 mg/kg. Thus, groups of mice were given two treatments of Compound 1 intraperitoneally one day before antigen treatment (day-1) and two days following antig tment (day +2). The doses of Compound 1 were either 100 mg/kg, 75 mg/k mg/kg in 100 μl PBS or saline control. On day 0, the four groups of mice were ed with 10 μg Fel d I alum intraperitonally. The priming and bleeding schedule for these four groups of mice were followed as described in priming #34.

As shown in Figs. 9 and 10, 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. TABLE 6

EXAMPLE 7

Priming #42

The priming and bleeding schedule was performed as described in priming #34 with the exception that ovalbumin was used as the antigen. Groups of 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 μl of PBS or saline control. On day 0 the three groups of mice were injected with 50 μg ovalbumin alum intraperitoneally.

As shown in Fig. 12, 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

EXAMPLE 8

Priming #45

The following experiment compared itraperitoneally (i.p.) and subcutaneously (s.c.) administration of Compound 1 to see if there is a difference in response. Also, several small doses of Compound 1 were given over 3-9 days instead of just two days. Listed below are the groups of mice and their immunization schedule.

TABLE 8

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.

EXAMPLE 9

Many different primings were done using varying doses of Compound 1 and varying routes of administration of Compound 1. In our studies we saw some toxicity of Compound 1 at high doses. Toxicity was seen in 6/18 mice (33%) that received a total dose of 200 mg/kg i.p. Also, toxicity was seen in 2/6 mice (33%) that received a total dose of 90 mg/kg i.p. (0 mg/kg i.p., 9 days in a row). At a much lower degree, it appears as though 2/36 mice (5.5%) died from a total dose of 100 mg/kg given i.p. Also, 1/6 mice (16.7%) died after getting a total dose of 50 mg/kg i.p. (10 mg/kg, 5 days in a row). None of the mice that were given Compound 1 s.c. died. Therefore, subcutaneous administration of the histamine derivative and an acceptable carrier appears to be the preferred route of administration, however any method of administration which reduces toxicity is also preferred.

TABLE 9

Example 10

Monoclonal Affinity Purification of Fel d I

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

For the IgG assay Fel d I was coated onto Immulon 2 (Dynatech, Chantilly, VA) 96-well plates by incubation of 50 μl/well of 2 μg/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.

Plates were washed three times with PBS-T (1 X PBS + 0.05% Tween 20). Sera were diluted in PBS-T. After incubation at room temperature for one hour and washing with PBS-T, the bound mouse antibody was detected by incubation with biotinylated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL). Streptavidin conjugated to horseradish peroxidase (Southern Biotechnology Associates) was added to detect antigen bound biotinylated antibody-complexes. TMB peroxidase substrate (Dirkegaard and Perry, Gaithersburg, MD) was used according to die directions supplied and resulting O.D. (450 nm) values were determined using an ELISA reader (Bio-Tek model #310, Winooski, VT). The serum titer is determined by 25% of the positive control.

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.

Culture Conditions for Proliferation Assays

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% CO₂ in RPMI-1640 with 10% FCS (#F4884,

Sigma, St Louis, MO), 100 U/ml penicillin G, 10 μg/ml streptomycin, 10 mM glutamine, and 5 x 10^-5 M 2-ME. Cells were cultured in triplicate 0.2 ml wells for in 96 well plates at 4 x 10⁶ cells/ml. Proliferation was measured by tritiated thymidine incorporation on Day 7. The Effect of Histamine Congeners on Antibody Isotype

Recent work has demonstrated the ability of helper T cell subsets to augment different antibody isotypes. Murine TH₁ cells appear to stimulate the production of IgG2 while TH2 cells stimulate the production of IgG1 and IgE. Experiments are addressing whether 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

Example 1. The formula for Compound 3 is as follows:

His-NH-(CH₂)₅-CONH-phi-CF₃

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.

A separate group of 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

(Fig. 17). Compound 3 appears to decrease the H-Mb specific IgG2a and IgG2b.

This implies that the target of Compound 3 activity may be part of the TH1 pathway.

In contrast, Compound 1 does not effect the H-Mb specific IgG2a or IgG2b. The decrease in IgG found after Compound 1 treatment (Fig. 15) 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.

The Effect of Histamine Congeners on T Cell Proliferation

Receptors for the Histamine congeners are present on most cells involved in the immune response. Experiments have been conducted to define the target of action of coumpound 1 and Compound 3. Mice were primed with Mb and treated with drug on day (-2) and (-1) (iv). Draining lymph nodes were harvested and antigen specific proliferation was measured (Fig. 18). Antigen specific T cell proliferation is decreased by treatment of the mice with Compound 3. The data implies that the stimulation of specific T cell activation is affected by Compound 3. This appears to be consistent with the antibody isotype effect of Compound 3. The TH₁ activity is responsible for the majority of T cell proliferation in vitro.

Example 11

The Effects of Histamine Autacoids on an Autoimmune Disease Model

Insulin-dependent diabetes mellitus (IDDM or 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) develops a type of pancreatic lesion that closely resembles Type I diabetes in man. Many experiments using immunosuppressive drugs that showed efficacy in NOD mice predicted the value of immunosupprcssives in people susceptible to type I diabetes. Unfortunately, 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.

The following experiments focus on the effects of compounds 1 and 3 on the progress of hyperglycemia and die development of insulitis. The experiments focus on the following issues: 1. Whether short courses (two doses) of either drug or the drugs combined alter the onset of hyperglycemia and occurrence of death; 2.

Whether protracted dosing of drug at weekly intervals alter the onset of IDDM in any more definitive way than short courses; and 3. Whether intermittent dosing of me two drugs retard or modify the onset or severity of the inflammatory lesion of the pancreas. It is believed that the immunosuppressive effects of the autacoid

(histamine congeners) 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.

The effects of short term treatment with compounds 1, 3 or 1 plus 3 were tested in groups of 10 female NOD mice. The drug (35 mg/Kg was given

subcutaneously) for 2 consecutive days for a cummulative dose of 70 mg/Kg of compounds 1 or 3 and 140 mg/Kg of combined compounds 1 and 3. The drug was given on days 90 and 91 (Fig. 19). Supression of the appearance of the

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.

The effects of long term treatment with histamine congeners on the development of hyperglycemia in NOD mice was tested in groups of 10 mice per group. The drugs were administered subcutaneously as Compound 1, or 3 or 1 plus 3 in the same doses used above. Drug or control treatment was started on day 76 and repeated at the same dose/Kg 14 days later and then at weekly intervals. Fig. 20 shows the absence of any disease in the group treated with compounds 1 plus 3 up to day 140.

In summary. Examples 1-11 show that histamine congeners are potent immunosuppressants and each has a different potential mechanism of

immunomodulation. 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.

Histamine congeners have similar immunosuppressive effects in the mouse model of human IDDM. Congener treatment delays the onset of hyperglycemia and insulitis in NOD mice. Since treatment with compounds 1 and 3 but not Compound 1 alone results in the greatest effect so far determined, these results indicate that: 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 TH₁ cell.

Although this invention has been described with reference to its preferred embodiments, other embodiments can achieve the same results. Variations and modifications to the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents that follow in the true spirit and scope of the invention.

Claims (38)

Claims

1. - A method for inhibiting at least a portion of an antigen specific antibody response by the immune system of a mammal comprising administering to said mammal an effective amount of a composition comprising at least one histamine derivative having binding specificity for at least one histamine receptor, said histamine derivative characterized by being mono-substituted at the side chain amine of a histamine molecule with a substituent having an aliphatic chain of from 2 to 10 carbon atoms, wherein the alpha-carbon of the chain is methylene or is substituted witii oxo or alkyl of from 1 to 3 carbon atoms, said chain terminating in hydrogen or carboxamido, wherein the carbo-amido nitrogen is substituted with alkyl of from 1 to 6 carbon atoms, tolyl, or trifluoromethylphenyl, with the proviso that when said chain terminates in hydrogen, the chain length is 5 or 6 atoms.

2. A method for inhibiting at least a portion of an antigen specific antibody response by the immune system of a mammal comprising administering to said mammal an effective amount of a composition comprising at least one histamine derivative having binding specificity for at least one histamine receptor, said histamine derivative having the formula

His-NH-X-(CH₂)_n Y.(HA)_b wherein:

X is CO or CHR, where R is an alkyl group of from 1 to 3 carbon atoms; n is an integer of from 2 to 6;

Y is CH₃ or CONHZ, wherein Z is H; (CH₂)_mCH₃, where m is 1 to 4;

substituted phenyl, where the substituent is methyl or trifluoromethyl;

A is a physiologically acceptable counterion; and

b is an integer of from 0 to 2.

3. A method for inhibiting at least a portion of an antigen specific antibody response by the immune system of a mammal comprising administering to said mammal an effective amount of a composition comprising at least one histamine derivative having binding specificity for a histamine receptor, said histamine derivative having the formula His-NH-X¹-(CH₂)_n ¹ -phi-D.(HA)_b

wherein:

n' is 2 to 5;

X is CO, CH₂ or CHCH₃;

D is methyl or trifluoromethyl;

phi is phenylene;

A is a physiologically acceptable counterion; and

b' is an integer of from 1 to 2.

4. The method of claim 1 wherein said histamine derivative is selected from the group consisting of:

His-NH-Q.(HA)b wherein A is a physiologically acceptable counterion such as acetate, chloride, sulfate, phosphate, and the like, preferably chloride;

wherein 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; and

wherein Q is defined as:

-CO-(CH₂)₃-CO-NH-phi-CH₃

-CH₂-(CH₂)₄-CH₃

-CH₂-(CH₂)₃-CO-NH-phi-CH₃

-CO-(CH₂)₂-CO-NH-phi-CH₃

-CHCH₃-(CH₂)₂-CO-NH-phi-CH₃

-CHCH₃-(CH₂)₁-CO-NH-phi-CF₃

-CHCH₃-(CH₂)₂-CO-NH-phi-CF₃

-CHCH₃-(CH₂)₃-CO-NH-phi-CH₃

-CHCH₃-(CH2)₄-CO-NH-phi-CH₃

-CHCH₃-(CH₂)₅-CO-NH-phi-CH₃

-CHCH₃-(CH₂)₃-CO-NH-phi-CF₃

-CHCH₃-(CH₂)₄-CO-NH-phi-CF₃

-CHCH₃-(CH₂)₄-CO-NH-(CH₂)₃-CH₃

-CHCH₃-(CH₂)₃-CO(BOC)_{0- 1}-N(H)_1-0-Phe-Gly-NHCH₃ where BOC is the t-butyloxycarbonyl blocking group.

5. The method of claim 1 wherein said histamine derivative comprises

CH(CH₃)-(CH₂₎₄-CONH-phi-CF₃

or

CH(CH₃)-(CH₂₎₃-CONH-phi-CF₃

6. The method of claim 1 wherein production of IgE antibodies by the immune system of the mammal is inhibited by at least about 30%.

7. The method of claim 1 wherein production of IgG antibodies by the immune system of the mammal is inhibited by at least about 60%.

8. The method of claim 1 wherein production of IgG and IgE antibodies by the immune system of the mammal is substantially inhibited.

9. The method of claim 1 further comprising the step of administering to the mammal an antigen or an immunogenic portion thereof to specifically inhibit the antigen specific antibody response to said antigen or said immunogenic portion thereof.

10. The method of claim 1, wherein the composition comprises said at least one histamine derivative in a concentration of less than or equal to 10^-3M.

11. The method of claim 9, wherein the composition comprises said at least one histamine derivative in a concentration of less than or equal to 10^-3M.

12. The method of claim 1, wherein the composition comprises said at least one histamine derivative administered to the mammal in a dosage of less than or equal to 10mg/kg.

13. The method of claim 1 wherein the composition is administered

subcutaneously.

14. The method of claim 9 wherein the composition is administered

subcutaneously.

15. A method for inhibiting at least a portion of an antigen specific antibody response to a predetermined antigen by the immune system of a mammal sensitive to said predetermined antigen and which has been producing antibodies to said predetermined antigen comprising administering to said mammal an effective amount of a composition comprising at least one histamine derivative having binding specificity for at least one histamine receptor, said histamine derivative characterized by being mono-substituted at the side chain amine of a histamine molecule with a substituent having an aliphatic chain of from 2 to 10 carbon atoms, wherein the alpha-carbon of the chain is methylene or is substituted with oxo or alkyl of from 1 to 3 carbon atoms, said chain terminating in hydrogen or carboxamido, wherein the carboxamido nitrogen is substituted with alkyl of from 1 to 6 carbon atoms, tolyl, or trifluoromethylphenyl, with the proviso that when said chain terminates in hydrogen, the chain length is 5 or 6 atoms, wherein further production of antibodies to said predetermined antigen is at least partially inhibited.

16. The method of claim 15 wherein said composition comprising at least one histamine derivative is administered in an amount effective to substantially inhibit the production of antibodies to said predetermined antigen.

17. The method of claim 15 further comprising the step of administering to said mammal said predetermined antigen or an immunogenic portion thereof to

specifically inhibit the antigen specific antibody response to said predetermined antigen or said immunogenic portion thereof.

18. The method of claim 15 wherein the predetermined antigen is a protein allergen.

19. The method of claim 17 wherein the predetermined antigen is a protein allergen.

20. The method of claim 19 further comprising the step of administering to said mammal a peptide derived from said protein allergen, said peptide comprising at least one T cell epitope of said protein allergen.

21. The method of claim 15 wherein the predetermined antigen is a protein allergen and further comprising the step of administering to said mammal a peptide derived from said protein allergen, said peptide comprising at least one T cell epitope of said protein allergen.

22. A method of treating sensitivity to an antigen in an individual, comprising administering to the individual a therapeutically effective amount of a therapeutic composition comprising at least one histamine derivative having binding specificity for at least one histamine receptor, said histamine derivative characterized by being mono-substituted at the side chain amine of a histamine molecule with a substituent having an aliphatic chain of from 2 to 10 carbon atoms, wherein the alpha-carbon of the chain is methylene or is substituted with oxo or alkyl of from 1 to 3 carbon atoms, said chain terminating in hydrogen or carboxamido, wherein the carbo-amido nitrogen is substituted with alkyl of from 1 to 6 carbon atoms, tolyl, or

trifluoromethylphenyl, with the proviso that when said chain terminates in hydrogen, the chain length is 5 or 6 atoms and a pharmaceutically acceptable carrier or diluent

23. The method of claim 22 wherein the therapeutic composition is administered at least once a year.

24. The method of claim 23 wherein the therapeutic composition is administered up to four times a year.

25. The method of claim 22 further comprising the step of administering to said individual said antigen or an immunogenic portion thereof to specifically inhibit the antigen specific antibody response to said antigen or said immunogenic portion thereof.

26. The method of claim 22 wherein the antigen is an autoantigen.

27. The method of claim 25 wherein the antigen is an autoantigen.

28. A method of treating sensitivity to an protein allergen in an individual, comprising administering to the individual a therapeutically effective amount of a therapeutic composition comprising at least one histamine derivative having binding specificity for at least one histamine receptor, said histamine derivative characterized by being mono-substituted at the side chain amine of a histamine molecule with a substituent having an aliphatic chain of from 2 to 10 carbon atoms, wherein the alpha-carbon of the chain is methylene or is substituted with oxo or alkyl of from 1 to 3 carbon atoms, said chain terminating in hydrogen or carboxamido, wherein the carbo-amido nitrogen is substituted with alkyl of from 1 to 6 carbon atoms, tolyl, or trifluoromethylphenyl, with the proviso that when said chain terminates in hydrogen, the chain length is 5 or 6 atoms and a pharmaceutically acceptable earner or diluent.

29. The method of claim 28 further comprising the step of administering to said individual the protein allergen or an immunogenic portion thereof to specifically inhibit the allergen specific antibody response to said allergen or said immunogenic portion thereof.

30. The method of claim 28 further comprising the step of administering to said individual a peptide derived from said protein allergen, said peptide comprising at least one T cell epitope of said protein allergen.

31. The method of claim 29 further comprising the step of administering to said individual a peptide derived from said protein allergen, said peptide comprising at least one T cell epitope of said protein allergen.

32. The method of claim 30 further comprising the step of administering to said individual a peptide derived from said protein allergen, said peptide comprising at least two T cell epitopes of said protein allergen.

33. The method of claim 31 further comprising the step of administering to said individual a peptide derived from said protein allergen, said peptide comprising at least two T cell epitopes of said protein allergen.

34. The method of claim 30 wherein the protein allergen is 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 Ouercus; 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.

35. The method of claim 30 wherein the protein allergen is selected from the group consisting of: Der p I; Der p II; Der f I; Der f II; Amb a I.I; Amb 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.

36. A method for inhibiting a portion of an antigen specific antibody response by the immune system of a mammal comprising the steps of:

1) administering to said mammal a composition comprising at least one histamine derivative having binding specificity for at least one histamine receptor, said histamine derivative characterized by being mono-substituted at the side chain amine of a histamine molecule with a substituent having an aliphatic chain of from 2 to 10 carbon atoms, wherein the alpha-carbon of the chain is methylene or is substituted with oxo or alkyl of from 1 to 3 carbon atoms, said chain terminating in hydrogen or carboxamido, wherein the carboxamido mtrogen is substituted with alkyl of from 1 to 6 carbon atoms, tolyl, or trifluoromethylphenyl, with the proviso that when said chain terminates in hydrogen, the chain length is 5 or 6 atoms; and

2) administering to the mammal an antigen or an immunogenic portion thereof to specifically inhibit the antigen specific antibody response to said antigen or said immunogenic portion thereof.

37. The method of claim 26 further comprising the step of administering to said individual a peptide derived from said autoantigen, said peptide comprising at least one T cell epitope of said autoantigen.

38. The method of claim 22 wherein the predetermined antigen is an autoantigen and further comprising at least one T cell epitope of said autoantigen.