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Title:  Bystander suppression of type I diabetes by oral administration of glucagon

United States Patent:  6,645,504

Issued:  November 11, 2003

Inventors:  Weiner; Howard (Brookline, MA); Miller; Ariel (Haifa, IL); Zhang; Zhengyi (Needham, MA); Al-Sabbagh; Ahmad (Norwood, MA)

Assignee:  AutoImmune Inc. (Lexington, MA)

Appl. No.:  468996

Filed:  June 6, 1995

Abstract

Described are methods for treating or preventing type I diabetes and insulitis by oral administration of the bystander antigen glucagon. The methods involve oral administration of glucagon in an amounts that are effective to treat or prevent type I diabetes or insulitis.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected and surprising discovery that oral or enteral administration (or administration by inhalation) of certain antigens (called "bystander antigens" and defined below) causes T-cells to be elicited that in turn suppress cells that contribute to immune attack of the organ or tissue involved in an autoimmune disease. The T-cells elicited by the bystander antigen mediate the release of transforming growth factor beta (TGF-.beta.) which suppresses the cells contributing to the immune attack that are found in the same vicinity.

For this type of suppression mechanism to work, it is not necessary that the TGF-.beta. releasing T-cells recognize the disease-contributing cells. All that is necessary is that both types of cells be found in the same vicinity when TGF-.beta. is released. One way to achieve this is to use as the bystander antigen an antigen that (a) has the ability to elicit T-cells that cause release of TGF-.beta. and (b) is itself specific to the tissue or organ under attack so that the suppressor T-cells that cause release of TGF-.beta. (and that are elicited pursuant to oral administration of the bystander antigen) will be directed to the same organ or tissue which is also a location where the disease-promoting cells are concentrated.

The bystander antigens may but do not need to be autoantigens, i.e. they do not need to be the same antigen(s) that is (are) under attack by the disease-inducing cells. It is an interesting feature of the present invention that oral administration of a bystander antigen can stave off tissue damage done by cells specific for another antigen or antigen fragment. This second antigen (or fragment) does not even need to have been identified.

Therefore, in one aspect the present invention is directed to a method for treating an autoimmune disease in a mammal, the method comprising administering to said mammal an effective amount for treating said disease of a bystander antigen, said antigen eliciting the release of transforming growth factor beta (TGF-.beta.) at a locus within the body of said mammal wherein T cells contributing to autoimmune response are found to suppress the T-cells contributing to said response.

In another aspect, the present invention is directed to compositions and dosage forms comprising amounts of a bystander antigen effective to treat an autoimmune disease in a mammal.

In yet another aspect, the present invention provides a pharmaceutical inhalable dosage form for treating an autoimmune disease in a mammal, the form comprising an effective amount for treating said disease of a bystander antigen, said antigen upon administration eliciting the release of transforming growth factor beta (TGF-.beta.) at a locus within the body of said mammal wherein T cells contributing to autoimmune response are found to suppress the T-cells contributing to said response; and a pharmaceutically acceptable carrier or diluent.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents, and literature references cited in this specification are hereby incorporated by reference in their entirety. In case of inconsistencies, the description including the definitions and interpretations of the present disclosure will prevail.

Definitions

The following terms used in this disclosure shall have the meaning ascribed to them below:

(a) "Bystander antigen" or "bystander" is a protein, protein fragment, peptide, glycoprotein, or any other immunogenic substance (i.e. a substance capable of eliciting an immune response) that (i) upon oral or enteral administration (or administration by inhalation) elicits suppressor T-cells that cause TGF-.beta. to be released and thereby suppress cells that contribute to destruction of tissue during an autoimmune disease and even when the destructive cells are specific to a different immunogenic substance. Preferably, the suppressor T-cells elicited by the bystander will be targeted to the same tissue that is under attack during an autoimmune disease. The term therefore encompasses but is not limited to antigens capable of causing the foregoing release of TGF-.beta. and specific to the tissue or organ under attack in said autoimmune disease. The term also encompasses autoantigens and fragments or analogs thereof that have the ability to elicit such T-cell suppressors upon oral or enteral administration or upon inhalation. Thus, "bystander" is not coextensive with "autoantigen" as the latter is defined herein; an "autoantigen" is not also a "bystander" unless upon ingestion or inhalation it suppresses autoimmune response via the elicitation of T-suppressors that cause release of TGF-.beta. as described above.

(b) "Bystander suppression" is suppression of cells that contribute to autoimmune destruction by the release of the immunosuppressive factor TGF-.beta., this release being in turn mediated by suppressor T-cells elicited by the ingestion or inhalation of a bystander antigen and recruited to the site where cells contributing to autoimmune destruction are found. The result is downregulation of the specific autoimmune response.

(c) "Mammal" is defined herein as any organism having an immune system and being susceptible to an autoimmune disease.

(d) "Autoimmune disease" is defined herein as a malfunction of the immune system of mammals, including humans, in which the immune system fails to distinguish between foreign substances within the mammal and/or autologous tissues or substances and, as a result, treats autologous tissues and substances as if they were foreign and mounts an immune response against them.

(e) "Autoantigen" is any substance or a portion thereof normally found within a mammal that, in an abnormal situation, is no longer recognized as part of the mammal itself by the lymphocytes or antibodies of that mammal, and is therefore the primary target of attack by the immunoregulatory system as though it were a foreign substance. The term also includes antigenic substances which induce conditions having the characteristics of an autoimmune disease when administered to mammals.

(f) "Treatment" is intended to include both the prophylactic treatment to prevent an autoimmune disease (or to prevent the manifestation of clinical or subclinical, e.g., histological symptoms thereof), as well as the therapeutic suppression or alleviation of symptoms after the onset of such autoimmune disease.

(g) "Synergists" are defined herein as substances which augment or enhance the suppression of the clinical (and/or histological) manifestation of autoimmune diseases when administered orally or by inhalation in conjunction with the administration of a bystander antigen and/or an autoantigen. As used in the preceding sentence, and elsewhere in this specification, "in conjunction with" (also referred to herein as in association with) means before, substantially simultaneously with or after oral or aerosol administration of autoantigens and/or bystander antigens. Naturally, administration of the conjoined substance should not precede nor follow administration of the autoantigen or bystander antigen by so long an interval of time that the relevant effects of the substance administered first have worn off. Therefore, the synergists should be administered within about 24 hours before or after the autoantigen or bystander antigen, and preferably within about one hour.

(h) "Oral" administration includes oral, enteral or intragastric administration.

(i) A disease having the "characteristics" or "symptoms" of a particular autoimmune disease refers to a spontaneous or induced disease state that presents with specific inflammation of the same organ or tissue as that afflicted in the autoimmune disease. An example of an induced state is EAE, a model for multiple sclerosis. An example of a spontaneous state is diabetes developed by NOD mice.

Description of Bystander Suppression

It has now unexpectedly been discovered that the oral or by-inhalation administration of bystander antigens is an effective treatment for an autoimmune disease. At least cell-mediated autoimmune diseases can be treated using the methods and pharmaceutical formulations of the present invention.

Suppression mediated by oral administration of bystander antigens is brought about by elicitation of suppressor T-cells that release an immunosuppressive factor, transforming growth factor-beta (TGF-.beta.). TGF-.beta. is not specific for the antigen triggering the suppressor cells that release it, even though these suppressor T-cells release TGF-.beta. only when triggered by the orally administered (or inhaled) antigen. Recruitment of the suppressor T-cells to a locus within a mammal where cells contributing to the autoimmune destruction of an organ or tissue are concentrated allows for the release of TGF-.beta. in the vicinity of the disease-causing cells and suppresses (i.e. shuts down) these cells. The ability of TGF-.beta. to suppress these "destructive" cells is independent of the antigen for which the destructive cells may be specific.

The preferred way to accomplish suppression of the destructive cells is to select for oral administration to the mammal an antigen which is not only capable of eliciting suppressor T-cells capable of releasing TGF-.beta. but which is capable of targeting these suppressor T-cells to a location within the mammal's body where destructive cells are found in high concentration. The preferred and most efficient target for the suppressor T-cells is the organ or tissue under immune attack in the particular autoimmune disease involved because the destructive cells will be concentrated in the vicinity of that organ or tissue. Hence, it is preferred that the bystander antigen (to which the suppressor T-cells are specific) be itself an antigen specific to the tissue or organ under attack. Thus, the bystander antigen may be an autoantigen or preferably a non-disease inducing fragment or analog of an autoantigen (there is evidence that the parts or epitopes of autoantigens that are involved in inducing disease or in tissue destruction are not the same as those involved in bystander suppression; See Example 6 below). More important, however, the bystander may be another antigen that is not an autoantigen; hence, the autoantigen (or autoantigens) involved need not be identified.

In more detail, the mechanism of bystander suppression according to the present invention for a tissue-specific bystander antigen is as follows: After a tissue-specific bystander antigen is administered orally (or enterally, i.e., directly into the stomach) or by inhalation, it passes into the small intestine, where it comes into contact with the so-called Peyers Patches, which are collections of immunocytes located under the intestinal wall. These cells, are in turn in communication with the immune system, including the spleen and lymph nodes. The result is that suppressor (CD8+) T-cells are induced and recruited to the area of autoimmune attack, where they cause the release of TGF-.beta., which can non-specifically downregulate the B-cells as well as the activated CD4+T-cells directed against the mammal's own tissues. Despite the non-specific nature of the activity of TGF-.beta., the resulting tolerance is specific for the autoimmune disease by virtue of the fact that the bystander antigen is specific for the tissue under attack and suppresses the immune cells that are found at or near the tissue being damaged.

Another instance of bystander suppression within the scope of the invention involves oral administration of an antigen that is neither an autoantigen nor specific to a tissue or organ under attack. To activate bystander suppression, injection with the same antigen has to take place. The ingested or inhaled antigen elicits formation of suppressor T-cells which are targeted to the microenvironment, pathway or inflamed tissue (depending on where the injected antigen localizes) where they cause the release of TGF-.beta.. Once released, TGF-.beta. suppresses all immune attack cells including the tissue-destructive cells.

TGF-.beta.

TGF-.beta. affects cells of the immune system (e.g., T and B lymphocytes) thereby influencing inflammatory responses. T-lymphocytes (and other cells) produce TGF-.beta.; it is released relatively late in the cascade of immune system response events (after T-cell activation) and is highly suppressive for both T- and B-cell proliferation. Numerous normal tissues have the ability to produce TGF-.beta.. These include human platelets, placenta, bovine kidney, bone, NK cells, B-cells, as well as CD4+ and CD8+ T-cells and activated macrophages. The isolation and biological properties of TGF-.beta. have been described in Transforming Growth Factor-.beta.s Chemistry, Biology, and Therapeutics, Piez, K. A. et al Eds, Ann. N.Y. Acad. Sci. 593:1-217, 1990.

Although TGF-.beta. was initially identified as a growth factor, it soon became clear that it was a substance having many and important immunoregulatory properties including inhibition of B- and T-cells and inhibition of the activity of CD4+ cells more than that of CD8+ cells, both in rodents and humans. TGF-.beta. is also known to antagonize inflammatory cytokines such as tumor necrosis factor (TNF) and gamma interferon (IFN-.gamma.), block cytotoxic lymphocyte activity and inhibit the induction of receptors for Interleukin-1 (IL-1) and Interleukin-2 (IL-2) thereby rendering cells unresponsive to these cytokines. TGF-.beta. is a protein which has a molecular weight of 25 kD and is composed of two identical 12.5 kD subunits that are held together by a number of interchain disulfide bonds. At least two forms of TGF-.beta. exist: active and latent. Active TGF-.beta. has a short half-life and a small volume distribution whereas latent TGF-.beta. has an extended half-life and a larger volume distribution. Two isoforms of TGF-.beta. exist, TGF-.beta.1 and TGF-.beta.2. It is believed that TGF-.beta.1 is involved in bystander suppression.

Animal Models

Throughout the present specification, reference is made to various model systems that have been developed for studying autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE) has been studied in mice and other mammalian species as a model for Multiple Sclerosis (MS). Those of ordinary skill in the art recognize that virtually all potential immune therapies for MS are first tested in this animal model system. The disease is induced by parenteral administration of myelin basic protein (MBP) or proteolipid protein (PLP) and an adjuvant (such as Freund's Complete Adjuvant, FCA). This treatment, with either antigen, induces both a monophasic and an exacerbating/remitting form of demyelinating disease (depending on the species and details of administration). The induced disease has the characteristics of the autoimmune disease MS.

Parenteral administration of Mycobacterium tuberculosis with Freund's Complete Adjuvant in oil into the dorsal root tail of susceptible mammals induces a disease with the characteristics of human rheumatoid arthritis. In like manner, parenteral administration of Type II collagen with an adjuvant will also induce a disease with the characteristics of human rheumatoid arthritis.

The administration to Lewis rats of S-antigen or IRBP-antigen with an adjuvant induces autoimmune uveoretinitis, whereas diabetes develops spontaneously in the NOD Mouse and the BB Rat.

One or more of the above disclosed model systems may be employed to demonstrate the efficacy and improved treatment provided by the present invention. In fact, the animal models are particularly suitable for testing therapies involving bystander suppression precisely because this mechanism allows suppression of all immune attack cells regardless of the antigen to which they are specific and is therefore unaffected by many of the actual or potential differences between a human autoimmune disorder and an animal model therefor.

The above animal models can be used to establish the utility of the present invention in mammals (including humans). For example, the present inventors orally administered a multiple sclerosis autoantigen, bovine myelin, to humans in a double-blind study and found that a certain patient subset received a considerable benefit from this treatment. In addition, rheumatoid arthritis symptoms, such as joint tenderness, AM stiffness, grip strength, etc., were successfully suppressed in humans receiving oral collagen (0.1-1.0 mg single dose daily). Finally, human trials with oral S-antigen showed very encouraging results for uveoretinitis. All of these human trials were attempted based on animal data using the appropriate disease model. Thus, the predictive value of animal models for therapeutic treatment of autoimmune diseases has been substantially enhanced.

Bystander Antigens

Bystander antigens not specific to the tissue under attack during autoimmune disease can be identified among nontoxic antigenic substances by using the same assay system as was used for OVA in e.g. Example 1.

Bystander antigens specific to a tissue or organ can be easily identified by testing the ability of such specific antigens to cause release of TGF-.beta., which can be detected. For example, one or more potential tissue specific bystander antigens can be purified using well-known antigen purification techniques from an organ or tissue that is the target of autoimmune attack.

Bystander antigens and autoantigens (as well as fragments and analogs of any of them) can also be obtained using recombinant DNA technology, in bacterial, yeast, insect (e.g. psacalan virus) and mammalian cells using techniques well-known to those of ordinary skill in the art. Amino acid sequences for many potential and actual bystander antigens are known (disease-inducing epitopes should preferably not be used): See, e.g., Hunt, C. et al PNAS (USA), 82:6455-6459, 1985 (heat shock protein hsp70); Burkhardt, H., et al.,Eur. J. Immunol. 21:49-54, 1991 (antigenic collagen II epitope); Tuohy, V. K., et al., J. Immunol. 142:1523-1527, 1989 (encephalitogenic determinant of mouse PLP); Shinohara, T. et al., In Progress in Retinal Research, Osborne, N. & Chader, J. Eds, Pergamon Press 1989, pp. 51-55 (S-antigen); Donoso, L. A., et al., J. Immunol. 143:79-83, 1989 (IRBP); Borst, D. E., et al., J. Biol. Chem. 264:115-1123, 1989 (IRBP); Yamaki, K. et al., FEBS 234:39-43, 1988 (S-antigen); Donoso, L. A. et al., Eye Res. 7:1087, 1988 (IRBP); Wyborski, R. J., et al., Mol. Brain Res. 8:193-198, 1990 (GAD).

The amino acid sequences for bovine PLP (SEQ. ID NO:1); bovine MBP (SEQ. ID NO:5), human MBP (SEQ. ID NO:4), chicken MBP (SEQ. ID NO:9), rat MBP (SEQ. ID NO:8), rabbit MBP (SEQ. ID NO:6), guinea pig MBP (SEQ. ID NO:7); human collagen alpha-1 (II) (SEQ. ID NO:10) and bovine collagen alpha-1 (II) (SEQ. ID NO:11) and bovine collagen alpha-1 (I) (SEQ. ID NO:12); and human insulin (SEQ. ID NOS: 2 and 3) are taken from published sources.

In addition, some tissue-specific antigens are commercially available: e.g. insulin, glucagon, myelin basic protein, collagen I, collagen II, etc.

The potential bystander can then be fed to mammals and spleen cells or circulating T-cells from, e.g. the blood or cerebrospinal fluid in the case of EAE or MS, from these mammals can be removed, and stimulated in vitro with the same antigen. T-cells elicited by stimulation can be purified and supernatants can be tested for TGF-.beta. content quantitatively and/or qualitatively using e.g. a suitable commercially available polyclonal or preferably monoclonal antibody raised against TGF-.beta. or another known assay for TGF-.beta. detection such as that described in Example 1 below using a commercially available mink lung epithelial cell line. Such methods for testing for TGF-.beta. are described in detail in the Examples, below. Methods for ascertaining the bystander potential of peptides derived from autoantigens are also illustrated in the Examples.

Use of Bystander Antigens--Dosages

The tolerance induced by the bystander antigens of this invention is dose-dependent over a broad range of oral (or enteral) or inhalable dosages. However, there are minimum and maximum effective dosages. In other words, suppression of the clinical and histological symptoms of an autoimmune disease occurs within a specific dosage range which however varies from disease to disease, mammal to mammal and bystander antigen to bystander antigen. For example, when the disease is PLP- or MBP-induced EAE in mice, the suppressive dosage range when MBP is used as the bystander is from about 0.1 to about 1 mg/mouse/feeding (with feedings occurring about every other day (e.g., 5-7 feedings over a 10-14-day period). A most preferred dosage is 0.25 mg/mouse/feeding. For suppression of the same disease in rats, the MBP suppressive dosage range is from about 0.5 to about 5 mg/rat/feeding and the most preferred dosage is 1 mg/rat/feeding. The effective dosage range for humans with MS, when MBP is used, is between about 1 and about 100, preferably between about 1 and about 50 mg MBP per day (administered every day or on alternate days) with the optimum being about 30 mg/day.

For rheumatoid arthritis, the effective dosage range for humans receiving either Type I or II collagen is about 0.1 to about 1 mg/day. For adjuvant-induced arthritis in mice the effective collagen dosage range is about 3 to about 30 micrograms/feeding with the same feeding schedule as for EAE.

Ascertaining the effective dosage range as well as the optimum amount is well within the skill in the art. For example, dosages for mammals and human dosages can be determined by beginning with a relatively low dose (e.g., 1 microgram), progressively increasing it (e.g. logarithmically) and measuring the amount of TGF-beta in the blood and/or scoring the disease severity, according to well-known scoring methods (e.g., on a scale of 1 to 5, or by measuring the number of attacks, or by measuring joint swelling, grip strength, stiffness, vision, etc. depending on the type of disease). The optimum dosage will be the one generating the maximum amount of TGF-beta in the blood and/or cause the greatest decrease in disease symptoms. An effective dosage range will be one that causes at least a statistically significant attenuation of at least one symptom characteristic of the disease being treated.

The present invention can also be advantageously used to prevent the onset of an autoimmune disease in susceptible individuals at risk for an autoimmune disease. For example, methods for the identification of patients who are at risk for developing Type 1 diabetes are extant and reliable and have been recently endorsed by the American Diabetes Association (ADA). Various assay systems have been developed which (especially in combination) have a high predictive value assessing susceptibility to Type 1 diabetes (Diabetes Care 13: 762-775, 1990. Details of one preferred screening test are available to those of ordinary skill in the art (Bonifacio, E. et al., The Lancet 335: 147-149, 1990).

From a practical point of view, preventing the onset of most autoimmune diseases is not as important a measure as it is in the case of diabetes. MS, RA, AT and AUR are declared at an early stage, before substantial tissue damage has taken place; therefore preventive treatment of these diseases is not as important as in the case of diabetes.

A non-limiting list of autoimmune diseases and tissue- or organ-specific confirmed or potential bystander antigens effective in the treatment of these diseases when administered in an oral or inhalable form are set forth in Table 1 below. Administration of combinations of antigens listed for each individual disease is also expected to be effective in treating the disease.

        TABLE 1
        Autoimmune Disease         Bystander Antigen
        Type 1 Diabetes (While beta- Glucagon, insulin, GAD (gamma
        cell function is still     amino decarboxylase), heat
        present)                   shock protein
        Multiple Sclerosis         MBP, MBP fragments
                                   (especially non-inducing),
                                   PLP, PLP fragments
                                   (especially non-inducing)
        Rheumatoid Arthritis       Collagen, collagen fragments
                                   (especially non-inducing),
                                   heat shock protein
        Uveoretinitis              S-antigen, S-antigen
                                   fragments (especially non-
                                   inducing), IRBP
                                   (Interphotoreceptor Retinoid
                                   Binding Protein) and
                                   fragments thereof (especially
                                   non-inducing)

For any autoimmune disease, tissue extracts can be used as well as specific bystander antigens. For example, myelin has been used for MS and pancreatic extracts have been used for Type 1 diabetes. However, administration of one or more individual antigens is preferred.

Thus, according to the present invention, when treating Type 1 diabetes, an effective amount (determined as described above) of glucagon can be administered orally or by inhalation. Glucagon is specifically present in the pancreas. Glucagon, however, is not an autoantigen because it is pancreatic beta cells that are destroyed in the course of Type 1 diabetes whereas glucagon is found exclusively in alpha cells, a different cell type. Thus, glucagon is a "pure" bystander: it does not have any autoantigen activity.

Insulin definitely has bystander activity for Type 1 diabetes. It is not at present known whether insulin is also an autoantigen. However, whatever the mechanism of action, oral, enteral or inhalable insulin preparations are effective in suppressing diseases with the characteristics of Type 1 diabetes as per copending commonly assigned patent application Ser. No. 595,468.

For diseases having the characteristics of multiple sclerosis, non-inducing fragments of MBP, e.g. a peptide comprising guinea pig MBP amino acids 21-40 act as bystanders not only for MBP-induced diseases (i.e. when MBP is the primary target of autoimmune attack) but also for PLP-induced disease (when PLP is the primary target of autoimmune attack).

For rheumatoid arthritis and animal models therefor, Type-I and Type-II collagen have bystander activity.

For diseases having the characteristics of uveoretinitis, S-antigen has bystander activity.

Noninducing fragments of those bystander antigens that are also autoantigens are preferred. Such fragments can be determined using the overlapping peptide method of Example 3 (which is a general technique although in Example 3 it is described specifically with respect to identification of noninducing fragments of MBP).

The present inventors have also discovered that orally administered autoantigens and bystander antigens both possess epitopes which specifically induce the production and/or release of TGF-.beta.. Although immunodominant epitopes of e.g., MBP have previously been disclosed, i.e., those epitopes which a majority of patients' CD4+ T lymphocytes recognize and proliferate in response to, or which a majority of a patient's antibodies recognize, immunosuppressive epitopes, i.e., those that elicit the production and/or release of TGF-.beta., have not been disclosed or suggested before the present invention. Therefore, oral or by-inhalation administration of peptides encompassing these epitopes is expected to be more specific in eliciting bystander suppression than administration of the entire antigen without the risk of sensitizing the animal to disease-inducing or disease-propagating portions of an autoantigen. The immunosuppressive epitopes can be identified using the method described in Example 3 for the identification of MBP-peptide 21-40. (See also FIG. 14.)

The bystander antigens can be administered in conjunction with autoantigens (the combination being effective) to treat or prevent autoimmune diseases. Autoantigen administration is carried out as disclosed in U.S. patent application Ser. Nos. 460,852, 596,936, 454,486, 551,632, 502,559, 607,826 and 595,468 mentioned above. It is anticipated that co-administration of specific autoantigens (and preferably non-inducing fragments of autoantigens) with other bystander antigens will provide effective suppression of the autoimmune diseases.

In addition, synergists can be conjoined in the treatment to enhance the effectiveness of the above. Non-limiting examples of synergists for use in the present invention include bacterial lipopolysaccharides from a wide variety of gram negative bacteria such as various subtypes of E. coli and Salmonella (LPS, Sigma Chemical Co., St. Louis, Mo.; Difco, Detroit, Mich.; BIOMOL Res. Labs., Plymouth, Pa.), Lipid A (Sigma Chemical Co., St. Louis, Mo.; ICN Biochemicals, Cleveland, Ohio; Polysciences, Inc., Warrington, Pa.) and immunoregulatory lipoproteins, such as peptides covalently linked to tripalmitoyl-S-glycarylcysteinyl-seryl-serine (P3 C55) which can be obtained as disclosed in Deres, K. et al. (Nature, 342:561-564, 1989) or "Brauns" lipoprotein from E. coli which can be obtained as disclosed in Braun, V., Biochim. Biophys. Acta 435:335-337, 1976. LPS is preferred and Lipid A particularly preferred. Lipid A is particularly preferred for use in the present invention because it is less toxic than the entire LPS molecule. LPS for use in the present invention can be extracted from gram-negative bacteria and purified using the method of Galanes et al. (Eur. J. Biochem. 9:245, 1969) and Skelly, R. R., et al. (Infect. Immun. 23:287, 1979).

Formulations

In another aspect, the present invention also provides oral pharmaceutical formulations for treating mammals suffering from autoimmune diseases comprising an amount of a bystander antigen (as described below) effective to suppress the autoimmune disease. The formulations optionally further comprise a synergist as disclosed in copending U.S. patent application, Ser. No. 487,732, filed Mar. 2, 1990 in an amount effective (in conjunction with the bystander antigen of the present invention) to treat the clinical symptoms of specific autoimmune diseases. Synergists, when administered in conjunction with bystander antigens, cause an increase of cytokines PGE (prostaglandin-E) and IL-4 (interleukin-4) in the vicinity of the target organ.

Throughout this discussion, it will be understood that any statistically significant attenuation of even one symptom of an autoimmune disease pursuant to the treatment of the present invention is within the scope of the invention.

Each oral (or enteral) formulation according to the present invention may additionally comprise inert constituents including pharmaceutically acceptable carriers, diluents, fillers, solubilizing or emulsifying agents, and salts, as is well-known in the art. For example, tablets may be formulated in accordance with conventional procedures employing solid carriers well-known in the art. Capsules employed in the present invention may be made from any pharmaceutically acceptable material, such as gelatin, or cellulose derivatives. Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are also contemplated, such as those described in U.S. Pat. No. 4,704,295, issued Nov. 3, 1987; U.S. Pat. No. 4,556,552, issued Dec. 3, 1985; U.S. Pat. No. 4,309,404, issued Jan. 5, 1982; and U.S. Pat. No. 4,309,406, issued Jan. 5, 1982.

Examples of solid carriers include starch, sugar, bentonite, silica, and other commonly used carriers. Further non-limiting examples of carriers and diluents which may be used in the formulations of the present invention include saline, syrup, dextrose, and water.

It will be appreciated that the unit content of active ingredient or ingredients contained in an individual dose of each dosage form need not in itself constitute an effective amount, since the necessary effective amount can be reached by administration of a plurality of dosage units (such as capsules or tablets or combinations thereof).

The route of administration of the bystander antigens of the present invention is preferably oral or enteral. The preferred oral or enteral pharmaceutical formulations may comprise, for example, a pill or capsule containing an effective amount of one or more of the bystander antigens of the present invention with or without an effective amount of a synergist.

In general, when administered orally or enterally, the bystander antigen may be administered in single dosage form or multiple dosage forms.

The effective amount of a synergist, e.g. LPS or Lipid A, to be administered in conjunction with the bystander broadly ranges between about 0.15 and 15 mg per kg body weight of said mammal per day and preferably between about 0.3 and 12 mg per kg body weight of said mammal per day.

In an alternative preferred embodiment of the present invention the pharmaceutical formulations or dosage forms of the present invention can also be administered to mammals suffering from autoimmune diseases by inhalation, preferably in aerosol form. The inhalation mode of administration is preferably not through the nasal passages but through the bronchial and pulmonary mucosa. It is expected that lower amounts of the bystander antigens of the present invention will be required using aerosol administration for treating an autoimmune disease as it has been found when treating experimental autoimmune encephalomyelitis (EAE) with myelin basic protein (MBP) and adjuvant arthritis with collagen as disclosed in co-pending U.S. patent application Ser. No. 454,486 filed Dec. 20, 1989. The amounts of the bystander antigens of the present invention which may be administered in an aerosol dosage form would be between about 0.1 mg and about 15 mg per kg body weight of a mammal per day and may optionally include a synergist in amounts ranging between about 0.1 and about 15 mg per kg body weight of said mammal per day and may be administered in single dosage form or multiple dosage forms. The exact amount to be administered will vary depending on the state and severity of a patient's disease and the physical condition of the patient.

The aerosol pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing and emulsifying agents, and salts of the type that are well-known in the art. Examples of such substances include normal saline solutions, such as physiologically buffered saline solutions, and water.

The route of administration of the bystander antigens according to this alternate embodiment of the present invention is in an aerosol or inhaled form. The bystander antigens and related compounds of the present invention can be administered as dry powder particles or as an atomized aqueous solution suspended in a carrier gas (e.g. air or N2). Preferred aerosol pharmaceutical formulations may comprise for example, a physiologically-acceptable buffered saline solution containing between about 1 mg and about 300 mg of the bystander antigens of the present invention.

Dry aerosol in the form of finely divided solid particles of bystander antigens that are not dissolved or suspended in a liquid are also useful in the practice of the present invention. The bystander antigens may be in the form of dusting powders and comprise finely divided particles having an average particle size of between about 1 and 5 microns, preferably between 2 and 3 microns. Finely divided particles may be prepared by pulverization and screen filtration using techniques well known in the art. The particles may be administered by inhaling a predetermined quantity of the finely divided material, which can be in the form of a powder.

Specific non-limiting examples of the carriers and/or diluents that are useful in the aerosol pharmaceutical formulations of the present invention include water and physiologically-acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7.0-8.0. Additional non-limiting examples of suitable carriers or diluents for use in the aerosol pharmaceutical formulations or dosage forms of the present invention are disclosed in U.S. Pat. No. 4,659,696, issued Apr. 21, 1987, U.S. Pat. No. 4,863,720, issued Sep. 5, 1989 and U.S. Pat. No. 4,698,332, issued Oct. 6, 1987.

The pharmaceutical formulations of the present invention may be administered in the form of an aerosol spray using for example, a nebulizer such as those described in U.S. Pat. No. 4,624,251 issued Nov. 25, 1986; U.S. Pat. No. 3,703,173 issued Nov. 21, 1972; U.S. Pat. No. 3,561,444 issued Feb. 9, 1971 and U.S. Pat. No. 4,635,627 issued Jan. 13, 1971. The aerosol material is inhaled by the subject to be treated.

Other systems of aerosol delivery, such as the pressurized metered dose inhaler (MDI) and the dry powder inhaler as disclosed in Newman, S. P. in Aerosols and the Lung, Clarke, S. W. and Davia, D. eds. pp. 197-224, Butterworths, London, England, 1984, can be used when practicing the present invention.

Aerosol delivery systems of the type disclosed herein are available from numerous commercial sources including Fisons Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, N.J.) and American Pharmoseal Co. (Valencia, Calif.).

As will be understood by those skilled in the art, the exact dosage and frequency of administration of the bystander antigens of the present invention (in oral or aerosol form) is a function of the activity of the bystander antigen, as well as the ages sex, weight, and physical condition of the subject to be treated, and the concurrent administration or absence of other treatments. Consequently, adjustment of the dosages used and administration schedules must be determined based on these factors, and may need to be determined experimentally. Such determination, however, requires no more than routine experimentation, given the guidelines contained herein.

Claim 1 of 6 Claims

What is claimed is:

1. A method for suppressing an autoimmune response associated with an autoimmune disease in a human, the method comprising orally or enterally administering to said human an effective amount for abating said response of a composition comprising a bystander antigen, wherein said disease is Type I diabetes and said bystander antigen is glucagon.




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