WO2001052860A2 - Method and composition for modulating an immune response - Google Patents

Method and composition for modulating an immune response Download PDF

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Publication number
WO2001052860A2
WO2001052860A2 PCT/US2001/001678 US0101678W WO0152860A2 WO 2001052860 A2 WO2001052860 A2 WO 2001052860A2 US 0101678 W US0101678 W US 0101678W WO 0152860 A2 WO0152860 A2 WO 0152860A2
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method
subject
immunomodulator
inosine
administered
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PCT/US2001/001678
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French (fr)
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WO2001052860A3 (en
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George Hasko
Csaba Szabo
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Inotek Corporation
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Abstract

Disclosed is a method of inhibiting or preventing a condition associated with undesired secretion of a macrophage inflammatory protein using inhibitors of ATP-sensitive K+-channels, inhibitors of the Na+/H+ antiporter, or inosine.

Description

METHOD AND COMPOSITION FOR MODULATING AN IMMUNE

RESPONSE

FIELD OF THE INVENTION

The invention relates to a method and composition for treatment of a condition associated with undesired secretion of a macrophage inflammatory protein

BACKGROUND OF THE INVENTION

Various forms of inflammation are characterized by activation of macrophages

Macrophages are thought to induce and maintain inflammatory processes mainly by producing various products which, acting on other cells, bring about the deleterious consequences of inflammation For example, macrophages produce cytokines These proteins are central mediators in inflammatory processes, such as the local inflammatory processes characteristic of arthritis or colitis Cytokines produced by macrophages are also thought to be involved in systemic inflammatory processes, such as endotoxic shock. Macrophage products are more generally involved in pathophysiological mechanisms, such as plasma extravasation, inflammatory cell diapedesis, release of toxic free radicals, endothelial injury, and release of tissue degrading enzymes, which can result in tissue injury and, ultimately, organ failure

Tumor necrosis factor (TNF) is another cytokine associated with macrophage activation TNF is also thought to be involved in inducing most of the pathophysiological events characteristic of inflammation For example, it is a key cytokine in the toxic effect of endotoxin (LPS) and in the pathogenesis of septic shock Serum plasma levels of TNF are high after LPS administered to animals and human volunteers or in septic subjects, while administration of anti-TNF antibodies protects against the lethal effects of LPS and live bacteria in a variety of animal models Moreover, TNF can be a central target in rheumatoid arthritis

Interleukin 12 (IL)-12 is another macrophage product which has been shown to be involved in the induction of pathology in several inflammatory diseases These diseases include autoimmune diseases such as multiple sclerosis, inflammatory bowel disease, insulin dependent diabetes mellitus, and rheumatoid arthritis, and inflammatory states such as septic shock and the generalized Schwarzman reaction For example, administration of antι-EL-12 antibodies substantially reduces the incidence and severity of adoptively transferred experimental allergic encephalomvehtis, suggesting that endogenous IL-12 is involved in its pathogenesis Furthermore, in adjuvant-induced arthritis, the course of the disease is suppressed in IL-12 deficient mice or in mice treated with antι-mIL-12 antibodies

The chemokine macrophage inflammatory protein (MΙP)-lα and the CXC chemokine MTP-2 are additional proinflammatory proteins expressed by macrophages

SUMMARY OF THE INVENTION

The invention is based in part on the discovery that various small molecules inhibit the release of macrophage inflammatory proteins Accordingly, the invention provides a method of treating a subject having or at risk for a condition associated with undesired secretion of a macrophage inflammatory protein The method includes administering an immunomodulator that is an inhibitor of KΛTP channels, an inhibitor of a Na+ H+ transporter, or inosine, or an analog thereof

In one aspect, the invention includes a method of treating a subject having, or at πsk for, a condition associated with undesired secretion of a macrophage inflammatory protein The method includes administering to the subject an amount of an immunomodulator in an amount sufficient to treat or delay the onset of the condition, wherein the immunomodulator is selected from the group consisting of a KATP channel inhibitor, an inhibitor of a NaTFT exchanger, inosine, and an inosine analog

The condition can be, e g , inflammation, shock, or both The inflammation can be associated with a condition such as e g , diabetes mellitus (including autoimmune diabetes) adult respiratory distress syndrome, arthritis, vasicuhtis, autoimmune disease, lupus erythematosus, lleitis, ulcerative colitis, Crohn's disease, asthma, gingivitis, peπodontitis, ophthalmitis, endophthalmitis, nephrosis, AIDS-related neurodegeneration, stroke, neurotrauma, Alzheimer's disease, encephalomvehtis, cardio-myopathy, transplant rejection and cancer Examples of conditions associated with shock include shock caused by, or associated with, gram positive bacteria-mediated circulatory shock, gram negative bacteria-mediated circulatory shock, hemorrhagic shock, anaphylactic shock, systemic inflammation, pro- inflammatory cytokines, and systemic inflammatory response syndrome (SIRS)

The immunomodulator can be administered via, e.g., intravenous, intramuscular, subcutaneous, sublingual, oral, rectal or aerosol delivery Administration of the immunomodulator can be prophylactic, therapeutic, or both

The immunomodulator can be e.g., a KAτp channel-blocking inhibitor In some embodiments, the inhibitor inhibits a macrophage KATP channel An example of a KATP channel inhibitor is a sulphonylurea compound, such as glibenclamide

In some embodiments, the immunomodulator is an inhibitor of a Na+/H+ exchanger, e.g., a pyrazinoylguanidine derivative or a pteridine derivative An example of a pyrazinoylguanidine derivative immunomodulator is amiloride An example of a pteridine derivative immunomodulator is triamterene

In some embodiments, the immunomodulator is inosine or an inosine analog

In some embodiments, the immunomodulator is one or more of amiloride, inosine, glibenclamide, 5-(N,-N-dimethyl)-amiloride hydrochloride, 5-(N-ethyl-N-isopropyl)- amiloride; 5(N, N-hexamethylene)-amiloride, 5-(N-methyl-N-isobutyl)-amiloride), benzamil, tolbutamide, glipizide, 2,3-butanedione monoxime, meglitinide

The subject can be, e.g., a mammal, such as a rat, mouse, rabbit, guinea pig, hamster, cow, pig, horse, goat, sheep, dog, cat, non-human primate, or human

In a further aspect, the invention includes a method for treating or preventing diabetes, e.g., autoimmune diabetes, by administering to a patient in need of such treatment a safe and therapeutically effective amount of inosine, or an inosine receptor ligand, e.g., a compound which binds to an inosine binding site

The immunomodulator can be administered via, e.g. , intravenous, intramuscular, subcutaneous, sublingual, oral, rectal or aerosol delivery Administration of the immunomodulator can be prophylactic, therapeutic, or both The subject can be, e.g., a mammal, such as a rat, mouse, rabbit, guinea pig, hamster, cow, pig, horse, goat, sheep, dog, cat, non-human primate, or human

Also provided is a method of increasing insulin levels in a subject The method includes administering to a subject in need thereof an amount of inosine or a ligand for an inosine binding site in an amount sufficient to increase insulin levels in said subject. In preferred embodiments, administering the inosine or ligand to the subject "increases pancreatic insulin levels in the subject

The methods and pharmaceutical compositions described herein can used to inhibit or prevent secretion of inflammatory proteins such as TNF, IL-12, MlP-lα, and MIP-2. Because of the pivotal role of these proteins in the initiation and maintenance of disease process in inflammatory diseases, these cytokines are ideal targets for anti-inflammatory therapy in these disease states The methods and described herein can simultaneously inhibit release of multiple inflammatory proteins Because these inflammatory proteins act in distinct ways, higher therapeutic effectiveness can be obtained with the herein-described methods and compositions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety In case of conflict, the present specification, including definitions, will control In addition, the materials, methods, and examples are illustrative only and not intended to be limiting

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing showing the release of various cytokines over time following administration of inosine in mice.

FIG. 2 is a graph showing the number of mice surviving (y-axis) over time (x-axis) following exposure to challenge with LPS following pretreatment with drug vehicle (physiologic saline) or 100-mg/kg inosine.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for treating disorders associated with undesired secretion of macrophage inflammatory proteins. The invention is based in part on the observations that: (a) inhibitors of the ATP-gated K+ channel inhibit secretion of inflammatory cytokines; (b) inhibitors of the Na+/H+ transporter inhibit secretion of inflammatory cytokines; and (c) the nucleoside inosine inhibits secretion of inflammatory cytokines.

Accordingly, in one aspect, the invention provides a method of treating a subject having or at risk for a condition associated with undesired secretion of a macrophage inflammatory protein. By "at risk for" is meant a state that negatively impacts a subject such that it has an increased likelihood of developing a condition associated with undesired secretion of a macrophage inflammatory protein. "Undesired" as used herein is secretion of an inflammatory protein that causes, or is otherwise associated with, an undesired physiological reaction in the subject. Inflammatory proteins include proteins such as TNF, IL-12, MTP-lα, MTP-2, or IFN-γ.

The method includes administering to the subject an immunomodulator in an amount sufficient to treat, or delay the onset of, the condition. The immunomodulator preferably inhibits secretion of two or more macrophage inflammatory proteins. Alternatively, or in addition, the immunomodulator inhibits secretion of one or more macrophage inflammatory proteins while promoting expression of one or more anti-inflammatory proteins. An example of a macrophage anti-inflammatory protein is IL-10. In another aspect, the invention provides pharmaceutical compositions comprising one or more of the herein-described immunomodulators. The compositions can be used for treating a subject having or at risk for a condition associated with undesired secretion of the macrophage inflammatory protein As used herein, an "immunomodulator" is a compound that modulates an immune response by inhibiting expression or activity of one or more macrophage inflammatory proteins Expression can be inhibited, for example, by inhibiting secretion of the inflammatory proteins Examples of immunomodulators include a KATP channel-blocking inhibitor, an inhibitor of a NaTrT exchanger, and/or inosine, or analogs thereof. Preferably, the KATP channel-blocking inhibitor inhibits a macrophage KATP channel.

An example of a KATP channel-blocking inhibitor is a sulphonylurea compound.

Examples of sulphonylurea compounds include glibenclamide, glipizide, and tolbutamide. This class of drugs, which is used to treat non-insulin dependent diabetes mellitus, is activated by the antihypertensive agents diazoxide or minoxidil. Accordingly, in some embodiments, the KATP channel-blocking inhibitors are administered with one or more of these antihypertensive agents

Na+/H+ exchanger inhibitors can e.g., a pyrazinoylguanidine derivative or a pteridine derivative, or analogs thereof. Preferred pyrazinoylguanidine derivatives include amilioride or an amiliroide analog, e.g., 5-(N,-N-dimethyl)-amiloride hydrochloride; 5-(N-ethyl-N- isopropyl)-amiloride; 5(N, N-hexamethylene)-amiloride; 5-(N-methyl-N-isobutyl)-amiloride, benzamil, and 3,4-dichlorobenzamil Preferred pteridine derivatives include, e.g., triamterene

Preferred KATP channel inhibitors include, e.g. 2,3-butanedione monoxime; meglitinide, glipizide, tolbutamide, chlorprpopamide, tolazamide, gliclazide, and repaglinide

Examples of inosine analogs include, e.g , 8-bromo-inosine, and 8-chloroinosine. Inosine analogs include those which bind to an inosine binding site.

The immunomodulators can be provided in a pure form or in a pharmaceutically acceptable carrier and can be used to treat or prevent conditions and disorders associated with undesired secretion of one or more macrophage inflammatory proteins. For example, the immunomodulators may be used to treat, or to delay the appearance of, diseases associated with inflammation Examples of such diseases include chronic inflammatory disorders of the joints including arthritis, e g , rheumatoid arthritis and osteoarthritis, inflammatory bowel diseases such as lleitis, ulcerative colitis and Crohn's disease, inflammatory lung disorders such as asthma and chronic obstructive airway disease Other examples of disorders include inflammatory disorders of the eye such as corneal dystrophy, trachoma, onchocerciasis, uveitis, sympathetic ophthalmitis and endophthalmitis Disorders may also include chronic inflammatory disorders of the gum, e g , peπodontitis, tuberculosis, leprosy, inflammatory diseases of the kidney including glomerulonephritis and nephrosis, inflammatory disorders of the skin including sclerodermatitis, psoriasis and eczema, inflammatory diseases of the central nervous system, including AFDS-related neurodegeneration, stroke, neurotraua and Alzheimer's disease, encephalomyehtis and viral or autoimmune encephalitis, autoimmune diseases including immune-complex vascuhtis, systemic lupus and erythematodes, systemic lupus erythematosus (SLE), and inflammatory diseases of the heart such as cardiomyopathy Additional examples include adult respiratory distress syndrome, arthritis e g , rheumatoid arthritis, osteo arthritis vasiculitis, autoimmune disease, lupus erythematosus, lleitis, ulcerative colitis, Crohn's disease, asthma, gingivitis, peπodontitis, ophthalmitis, endophthalmitis, nephrosis, AIDS-related neurodegeneration, stroke, neurotrauma, Alzheimer's disease, encephalomyehtis, cardio-myopathy, transplant rejection, and cancer

Alternatively, or in addition, the condition can be shock Shock in the subject may be associated with an underlying condition such as septic shock, e g , gram positive bacteria- mediated circulatory shock, gram negative bacteria-mediated circulatory shock, hemorrhagic shock, anaphylactic shock, systemic inflammation, pro-inflammatory cytokines, and systemic inflammatory response syndrome (SIRS) The immunomodulators may also be used to prevent or treat circulatory shock, such as shock occurring as a result of gram-negative and gram positive sepsis, trauma, hemorrhage, burn injury, anaphylaxis, cytokine immunotherapy, liver failure, kidney failure or systemic inflammatory response syndrome

In some embodiments, the immunomodulator is used to treat or prevent diabetes mellitus in a subject The diabetic condition can be, e g , Type I or Type II diabetes The diabetic condition treated can be autoimmune diabetes Autoimmune diabetes is associated with a strong inflammatory component, and activation of macrophages, and infiltration of mononuclear cells into the pancreas The subsequent inflammatory processes bring about the deleterious consequences of inflammation diabetes, such as islet inflammation, islet cell destruction, insulin deficiency and hyperglycemia (Rabinovitch et al , Biochem Pharmacol 55.1139-49, 1998, Almawi et al , J Clin Endocrinol Metab 84 1497-502, 1999) Macrophage-produced cytokines can be important mediators in the intraislet inflammatory processes Accordingly, the herein-disclosed immunomodulators can be used to treat or prevent the development of a diabetic condition in a subject In some embodiments, the immunomodulator is inosine or a ligand that binds to an inosine-binding site In some embodiments, delivery of the immunomodulator is oral The immunomodulator can be administered to the subject prophylactically or therapeutically The subject can be, e.g., a mammal, such as a rat, mouse, rabbit, guinea pig, hamster, cow, pig, horse, goat, sheep, dog, cat, non-human primate, or human. The subject can have, or be at risk for developing, a condition associated with undesired secretion of one or more inflammatory cytokines

The immunomodulator can be administered to the subject by any route that elicits the desired response, while preferably minimizing any undesirable side effects Suitable routes can include intravenous, intramuscular, subcutaneous, sublingual, oral, rectal or aerosol delivery.

The macrophage immunomodulators are administered to a subject in need of treatment for the conditions described above in an effective amount As used herein,

''effective amount" defines that amount of pharmaceutical active which provides the desired therapeutic effect while providing an acceptable level of side effects (if any) for the subject

Pharmaceutical compositions

In another aspect the invention includes pharmaceutical, or therapeutic, compositions containing one or more immunomodulators described herein Pharmaceutical formulations may include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration, or for administration by inhalation or insufflation The formulations may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy All such pharmacy methods include the steps of bringing into association the active compound with liquid carriers or finely divided solid carriers or both as needed and then, if necessary, shaping the product into the desired formulation

Pharmaceutical formulations suitable for oral administration may conveniently be presented as discrete units, such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient, as a powder or granules, or as a solution, a suspension or as an emulsion The active ingredient may also be presented as a bolus electuary or paste, and be in a pure form, 1 e , without a carrier Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrant or wetting agents A tablet may be made by compression or molding, optionally with one or more formulational ingredients Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent The tablets may be coated according to methods well known in the art Oral fluid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives The tablets may optionallv be formulated so as to provide slow or controlled release of the active ingredient therein

Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteπostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents The formulations may be presented in unit dose or multi-dose containers, for example sealed ampoules and vials, and mav be stored in a freeze-dπed (lyophihzed) condition requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use Alternatively, the formulations may be presented for continuous infusion Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter or polyethylene glycol. Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges, comprising the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a base such as gelatin and glycerin or sucrose and acacia. For intra-nasal administration the compounds of the invention may be used as a liquid spray or dispersible powder or in the form of drops. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs.

For administration by inhalation the compounds are conveniently delivered from an insufflator, nebulizer, pressurized packs or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichiorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, the compounds may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form, in for example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.

When desired, the above-described formulations, adapted to give sustained release of the active ingredient, may be employed. The pharmaceutical compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressants or preservatives.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavoring agents.

Preferred unit dosage formulations are those containing an effective dose, as recited below, or an appropriate fraction thereof, of the active ingredient. For each of the aforementioned conditions, the macrophage immunomodulator ligands may be administered orally or via injection at a dose of from about 0 1 to about 250 mg/kg per day The dose range for adult humans is generally from about 5 mg to about 17 5 g/day, preferably about 5 mg to about 10 g/day, and most preferably about 100 mg to about 3 g/day Tablets or other unit dosage forms of presentation provided in discrete units may conveniently contain an amount which is effective at such dosage or as a multiple of the same, for instance, units containing about 5 mg to about 500 mg, usually from about 100 mg to about 500 mg

The pharmaceutical composition preferably is administered orally or by injection (intravenous or subcutaneous), and the precise amount administered to a subject will be the responsibility of the attendant physician However, the dose employed will depend upon a number of factors, including the age and sex of the subject, the precise disorder being treated, and its severity Also the route of administration may vary depending upon the condition and its severity

Examples

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims The following examples illustrate the characterization of the immunomodulators described herein on macrophages in vitro and in vivo in animal models of inflammation

Example 1 - Glibenclamide inhibits macrophage production of TNF and IL-12

To determine the effects of glibenclamide on proinflammatory selection, J774 1 macrophages were exposed to control (no drug) of glibenclamide ranging in concentration from 10-300μM TNF and IL-12 production by the macrophages was then measured The results are shown in Table 1 Glibenclamide, a selective inhibitor of K TP channels, blocks the production of both TNF and IL-12 (Table 1)

The effect on TNF and IL-12 macrophage production of an agent which opens KATP channels was also measured, the opener of these channels caused a substantial increase in the release of both of these cytokines (Table 2) These data point toward an important role of KATP channel activation in the regulation of macrophage function and suggest that inhibition of these channels may be an effective anti-inflammatory strategy

TABLE 1 - EFFECT OF GLLBENCLAMLDE ON TNF AND IL-12 PRODUCTION BY J 774.1

Figure imgf000014_0001

TABLE 2 - EFFECT OF DIAZOXLDE ON TNF AND LL-12 PRODUCTION BY J 774.1 MACROPHAGES STIMULATED WITH LPS/IFN-

Figure imgf000014_0002

To further examine the effect of glibenclamide on macrophage activation, the surface expression of MHC -II molecules in response to IFN-γ was measured using flow cytometry MHC II (I-Ad) expression was decreased by treatment with glibenclamide While IFN-γ exposure increased MHC II expression in peritoneal macrophages from 12 8 ± 1 7 (mean fluorescent intensity) to 55 6 ± 4 2, cotreatment of the cells with 100 μM glibenclamide decreased the expression of MHCII to 29 ± 2 3

We also tested whether the inhibition of costimulatory cytokine (TNF-α and 11-12) production by glibenclamide shifts the cytokine response from a Thl to a Th2 direction Our results demonstrated that glibenclamide decreases the production of the Thl cytokine IFN-γ induced by either LPS or anti-CD3 (Table 3) in mouse spleen cells However, glibenclamide was more potent in decreasing LPS-induced than anti-CD-3 -induced IFN-γ In contrast, this KATP channel blocker caused a concentration dependent augmentation of the production of the Th2 cytokine EL-4 (Table 3) Taken together, glibenclamide polarizes the cytokine response towards a Th2 response

TABLE 3 - EFFECT OF GLLBENCLAMEDE ON TL-4 AND LFN-γ PRODUCTION BY SPLEEN CELLS STIMULATED WITH LPS OR AN ANTIBODY TO ANTI-CD3

Figure imgf000015_0001

Example 2 - Glibenclamide suppresses inflammatory cytokine production in vivo

To assess the effect of glibenclamide in vivo, we utilized an endotoxemic mouse model, in which cytokine production was induced by intraperitoneal (i p ) LPS injection and TNF-α levels were measured from the plasma of the animals We measured TNF-α, because this cytokine appears first in vivo after LPS, while IL-12 has a delayed time-course Under these conditions, glibenclamide significantly suppressed the plasma TNF-α level induced by LPS- the TNF-α level in vehicle-pretreated mice was 1 08 ± 0 28 ng/ml, whereas TNF-α level in the glibenclamide-pretreated mice was 0 43 ± 0 09 ng/ml (n=S in both groups, p < 0 05).

Example 3 - Amilioride inhibits macrophage release of IL-12, MD?- I , and MLP-2

Na+/H+ exchangers, also called antiporters, are transmembrane transporters involved in multiple cellular functions, including the regulation of intracellular pH, the control of cell volume, and mitogenesis (Demaurex et al , J Exp Biol 196 389-404, 1994) To determine the effect of Na+/H+ antiporters on inflammatory cytokine production, stimulated macrophages were exposed to the antiporter amilioride at concentration of 0 to 300 μM, after which production of cytokines 11-12, MlP-lα and MTP-2 was measured The results are shown in Table 4 Amiloride was found to inhibit the release of IL- 12, MIP- la, and MTP-2 by J774 1 macrophages These results suggest that blockade of the Na+/H+ exchangers has anti-inflammatory effects

TABLE 4 - EFFECT OF AMLLORLDE ON LL-12, Mw-lα, AND MIP-2 PRODUCTION BY S 774.1 MACROPHAGES STIMULATED WITH LPSHFN-

Figure imgf000016_0001

Example 4 - Inosine inhibits in vitro macrophage release of IL-12 and TNF

To determine the effect of inosine on inflammatory cytokine production, stimulated macrophages were exposed to inosinethe antiporter amilioride at 0 to 1000 μM, after which production of cytokines 11-12 and TNF was measured The results are shown in Table 4 Inosine was found to inhibit the release of these cytokines These results suggest that inosine has anti-inflammatory effects TABLE 5 - EFFECT OF INOSINE ON IL-12 AND TNF PRODUCTION BY PERITONEAL MACROPHAGES STIMULATED WITH LPS/IFN-

Figure imgf000017_0001

Example 5 - Inosine inhibits inflammatory cytokine responses in vivo while increasing anti-inflammatory cytokine release

To determine whether inosine inhibits inflammatory cytokine release in vivo, we injected male BALB/c mice with inosine (100 mg/kg, i p ) followed 30 minutes later by an i.p injection of LPS (70 mg/kg) Plasma levels of the different cytokines were measured at various times (90 min, 2h, 4h, and 8h) after the LPS challenge

The results are shown in FIGS 1A-E Data are means SEM of n = 8 mice The asterisk in the figure indicates p < 0 05 Inosine was observed to suppress the production of TNF-α (FIG 1 A), IL-12 (FIG IB), IFN-γ (FIG 1 C), and MTP- 1 α (FIG ID), but to augment IL-10 (FIG IE) formation in endotoxemic mice These results were similar to inosine's effects on macrophage cytokine release in vitro Furthermore, inosine also suppressed the production of EFN-γ, which is also involved in the pro-inflammatory effects of LPS Taken together, these data demonstrate that inosine selectively and differentially alters the production of cytokines in vivo Namely (1) it inhibits the production of proinflammatory cytokines, but (2) potentiates the formation of the anti-inflammatory EL- 10

Example 6 - Inosine protects against lethal challenge of LPS in an in vivo model system

Because inosine skewed the cytokine response towards an anti-inflammatory profile, the ability of inosine to decrease LPS-induced lethality in a murine model system was investigated BALB/c mice were pretreated with drug vehicle (physiologic saline) or 100 mg/kg inosine 30 min before the injection of 70 mg/kg of i p LPS The results are shown in FIG 2 Survival was recorded at 24, 48, 72, and 96 h after the LPS injection Results from the summary of two different experiments are shown N= 16 animals in each group Inosine improved survival rate at 24-96h (p<0 05) Thus, inosine conferred significant protection in this endotoxemic model

Example 7 - Inosine inhibits the development of diabetes-associated symptoms in an in vivo model system

Autoimmune diabetes is associated with a strong inflammatory component, along with activation of macrophages and infiltration of mononuclear cells into the pancreas The subsequent inflammatory processes bring about the deleterious consequences of inflammation diabetes, such as islet inflammation, islet cell destruction, insulin deficiency and hyperglycemia (Rabinovitch et al , Biochem Pharmacol 15 1139-49, 1998, Almawi et al., J Clin Endocrinol Meatab 84 1497-502, 1999) Cytokines produced mainly by macrophages have been reported to be central mediators in the intraislet inflammatory processes

The effect of inosine was in a rat model of streptozitocin-induced diabetes was examined Mice were treated with streptozotocin (40 mg/kg dissolved in citrate buffer, pH) or vehicle (citrate buffer) i p for 5 consecutive days to induce diabetes Blood glucose was monitored over the following 21 days using a one-touch blood glucose meter (Lifescan) Blood glucose was measured on days 1, 7 and 21 from blood obtained from the tail vein Hyperglycemia was defined as non-fasting blood glucose level higher than 200mg/dL Mice were treated simultaneously with streptozotocin injection, throughout the 21 days of the experiments, and vehicle or inosine (100 mg/kg oral gavage, twice a day) Samples of pancreas were removed on day 21 and weighed before being placed into 6 mis of acid ethanol (23 7 0 45, ethanol dH 0 HC1) and homogenized The pancreas was incubated for 72 h at 4 °C before being centrifuged The insulin content of the supernatant determined using an ELISA assay

TABLE 6 shows mean and median glucose levels, and incidence of diabetes in streptozotocin (STZ) diabetic mice receiving vehicle or inosine treatment A "*" indicates significant reduction of circulating glucose or diabetes incidence in the inosine-treated streptozotocin rats, when compared to vehicle-treated streptozotocin rats (p<0 05) TABLE 6 - CHANGE IN GLUCOSE LEVELS IN STZ DIABETIC MICE RECEIVING VEHICLE OR INOSINE

Figure imgf000019_0001

The relative effect of vehicle and inosine on pancreatic insulin content in STZ diabetic-mice is shown in TABLE 7. A "*" in TABLE 7 indicates significant decreases in insulin content in response to streptozotocin when compared to control, and A "#" indicates significant preservation of pancreatic insulin content in the inosine-treated streptozotocin rats (p<0.05). Pancreatic insulin content at 21 days in streptozotocin diabetic mice receiving vehicle or inosine treatment.

TABLE 7 -PANCREATIC INSULIN CONTENT AT 21 DAYS LN STZ DIABETIC MICE RECEIVING VEHICLE OR INOSINE

Figure imgf000020_0001

Vehicle-treated animals developed diabetes, as demonstrated by progressive hyperglycemia (Table 6) and the suppression of pancreatic insulin content (Table 2). In contrast, animals treated with inosine showed significant reductions in the incidence of diabetes, and mean and median plasma glucose levels. They also showed a significant preservation of pancreatic insulin content (Table 7). These data indicate that inosine and insoine receptor ligands are capable of suppressing the development of diabetes.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

What is claimed is:
1. A method for treating diabetes mellitus in a subject, the method comprising administering to said subject a therapeutically effective amount of an immunomodulator selected from the group consisting of inosine and a ligand for an inosine binding site.'
2. The method of claim 1, wherein said immunomodulator administered to said subject by a route selected from intravenous, intramuscular, subcutaneous, sublingual, oral, rectal or aerosol delivery.
3. The method of claim 1, wherein said immunomodulator is administered at a dose of from about 0.1 to about 250 mg/kg/day in said subject.
4. The method of claim 1, wherein said subject is a human.
5. The method of claim 1, further comprising administering to said subject an immunomodulator selected from the group consisting of a KATP channel inhibitor and an inhibitor of a Na+/FT exchanger.
6. The method of claim 1, wherein said subject has or is at risk for Type I diabetes.
7. The method of claim 1, wherein said subject has or is at risk for Type II diabetes.
8. The method of claim 1, wherein said immunomodulator is administered prophylactically.
9. The method of claim 1, wherein said immunomodulator is administered therapeutically.
10. The method of claim 1, wherein said immunomodulator is inosine.
11. A method of increasing insulin levels in a subject, the method comprising administering to a subject in need thereof an amount of an immunomodulator sufficient to increase insulin levels in said subject, wherein said immunomodulator is selected from the group consisting of inosine and an analog of an inosine binding site.
12 The method of claim 11, wherein said immunomodulator is administered to said subject by a route selected from intravenous, intramuscular, subcutaneous, sublingual, oral, rectal or aerosol delivery
13. The method of claim 6, wherein administering said immunomodulator increases pancreatic insulin levels in said subject
14 The method of claim 6, wherein immunomodulator is administered at a dose of from about 0 1 to about 250 mg/kg/day in said subject
15. The method of claim 6, wherein said subject is a human
16. The method of claim 11, wherein said immunomodulator is administered prophylactically
17. The method of claim 11, wherein said immunomodulator is administered therapeutically
18 The method of claim 11, wherein said subject suffers from or is at risk for Type I diabetes.
19. The method of claim 11, wherein said subject suffers from or is at risk for Type II diabetes.
20. The method of claim 11, wherein said immunomodulator is inosine
PCT/US2001/001678 2000-01-24 2001-01-18 Method and composition for modulating an immune response WO2001052860A2 (en)

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