Treatment of inflammatory diseases

The present invention relates to the use of a combination of an inhibitor of the purine biosynthetic pathway and a compound susceptible to enzymatic degradation/metabolism by an enzyme involved in purine biosynthesis for the treatment and/or prevention of an inflammatory disease, a pharmaceutical composition comprising the same, a method for producing the pharmaceutical composition as well as methods for treating and/or preventing an inflammatory disease.

There is a continuous need for therapeutics for the treatment of inflammatory diseases. Treatment of inflammatory diseases is mostly based on interrupting the synthesis or action of mediators that drive the inflammatory response. Non-steroidal anti- inflammatory drugs (NSAIDs), steroids and anti-histamines were developed on this rationale. Such small- molecule drugs have provided a major treatment for inflammatory diseases, but are not without shortcomings. An alternative approach is the development of novel therapeutics based on endogenous mediators (or metabolites) and mechanisms that downregulate the inflammation response (Gilroy et al., 2004, Inflammatory Resolution: New Opportunities for Drug Discovery, Nature Reviews Drug Discovery 3, 401-416).

One treatment for inflammatory diseases suggested in the art includes the increase of endogenous adenosine. One suggested approach includes the administration of adenosine itself, which however resulted in systemic side effects (Hasko and Cronstein, 2004, Adenosine: an endogenous regulator of innate immunity, Trends in Immunology 2004, 33- 39). Adenosine itself is an approved treatment for supraventricular tachycardia, but its short plasma half- life of less than 5 seconds makes it unsuitable for most other diseases.

Another suggestion was the administration of adenosine receptor agonists (especially A2a and A3 receptors) that mimic adenosine action. However, again systemic side effects are expected due to the expression of the receptors in the heart and the brain resulting in cardiovascular and CNS side effects (Sitkovsky 2003, Use of the A(2A) adenosine receptor as a physiological immunosuppressor and to engineer inflammation in vivo. Biochemical Pharmacology 65 (4), 493-501).

Other suggestions are to modulate enzymes involved in adenosine nucleoside and nucleotide metabolism such as adenosine deaminase (Terasaka et al., 2005, Rational design of non-nucleoside, potent, and orally bioavailable adenosine deaminase inhibitors:

Predicting enzyme conformational change and metabolism. J. Med. Chem. 48 (15), 4750- 4753), AMP deaminase (Kasibhatla et al., 2001, AMP deaminase inhibitors.5. Design, synthesis, and SAR of a highly potent inhibitor series. J. Med. Chem. 44 (4), 613-618), and adenosine kinase (Ugarkar et al., 2003, Adenosine kinase inhibitors.3. Synthesis, SAR, and anti- inflammatory activity of a series of 1-lyxofuranosyl nucleosides, J. Med. Chem. 46 (22), 4750-4760). None of these inhibitors has been approved for treatment yet.

Furthermore, it has been suggested to treat patients with methotrexate in order to stimulate adenosine release. However, side effects as well as a slow onset were observed (Cronstein, 2005, Low-Dose Methotrexate: A mainstay in the treatment of rheumatoid arthritis. Pharmacological Reviews 57, 163-172).

In the art, also the administration of AICA riboside (5-aminoimidazole-4-carboxamide ribonucleoside; AICAR) or AICA ribotide (5-aminoimidazole-4-carboxamide ribonucleotide, ZMP) has been suggested. A major drawback, however, of AICAR is the low oral bioavailability of AICAR due to rapid metabolism. Poor bioavailability was shown (<5%) when administered orally in solution (Dixon et al., 1991, AICA riboside: Safety, tolerance, and pharmacokinetics of a novel adenosine-regulating agent. J. Clin. Pharmacol. 31, 342-347). Furthermore, AICAR is metabolized to uric acid through normal purine pathways (Dixon et al., 1993. Acadesine (AICA riboside): Disposition and metabolism of an adenosine-regulating agent. J. Clin. Pharmacol. 33, 955-958).

It was reported that AICAR can exhibit anti-inflammatory properties, presumably by activating AMP-activated kinase (AMPK), a kinase known to be activated by AICAR (Giri et al., 2004, 5-Aminoimidazole-4-carboxamide-l-β-4-ribofuranoside inhibits proinflammatory reponse in glial cells: a possible role of AMP-activated kinase. The Journal of Neuroscience 24(2), 479-487). The application of AICAR inhibited the lipopolysaccharide-(LPS)-induced expression of proinflammatory cytokines in several cell types and in an animal model of inflammation. In the animal experiments rats were intraperitoneally (i.p) injected with AICAR before LPS treatment. The authors suggest that AMPK may be exploited as a target for anti-inflammatory drugs such as AICAR. However they point out that AICAR has a high clearance and is poorly bioavailable with oral administration.

In addition it was demonstrated by the same research group that AICAR showed beneficial effects in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (Nath et al., 2005, 5-Aminoimidazole-4-carboxamide ribonuleoside: a

novel immunomodulator with therapeutic efficacy in experimental encephalomyelitis. J. Immunology 175(1), 566-574).

WO 89/00854 suggests the use of various purine nucleosides including AICAR or ribavarin alone or in combination with various other agents including methotrexate or succinylaminoimidazole carboxamide riboside for enhancing extracellular adenosine e.g. in arthritis. Several administration modes are suggested including intravenous, oral, rectal or topical administration. The examples are directed to subcutanous or intravenous administration.

The present invention relates to the use of a combination of

a) an inhibitor of the purine bio synthetic pathway, and b) a compound susceptible to enzymatic degradation/metabolism by an enzyme involved in purine biosynthesis

for the preparation of a medicament for oral administration for the treatment and/or prevention of an inflammatory disease, preferably selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, psoriasis, Crohn's disease, systemic lupus erythematosis, ulcerative colitis, atopic dermatitis, multiple sclerosis, myasthenia gravis, ankylosing spondylitis, inflammatory bowel disease, and uveitis.

Especially, the present invention relates to the use of a combination of

a) an inhibitor selected from the group consisting of methotrexate, amethopterin, 7- hydroxy-methotrexate, MX-68 (CAS RN 156 579-02-1), and sulfasalazine, and b) the compound AICA riboside

for the preparation of a medicament for oral administration for the treatment and/or prevention of an inflammatory disease, preferably selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, psoriasis, Crohn's disease, systemic lupus erythematosis, ulcerative colitis, atopic dermatitis, multiple sclerosis, myasthenia gravis, ankylosing spondylitis, inflammatory bowel disease, and uveitis.

In a third aspect aspect, the invention relates to the use of a compound susceptible to enzymatic degradation/metabolism by an enzyme involved in purine biosynthesis, preferably AICA riboside, for the preparation of a medicament for oral administration for the treatment and/or prevention of an inflammatory disease, preferably selected from the

group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, psoriasis, Crohn's disease, systemic lupus erythematosis, ulcerative colitis, atopic dermatitis, multiple sclerosis, myasthenia gravis, ankylosing spondylitis, inflammatory bowel disease, and uveitis, wherein said compound is administered simultaneously, separately or sequentially with an inhibitor of the purine biosynthetic pathway, preferably methotrexate, amethopterin, 7-hydroxy-methotrexate, MX-68 (CAS RN 156 579-02-1), and sulfasalazine.

In a fourth aspect, the invention relates to the use of an inhibitor of the purine biosynthetic pathway, preferably methotrexate, amethopterin, 7-hydroxy-methotrexate, MX-68 (CAS RN 156 579-02-1), and sulfasalazine, for the preparation of a medicament for oral administration for the treatment and/or prevention of an inflammatory disease, preferably selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, psoriasis, Crohn's disease, systemic lupus erythematosis, ulcerative colitis, atopic dermatitis, multiple sclerosis, myasthenia gravis, ankylosing spondylitis, inflammatory bowel disease, and uveitis, wherein said inhibitor is administered simultaneously, separately or sequentially with a compound susceptible to enzymatic degradation/metabolism by an enzyme involved in purine biosynthesis, preferably AICA riboside.

Consequently, the invention focuses on the oral administration of two agents for the treatment of inflammatory diseases. Rheumatoid arthritis, osteoarthritis, psoriatic arthritis, psoriasis, Crohn's disease, systemic lupus erythematosis, ulcerative colitis, atopic dermatitis, multiple sclerosis, myasthenia gravis, ankylosing spondylitis, inflammatory bowel disease, and uveitis are generally known as inflammatory diseases.

"Inflammation" can be defined as a localized protective response elicited by injury or destruction of tissues, which servers to destroy, dilute, or wall off (sequester) both the injurious agent and the injured tissue. It is characterized in the acute form by the classical signs of pain, heat, redness, swelling and loss of function. Histologically, it involves a complex series of events, comprising a vascular response (including dilation of arterioles, capillaries, and venules, with increased permeability and blood flow, exudation of fluids and plasma proteins) and a cellular response including leucocyte migration into the inflammatory focus. It is usually divided into acute inflammation, chronic inflammation and repair. The cells that are present in the area of inflammation (inflammatory infiltrate) determine the classification. If the infiltrate is composed predominantly of neutrophils and some macrophages, it is called "acute inflammation". In case the infiltrate is composed of macrophages, lymphocytes, and/or plasma cells, it is classified as "chronic inflammation".

Acute and chronic inflammations are related in time as neutrophils will precede macrophages and lymphocytes in a typical inflammatory response (Review: Chapter 2, "Acute and Chronic Inflammation" in "Robbins Pathologic Basis of Disease" by Cotran, Kumar, Collins and Robbins; Saunders Company, 6 ^th edition, 1999, ISBN: 072167335X).

According to the present invention, the term "treating" or "treatment" refers to all actions where an amelioration of the respective disease is obtained. Especially, these terms also refer to conditions where the outbreak of the respective disease is prevented.

In the present invention the first agent is an inhibitor of the purine biosynthetic pathway. In the de novo purine biosynthetic pathway, the purine ring is synthesized in mammals utilizing amino acids as carbon and nitrogen donors and CO ₂ as carbon donor. The de novo pathway for purine nucleotide synthesis consists often metabolic steps and leads to inosine 5 '-monophosphate (IMP). IMP is the common precursor for AMP and GMP synthesis. AMP and GMP are then converted into ATP and GTP, respectively.

The committed step in the de novo synthesis of purine nucleotides is the formation of 5- phosphoribosylamine from 5 -phosphoribosyl-1 -pyrophosphate (PRPP) and glutamine. The first five steps of the pathway lead to the formation of 5-aminoimidazole ribonucleotide. The essence of these five reactions is (1) displacement of pyrophosphate by the side chain amino group of glutamine (glutamine PRPP amidotransferase), (2) addition of glycine (GAR synthetase), (3) formylation by NlO-formyltetrahydrofolate (GAR transformylase), (4) transfer of a nitrogen atom from glutamine (FGAM synthetase), and (5) dehydration and ring closure (AIR synthetase). The second phase of the pathway transforms 5- aminoimidazole ribonucleotide into inosinate. The essence of these steps is (6) carboxylation (AIR carboxylase), (7) addition of aspartate (SAICAR synthetase), (8) elimination of fumarate (adenylosuccinate lyase), (9) formylation by NlO- formyltetrahydrofolate (AICAR transformylase), and (10) dehydration and ring closure (IMP cyclohydrolase). The vertebrate enzymes catalysing steps 9 and 10 are present on a single polypeptide chain (ATIC, see below) (Chapter 29 "Biosynthesis of Nucleotides", pp. 739-762, in Biochemistry by L. Stryer, fourth edition, 1995, W.H. Freeman and Company, New York).

In addition to the de novo synthesis of purine rings, cells have the capability to recycle ("salvage") purine bases and nucleosides through so-called salvage pathways. These pathways utilize the preformed bases or nucleosides by contrast to the new synthesis in the de novo pathway. Throughout this invention, the purine biosynthetic pathway refers to the de novo pathway.

Preferably, the inhibitor is selected from the group consisting of methotrexate, amethopterin, 7-hydroxy-methotrexate (a methotrexate metabolite), MX-68 (CAS RN 156 579-02-1), and sulfasalazine. MX-68 is a methotrextae derivative described in J. Med. Chem. 1997 Jan 3;40(l):105-l l l.

Methotrexate and its major metabolite 7-hydroxy-methotrexate are taken up by cells and polyglutamated. Methotrexate polyglutamates have been shown to be even more active than the parent drug as inhibitors of a variety of folate-dependent enzymes, for example AICAR transformylase (Allegra et al., 1985, Proc. Natl. Acad. Sci. USA 82, 4881-4885).

Most preferred is methotrexate. Methotrexate has been extensively tested for clinical applications and methods for formulating and administering Methotrexate are abundantly available to those skilled in art.

Alternatively, the following inhibitors may be used:

FMTX (3'-fluoromethotrexate), PT-523 (CAS RN 113857-87-7), Trimetrexate (CAS RN 52128-35-5), MDAM (CAS RN 176857-41-3), ZD-9331/BGC-9331 (CAS RN 153537-73- 6), Nolatrexed (CAS RN 147149-76-6; AG-337, Thymitaq), Pemetrexed (CAS RN 150399-23-8), Lometrexol (CAS RN 106400-81-1), Piritrexim (CAS RN 72732-56-0), Edatrexate (CAS RN 80576-83-6), Raltitrexed (CAS RN 112887-68-0), NSC30171 (J. Med. Chem. 2004 Dec 30;47(27):6681-90), 326203-A (Li et al., 2004, J. Biol. Chem. 279(48):50555-50565), BW1540 (Cheong et al., 2004, J. Biol. Chem. 279(17): 18034-45), BW2315 (Cheong et al., J. Biol. Chem 279(17): 18034-45), TNP351(CAS RN 125991-51-7).

The second agent is a compound susceptible to enzymatic degradation/metabolism by an enzyme involved in purine biosynthesis.

Many pharmaceutically effective compounds are metabolically processed, some being activated while others are inactivated or their pharmacokinetic characteristics such as, for example, efficacy, specificity, half life, clearance rate, side effects, and/or drug targeting are modified. Most often metabolic modification, in particular degradation, is undesired. Extensive efforts have been undertaken to control metabolic degradation ranging from chemical modification of the compounds (e.g. prodrugs), the modification of the pharmaceutical formulation or the mode of administration, as well as the use of enzyme inhibitors.

Generally speaking, the term "degradation" relates to the modification of compounds that results in a partial or complete loss of the biological activity of the unmodified compound by e.g. reduction in molecular size (e.g. cleavage), addition of moieties (e.g. sugar moieties) that increase body clearance, reduction or oxidation of functional groups, etc.

The term "metabolism" of a pharmaceutically effective compound in the context of the present invention with respect to intermediates (metabolites) relates to the enzymatic conversion into the next (downstream) intermediate of the pathway or, in case of reversible reactions, into the previous (upstream) intermediate of the pathway.

In the context of this invention, "compounds" can be metabolic intermediates of biosynthetic pathways such as the de novo purine synthesis pathway: 5-Phophoribosyl-l- pyrophosphate (PRPP), 5-phosphoribosylamine (PRA), 5-phosphoribosylglycinamide (GAR), 5-phosphoribosylformylglycinamide (FGAR), 5-phosphoribosylformylglycin- amidine (FGAM), 5-Phophoribosyl-5-aminoimidazole (AIR), 5-Phophoribosyl-5-amino- imidazole-4-carboxylic acid (CAIR), 5-Phophoribosyl-5-aminoimidazole-4-N-succino- carboxamide (SAICAR), 5-Phophoribosyl-5-aminoimidazole-4-carboxamide (AICA ribotide).

Preferably, the compound is AICA riboside (5-aminoimidazole-4-carboxamide ribonucleoside; AICAR). However, throughout the invention, instead of AICA riboside, 5- Phophoribosyl-5-aminoimidazole-4-carboxamide (AICA ribotide; ZMP) can also be used.

Throughout the invention, the compounds can not only be synthetic drugs but also natural metabolites (e.g. intermediates in biosynthesis or biodegradation pathways).

In a preferred embodiment, said enzyme is AICAR transformylase.

AICARFT (5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase; EC 2.1.2.3; synonym AICAR transformylase) and IMPCHase (IMP cyclohydrolase; EC 3.5.4.10) catalyze the penultimate and final steps, respectively, of the de novo purine biosynthetic pathway. Rayl et al. cloned a cDNA for human AICARFT/IMPCHase from a hepatoma cDNA library (Rayl et al., 1996, J. Biol. Chem. 271: 2225-2233).

Both enzymatic activities are present in the same protein, designated ATIC, in all species of prokaryotes and eukaryotes studied. The human ATIC cDNA encodes a deduced 591- amino acid protein that is 81% identical to the chicken sequence. Rayl et al. (1996) created truncation mutants of the cDNA and measured their enzymatic properties. In this way they

were able to localize the AICARFT activity within the amino-terminal 223 amino acids and the IMPCHase activity to the carboxyl-terminal 406 residues.

Throughout the invention, the inhibitor a) and the compound b) are for simultaneous, separate and/or sequential administration.

Depending one the overall mode of administration or merely from a standpoint of convenience, the coadministered drugs may be administered at different times in different formulations and in different locations.

Throughout the invention, the terms "combined" or "in combination" or "combination" as used herein in the context of an inhibitor of the purine biosynthetic pathway (e.g. methotrexate) being combined with at least one compound susceptible to enzymatic degradation/metabolism (e.g. AICA-riboside or AICA ribotide) relate to the functional combination of the inhibitor (Methotrexate) as an AICAR-transformylase inhibitor together with at least one therapeutic or prophylactic compound.

"Combined" or "in combination" or "combination" should be understood as a functional coadministration only, wherein some or all compounds may be administrated separately, in different formulations, different modes of administration (subcutaneous, intravenous, intravenous, oral, etc.), and different times of administration. The mode of administration, the formulation, the time(s) of administration, and other pharmacologically related measures for each compound separately or both compounds together depend on the properties of inhibitor (methotrexate) and those desired compound(s) that are to be coadministered.

The compound and/or the inhibitor are administered orally. This means that the substances are formulated such that they are suitable for oral administration. The skilled person is aware of such methods of formulation (see e.g. Remington's Pharmaceutical Sciences by E.W. Martin, 1975, 15 ^th edition; Mack Publishing Co., ppp.1405-1412 and pp. 1461-1487). In general, oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, or magnesium carbonate.

In a further preferred embodiment, the coadministration of the inhibitor, preferably methotrexate and at least one pharmaceutically effective compound is performed in one formulation, and/or in the same mode and/or at the same time of administration.

Generally speaking, for combinational therapy, Methotrexate and a compound susceptible to enzymatic degradation/metabolism by an enzyme involved in purine biosynthesis can be administered together at the same time of administration and in the same formulation, and at the same location, e.g. orally.

In a further preferred embodiment, the inhibitor a) is administered before the compound b). In this case, it is also envisaged that the administration of the inhibitor a) may be continued during the administration of the compound b).

The skilled person would be able to determine the amounts to be administered based on his professional experience. Preferably, the inhibitor, preferably methotrexate, is administered in an amount of less than 10mg/day, preferably less than lmg/day, and said compound, preferably AICA riboside, is administered in an amount of less than 500mg/day, preferably less than 100mg/day.

If the inhibitor is Sulfasalzine, higher doses, possibly up to 3 g/day, are preferred.

In a preferred embodiment of the invention, iurther folic acid is used, preferably at a dose of less than 5mg/day. Preferably, folic acid is also administered orally.

The administration of folic acid is expected to decrease Methotrexate-induced side effects due to inhibition of other folate-dependent enzymes than AICAR transformylase. Folic acid is widely used together with Methotrexate for the treatment of rheumatoid arthritis. Folic acid had been extensivly tested for clinical applications and methods for formulating and administering folic acid are abundantly available to those skilled in art (Whittle and Hughes, 2004. Folate supplementation and methotrexate treatment in rheumatoid arthritis: a review. Rheumatology 43 (3), 267-271).

The invention further relates to a pharmaceutical composition comprising either together or in separate dosage forms

a) an inhibitor of the purine bio synthetic pathway, and b) a compound susceptible to enzymatic degradation/metabolism by an enzyme involved in purine biosynthesis, and c) an excipient suitable for oral administration.

With respect to the pharmaceutical composition of the invention, all embodiments defined above for the uses of the invention also apply.

The invention further provides a method for producing the pharmaceutical composition of the invention, wherein the inhibitor a), the compound b) and the excipient c) are formulated to a pharmaceutical composition. With respect to formulation and amounts, the embodiments as defined above for the uses of the invention also apply to this method of the invention.

The invention further provides a method of treating and/or preventing an inflammatory disease, preferably selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, psoriasis, Crohn's disease, systemic lupus erythematosis, ulcerative colitis, atopic dermatitis, multiple sclerosis, myasthenia gravis, ankylosing spondylitis, inflammatory bowel disease, and uveitis in a mammal, which method comprises orally administering to the mammal an effective amount of

a) an inhibitor of the purine bio synthetic pathway, and b) a compound susceptible to enzymatic degradation/metabolism by an enzyme involved in purine biosynthesis.

Furthermore, the invention relates to a method of treating and/or preventing an inflammatory disease, preferably selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, psoriasis, Crohn's disease, systemic lupus erythematosis, ulcerative colitis, atopic dermatitis, multiple sclerosis, myasthenia gravis, ankylosing spondylitis, inflammatory bowel disease, and uveitis in a mammal, which method comprises orally administering to the mammal an effective amount of

a) an inhibitor selected from the group consisting of methotrexate, amethopterin, 7- hydroxy-methotrexate, MX-68 (CAS RN 156 579-02-1), and sulfasalazine, and b) the compound AICA riboside.

With respect to these methods of the invention, the same applies as for the above defined uses of the invention.

Throughout the invention, the administration of two compounds in a therapeutic effective amount includes that one or each of the compounds is administered in a subtherapeutic amount, i.e. that the amount of each compound on its own is not sufficient to provide a therapeutic effect, but that the combination of the compounds results in the desired therapeutic effect. However, it is also included that each of the compounds on its own is administered in a therapeutically effective amount.

The invention is further illustrated by the following examples, which are not intended to be limiting for the scope of the present invention. Inter alia, the examples demonstrate the beneficial effect of treatment with Methotrexate on the bioavailability of orally administered AICAR (see Example 5). Furthermore, Example 6 demonstrates a beneficial effect of Methotrexate and AICAR in an air pouch model of inflammation.

Since Methotrexate is the prototype of an inhibitor of the purine biosynthetic pathway, and since AICAR is known to be a compound susceptible to enzymatic degradation/metabolism by an enzyme involved in purine biosynthesis, it is believed that the findings for both agents can be generalized. This generalization does not only include the generalization from Methotrexate to amethopterin, 7-hydroxy-methotrexate (a methotrexate metabolite), MX-68 (CAS RN 156 579-02-1), and sulfasalazine, but it is also believed that these findings can be extended to each of said inhibitors and to each of said compounds.

Examples

Example 1: Pharmacokinetics

Determination of the enhancement of the oral bioavailability of AICAR by Methotrexate

Groups of 5 rats are dosed with a single oral dose of methotrexate at a dose of 2.5 mg/kg by oral gavage each day for 5 days. 2 hours after the last dose of methotrexate, AICAR at a dose of 100mg/Kg is given by oral gavage. Urinary excretion of AICAR is measured according to the method of Baggott and colleagues (Arthritis & Rheumatism Volume 41, Issue 8, Pages 1407 - 1410, 1998).

Control rats are dosed with an equivalent volume of saline and urinary excretion of AICAR similarly measured.

Enhancement of oral bioavailability is recorded as the difference between urinary excretion of AICAR in treated and control rats.

Determination of the enhancement of the oral bioavailability of AICAR by Sulfasalazine

Groups of 5 rats are dosed with a single oral dose of sulfasalazine at a dose of 100 mg/kg by oral gavage each day for 5 days. 2 hours after the last dose of sulfasalazine AICAR at a dose of 100mg/Kg is given by oral gavage. Urinary excretion of AICAR is measured according to the method of Baggott and colleagues (Arthritis & Rheumatism Volume 41, Issue 8 , Pages 1407 - 1410, 1998).

Control rats are dosed with an equivalent volume of saline and urinary excretion of AICAR similarly measured.

Enhancement of oral bioavailability is recorded as the difference between urinary excretion of AICAR in treated and control rats.

Example 2

Determination of IC50 values of test compounds

An AICAR transformylase enzyme assay was described preciously (Xu et al., 2004, J. Biol. Chem. 279, 50555-50565). An AICAR transformylase inhibition assay to determine IC ₅₀ values of test compounds can be performed in a solution containing 25 nM ATIC enzyme, 50 microM AICAR, and 8.5 microM N ¹⁰ -formyltetrahydrofolate (10-f-ThF).

Example 3

Treatment of TNBS induced colitis in rats with a combination of Methotrexate and AICAR.

Treated rats are dosed for 7 days with an oral gavage of 2.5mg/Kg of methotrexate and an oral gavage of 100mg/Kg AICAR. On day 5 trinitrobenzenesulphonic acid (TNBS) is administered rectally to rats (25mg in ImI of 50% ethanol). Untreated rats are dosed for 7 days with an equivalent volume of vehicle. On day 5 TNBS is administered rectally.

The rats that survived the disease 48 hr after induction are terminated under ether anesthesia and 10cm segments of the distal colon are evaluated for macroscopic histological damage. Segments are scored from 0 (no damage) to 10 (maximal damage) by a naked-eye examination of areas of mucosal discoloration, erosion, exudation, ulceration, bowel wall thickening and percentage of damaged area.

The effect of treatment is assessed by the number of surviving animals and macroscopic histological damage score.

Example 4

Treatment of collagen-induced arthritis with a combination of Methotrexate and AICAR.

The combination of methotrexate and AICAR is evaluated in BALB/c mice, 6-8 weeks of age, in which arthritis is induced by monoclonal antibodies (mAbs) raised against type II collagen, plus lipopolysaccharide (LPS). The animals are administered intravenously with a combination of 4 different mAbs totaling 4 mg/mouse on day 0, and followed by intravenous 25microg of LPS 72 hours later (day 3). From day 3, one hour after LPS application, methotrexate (2.5mg/kg) and AICAR (100mg/kg) are administered orally once daily for 15 days. For each animal, increase in volume of both hind paws is measured using a plethysmometer with water cell (12 mm diameter) on days 0, 5, 7, 10, 14 and 17. Percent inhibition of increase in volume is calculated. Reduction of edema in the hind paws by 30% or more is considered significant.

Example 5: Effect of Methotrexate treatment on the oral bioavailability of AICAR

The data of this experiment demonstrate the beneficial effect of treatment with Methotrexate on the bioavailability of orally administered AICAR. Rats treated with Methotrexate and AICAR (group 1) showed higher AICAR plasma concentrations compared to control groups treated with Methotrexate alone (group 2), AICAR alone (group 3) or saline controls (group 4).

Study protocol: Treatment of animals

Group 1

5 rats (Lewis) were treated with methotrexate ( Calbiochem, San Diego, CA, USA; catalogue number 454125) for 5 days at lmg/Kg (1ml of a solution in normal saline -

0.9%) intraperitoneal (ip). Two hours after the last dose of methotrexate, AICAR (AICA- Riboside, Calbiochem, San Diego, CA, USA; catalogue number 123040, 100 mg/Kg) was dosed as an oral gavage in normal saline (1 ml). Blood samples were taken 4 hours (hr) post dose and plasma separated and frozen at -20°C until analysis.

Group 2

5 rats (Lewis) were treated with methotrexate for 5 days at lmg/Kg ip (1ml of a solution in normal saline - 0.9%). Two hours after the last dose of methotrexate, normal saline was dosed as an oral gavage (1 ml). Blood samples were taken 4hr post dose and plasma separated and frozen at -20°C until analysis.

Group 3

5 rats (Lewis) were treated ip with ImI of normal saline for 5 days. Two hours after the last dose of saline, AICAR (100 mg/Kg) was dosed as an oral gavage in normal saline (1 ml). Blood samples were taken 4hr post dose and plasma separated and frozen at -20°C until analysis.

Group 4

5 rats (Lewis) were treated ip with ImI of normal saline for 5 days. Two hours after the last dose of saline, normal saline (1 ml) was dosed as an oral gavage. Blood samples were taken 4hr post dose and plasma separated and frozen at -20°C until analysis.

Sample Preparation

Plasma samples and standards (0.5ml) were each transferred to an Amicon Centrifree Micropartitioning Cartridge (No. 4104; Amicon Div., W.R. Grace & Co., Danvers, MA 01923) which has a molecular weight (m.w.) cut off at approxilately 30,000. The ultrafiltration devices were centrifuged at 2000 x g for 30 minutes using a fixed angle centrifuge rotor. The clear, almost colourless ultrafiltrated samples were then transferred to a Waters WISP 712 Autosampler for chromatography.

Sample Analysis

Reverse-phase HPLC was performed at room temperature using a Beckman Ultrasphere ODS column (5 um; 250 x 4.6 mm) with a mobile phase of 1.5% methanol in 1OmM ammonium phosphate (pH3) at a flow rate of 1.Oml/min. AICAR has a retention time of 6 minutes. Detection was at 270nm using a Waters model 484 UV detector and a Hewlett- Packard Model 3390A integrator.

Results

Compound levels of AICAR in plasma (ng/ml; nd = not detected)

Table 1:

Example 6: Air-pouch model for inflammation

Injection of air into the dorsal surface of rodents provides a cavity in which the inflammatory response can be studied. Over a period of days this leads to the development of a lining of granulation tissue consisting of fibroblasts, macrophages, and mast cells. The formation of the lining tissue allows a response to inflammatory stimuli and irritants injected into the air pouch which can be quantified by analysing the fluid exudate and the number of infiltrating inflammatory cells. The air-pouch model has been used for the study of various types of inflammation and the effect of anti- inflammatory drugs (Cronstein et al., 1993, J. Clin. Invest. 92, 2675-2682). In this experiment the effect of Methotrexate alone was compared to Methotrexate plus AICAR.

Induction of air pouches and carrageenan-induced inflammation

To induce an air pouch mice (female Balb/c, weight approx. 25 gram, 8 animals per group; Harlan) were injected subcutaneously (s.c.) with 3ml of air on the back on day -6 and -3. At time point zero the air pouch was injected with 1 ml of a 2% (weight/volume) suspension of carrageenan in saline and mice were returned to their cages. At the end of 4 hours the animals were sacrificed and ImI of normal saline was injected into the the pouch. The contents of the pouch were aspirated and cells were counted in samples using a Coulter Counter.

Study protocol: Treatment of animals

Group 1 : Control

Eight mice (female Balb/c) were treated with normal 0.9% saline once every seven days for three weeks (1 ml; intraperitoneal (ip)). Vehicle (1 ml of normal saline) was dosed as an oral gavage 60 minutes before injection of carrageenan. Air pouch exudates were collected as described above and cell numbers were counted.

Group 2: Methotrexate treatment

Eight mice (female Balb/c) were treated with Methotrexate (Calbiochem, catalogue number 454125) once every seven days for three weeks at 0.05 mg/kg (1 ml of a solution in normal 0.9% saline; intraperitoneal (ip)). Vehicle (1 ml of normal saline) was dosed as an oral gavage 60 minutes before injection of carrageenan. Air pouch exudates were collected as described above and cell numbers were counted.

Group 3: Methotrexate and AICAR treatment Eight mice (female Balb/c) were treated with Methotrexate (Calbiochem, catalogue number 454125) once every seven days for three weeks at 0.05 mg/kg (1 ml of a solution in normal 0.9% saline; intraperitoneal (ip)). AICAR (100 mg/kg as a solution in 1 ml of normal saline; Calbiochem, catalogue number 123040) was dosed as an oral gavage 60 minutes before injection of carrageenan. Air pouch exudates were collected as described above and cell numbers were counted.

Results

The result shows that the number of infiltrated cells in the air pouch exudates is lower for the animals treated with Methotrexate and AICAR (group 3) compared to Methotrexate alone (group 2) or the control group demonstrating a beneficial effect of the combination of Methotrexate and AICAR treatment.

Table 2: Cell numbers in air pouch exudates