EA040544B1 - PHARMACEUTICAL COMPOSITION FOR TREATMENT OF GASTROINTESTINAL INFLAMMATORY DISEASE, METHOD OF TREATMENT - Google Patents

Description

The field to which the invention relates

The invention is directed to pharmaceutical compositions comprising naphthyridine compounds, methods of using such compositions for the treatment of inflammatory diseases.

Prior Art

Ulcerative colitis is a chronic inflammatory disease of the colon. This disease is characterized by inflammation and ulceration of the mucous membrane of the rectum and large intestine. Common symptoms include diarrhea, bloody stools, and abdominal pain. The clinical course is intermittent, characterized by alternating periods of exacerbation and remission. This disease appears to be more common in developed countries than in developing countries. An estimated 1.2 million people in major industrialized countries have ulcerative colitis and this number is expected to increase with population growth. Patients with ulcerative colitis have an increased risk of developing colorectal cancer (eg, Danese et al. N. Engl. J. Med., 2011, 365, 1713-1725).

Although there are a number of therapeutic agents for promoting and maintaining remission of ulcerative colitis (UC) in patients, none of them is ideal. Sulfasalazine-based treatments are often effective in mild UC, but are significantly less effective in moderate to severe disease. Corticosteroids are often used to provide rapid induction of remission in patients with moderate to severe UC. However, long-term use of steroids to maintain remission is discouraged because it is associated with long-term side effects (eg, osteoporosis and bone fractures, infections, cataracts, slow wound healing, and suppression of adrenal hormone production). Systemic immunosuppressants such as azathioprine, cyclosporine, and methotrexate have a slow onset of action and limited efficacy in patients with moderate to severe UC, but long-term use may be problematic due to consequences such as prolonged systemic immunosuppression (eg, increased risk of infections and lymphomas). Anti-TNFa antibodies (eg, infliximab and adalimumab), although expensive and requiring subcutaneous or intravenous administration, are effective in approximately 60-70% of UC patients with moderate to severe disease. However, up to one third of patients do not respond well to treatment, while another third of those initially responding develop resistance within weeks (Allez et al., J. Crohn's Cilitis, 2010, 4, 355-366; Rutgeerts et al. al., N. Engl. J. Med., 2005, 353, 2462-2476). A more recently approved UC therapy, the anti-a ₄ to ₇ integrin antibody vedolizumab, is effective in patients with moderate to severe UC, although the parenteral route is not optimal and the consequences of long-term immunosuppression through this mechanism remain to be elucidated. Despite existing therapeutics, about 10-20% of UC patients still require colectomy within 10 years of diagnosis (Targownik et al., Am. J. Gastroenterol., 2012, 107, 1228-1235). Clearly, there remains an unmet medical need for an effective therapy to promote and maintain remission of moderate to severe UC without the safety concerns associated with chronic systemic immunosuppression.

Although the mechanism underlying ulcerative colitis has not been fully elucidated, it is believed that environmental factors in genetically predisposed individuals cause an inappropriate (over) response of the immune system to the gut microbiota, leading to colonic inflammation, tissue damage, and associated symptoms characteristic of gut microbiota. diseases.

Although the exact pathogenesis of UC has not been established, it is clear that pro-inflammatory cytokines play a key role in the immunological response (Strober et al., Gastroenterol., 2011, 140, 1756-1767). Many of the pro-inflammatory cytokines most commonly elevated in UC (eg, IL-4, IL-6, IL-13, IL-15, IL-23, IL-24, IFNy, and leptin) use the JAK family of tyrosine kinases (i.e., .JAK1, JAK2, JAK3 and Tuk2) for signal transmission. Binding of a ligand to a cytokine receptor triggers autophosphorylation of its associated JAK, which in turn leads to phosphorylation of the signal transmitter and transduction activator protein (STAT). Various STATs form hetero- or homodimers and promote transcription of their target genes in the cell nucleus to regulate functions such as cell growth, differentiation and death (Clark et al., J. Med. Chem., 2014, 57, 5023-5038).

Inhibition of JAK family enzymes can inhibit the signaling of many key pro-inflammatory cytokines. Thus, JAK inhibitors may be useful in the treatment of ulcerative colitis and other inflammatory diseases such as Crohn's disease, allergic rhinitis, asthma, and chronic obstructive pulmonary disease (COPD). However, due to the modulating effect of the JAK/STAT pathway on the immune system, systemic exposure to JAK inhibitors may have an adverse systemic immunosuppressive effect. Therefore, it would be desirable to provide new JAK inhibitors that have an effect at their site of action without significant systemic effects. In particular, for the treatment of gastrointestinal inflammatory diseases such as ulcerative colitis, it would be desirable to provide novel JAK inhibitors that can be administered orally and achieve therapeutically relevant effects in the gastrointestinal tract with minimal systemic exposure.

The essence of the invention

Accordingly, the invention provides a pharmaceutical composition for the treatment of a gastrointestinal inflammatory disease comprising a compound of the formula

or a pharmaceutically acceptable salt thereof; microcrystalline cellulose; lactose; magnesium stearate.

In another aspect, the invention provides a method of treating a gastrointestinal inflammatory disease in a human, comprising administering to a human a pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula

or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, wherein the Cmax in the subject is less than 100 ng/mL following administration to a human of a single dose containing up to 1000 mg of the compound, or less than 30 ng/mL after administration to a human of a dose containing up to 300 mg connections per day for 14 days.

Detailed description of the invention

The present invention provides a pharmaceutical composition for the treatment of a gastrointestinal inflammatory disease, comprising a compound of the formula

or a pharmaceutically acceptable salt thereof; microcrystalline cellulose; lactose; magnesium stearate.

In a specific aspect, the invention provides a pharmaceutical composition wherein the compound is in free base form.

In another specific aspect, the invention provides a pharmaceutical composition wherein the compound is present in an amount of 1 to 400 mg.

In another specific aspect, the invention provides a pharmaceutical composition wherein the compound is present in an amount of 5 to 300 mg.

In another specific aspect, the invention provides a pharmaceutical composition wherein the compound is present in an amount of 20 to 70 mg.

In another specific aspect, the invention provides a pharmaceutical composition in which the compound is present in an amount of 10 mg.

In another specific aspect, the invention provides a pharmaceutical composition in which the compound is present in an amount of 20 mg.

In another specific aspect, the invention provides a pharmaceutical composition in which the compound is present in an amount of 40 mg.

In another specific aspect, the invention provides a pharmaceutical composition in which the compound is present in an amount of 100 mg.

The present invention provides a method for treating a gastrointestinal inflammatory disease in a human, comprising administering to a human a pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula

- 2 040544

or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, wherein the Cmax in the subject is less than 100 ng/mL following administration to a human of a single dose containing up to 1000 mg of the compound, or less than 30 ng/mL after administration to a human of a dose containing up to 300 mg connections per day for 14 days.

In a specific aspect, the invention provides a method of treatment wherein the subject's Cmax is less than 50 ng/mL following administration to the human of a single dose containing up to 1000 mg of the compound.

In another specific aspect, the invention provides a method of treatment wherein the subject is administered a single dose containing 1000 mg of a compound.

In another specific aspect, the invention provides a method of treatment wherein the subject is administered 300 mg of a compound per day for 14 days with a Cmax of less than 30 ng/mL.

In another specific aspect, the invention provides a method of treatment wherein the C _max is less than 15 ng/mL following administration to a human of a dose containing up to 300 mg of the compound per day for 14 days.

Definitions

In describing the present invention, including its various aspects and embodiments, the following terms have the following meanings, unless otherwise indicated.

The term "therapeutically effective amount" means an amount sufficient to effect treatment when administered to a patient in need of treatment.

The term treatment, as used herein, means the treatment of a disease, disorder, or medical condition (such as gastrointestinal inflammatory disease) in a patient, such as a mammal (particularly a human), which includes one or more of the following:

(a) preventing the occurrence of a disease, disorder or medical condition, i.e. preventing the occurrence of a disease or medical condition or prophylactically treating a patient who is predisposed to such a disease or medical condition;

(b) reducing the intensity of the disease, disorder or medical condition, i.e. eliminating or inducing regression of a disease, disorder or medical condition in a patient, including counteracting the effects of other therapeutic agents;

(c) suppression of the disease, disorder or medical condition, i.e. slowing or stopping the development of a disease, disorder or medical condition in a patient; or (d) alleviating symptoms of a disease, disorder, or medical condition in a patient.

The term "pharmaceutically acceptable salt" means a salt that is acceptable for administration to a patient or a mammal, such as a human (eg, salts that are acceptable for mammalian safety in a given administration schedule). Representative pharmaceutically acceptable salts include acetic, ascorbic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, edisylic, fumaric, gentisic, gluconic, glucuronic, glutamic, hippoucric, hydrobromic, hydrochloric, isethionic, lactic, lactobionic, maleic, malic, almond, methanesulfonic, mucinic, naphthalenesulfonic, naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic and xinafoic acids, etc. .

pharmaceutical compositions.

The compounds of the present invention and their pharmaceutically acceptable salts are typically used in the form of a pharmaceutical composition or formulation. Such pharmaceutical compositions can be administered to a patient by any suitable route of administration, including, but not limited to, oral, rectal, nasal, inhalation, topical (including transdermal) and parenteral routes of administration.

Accordingly, in one aspect relating to compositions, the invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a compound of the formula

- 3 040544

or a pharmaceutically acceptable salt thereof.

Optionally, such pharmaceutical compositions may contain other therapeutic agents and/or formulation agents, if desired.

Pharmaceutical compositions of the present invention typically contain a therapeutically effective amount of a compound of the formula

Those skilled in the art will appreciate, however, that a pharmaceutical composition may contain more than a therapeutically effective amount, i. bulk compositions, or less than a therapeutically effective amount, i. separate unit doses, intended for several injections to achieve a therapeutically effective amount.

Typically, such pharmaceutical compositions will contain from about 0.1% to about 95% by weight of the active substance; preferably from about 5 to about 70 wt.% and more preferably from about 10 to about 60 wt.% of the active substance.

Any conventional carrier or excipient can be used in the pharmaceutical compositions of the present invention. The choice of a particular carrier or excipient, or combinations of carriers or excipients, will depend on the route of administration used to treat a particular patient, or the type of medical condition or disease state. In this regard, obtaining a suitable pharmaceutical composition for a particular route of administration is within the competence of specialists in the field of pharmaceuticals. In addition, carriers or excipients used in the pharmaceutical compositions of the present invention are commercially available. As a further illustration, conventional formulation methods are described in Remington: The Science and Practice of Pharmacy, ^20th Edition, Lippincott Williams & White, Baltimore, Maryland (2000); and H.C. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, ^7th Edition, Lippincott Williams & White, Baltimore, Maryland (1999).

Representative examples of substances that can serve as pharmaceutically acceptable carriers include, but are not limited to, the following: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose such as microcrystalline cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycols; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic compatible substances used in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by intimately mixing or combining the active substance with a pharmaceutically acceptable carrier and one or more optional ingredients. The resulting homogeneous mixture can then be molded or loaded into tablets, capsules, pills, and the like. using traditional procedures and equipment.

The pharmaceutical compositions of the present invention are preferably packaged in unit dosage form. The term unit dosage form refers to a physically discrete unit suitable for administering a dose to a patient, i.e. each unit contains a predetermined amount of the active substance, calculated to provide the desired therapeutic effect, either alone or in combination with one or more additional units. For example, such unit dosage forms may be capsules, tablets, pills, and the like. or standard packs suitable for parenteral administration.

In one embodiment, the pharmaceutical compositions of the present invention are suitable for oral administration. Suitable pharmaceutical compositions for oral administration may be in the form of capsules, tablets, pills, lozenges, sachets, dragees, powders, granules; or as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup; and so on.; wherein each form contains a predetermined amount of the compound of the present invention as the active ingredient.

When intended for oral administration in solid dosage form (i.e., capsules, tablets, pills, etc.), the pharmaceutical compositions of the present invention will typically comprise the active agent and one or more pharmaceutically acceptable carriers such as citrate. sodium or dicalcium phosphate. Optionally or alternatively, such solid dosage forms may also include fillers or bulking agents such as starches, microcrystalline cellulose, lactose, dicalcium phosphate, sucrose, glucose, mannitol and/or silicic acid; binders such as carboxymethylcellulose, hydroxypropylmethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or gum arabic; humectants such as glycerin; disintegrants such as croscarmellose sodium, crospovidone, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and/or sodium carbonate; retarding dissolution agents such as paraffin; absorption accelerators such as quaternary ammonium compounds; wetting agents such as cetyl alcohol and/or glycerol monostearate; absorbents such as kaolin and/or bentonite clay; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and/or mixtures thereof; dyes; and buffer agents.

Release agents, wetting agents, coating agents, sweeteners, flavoring agents, preservatives and antioxidants may also be present in the pharmaceutical compositions of the present invention. Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodium sulfite, and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid, sorbitol, tartaric acid, phosphoric acid, and the like. Coating agents for tablets, capsules, pills and the like. include agents used for enteric coatings such as cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, methacrylic acid, methacrylic acid ester copolymers, cellulose acetate trimethyllate, carboxymethyl ethylcellulose, hydroxypropyl methylcellulose acetate succinate, and the like.

Pharmaceutical compositions of the present invention can also be formulated to provide slow or controlled release of the active substance using as an example hydroxypropyl methylcellulose in varying proportions or other polymeric matrices, liposomes and/or microspheres. In addition, the pharmaceutical compositions of the present invention may optionally contain opacifying agents and may be formulated so that they release the active ingredient only, or predominantly, in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of drug embedding compositions that can be used include polymeric materials and waxes. The active substance may also be in microencapsulated form, if appropriate, with one or more of the excipients described above.

Suitable liquid dosage forms for oral administration include, by way of illustration, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. Liquid dosage forms typically comprise an active agent and an inert diluent such as, for example, water, or other solvents, solubilizing agents, and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (particularly cottonseed, peanut, corn, wheat germ, olive, castor and sesame oils), oleic acid, glycerin, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Alternatively, some liquid compositions can be converted, for example by spray drying, to a powder that is used to prepare solid dosage forms using conventional procedures.

Suspensions, in addition to the active ingredient, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Alternatively, the pharmaceutical compositions of the present invention are formulated for administration by inhalation. Suitable pharmaceutical compositions for administration by inhalation will typically be in the form of an aerosol or powder. Such compositions are usually administered using well known delivery devices such as metered dose inhaler, dry powder inhaler, nebulizer

- 5 040544 or similar delivery device.

When administered by inhalation using a pressurized container, the pharmaceutical compositions of the present invention typically may include the active ingredient and a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In addition, the pharmaceutical composition may be in the form of a capsule or cartridge (eg gelatin) comprising a compound of the present invention and a powder suitable for use in a dry powder inhaler. Suitable powder bases include, by way of example, lactose or starch.

The following non-limiting examples illustrate representative pharmaceutical compositions of the present invention.

Oral solid dosage form in the form of a tablet.

Formula Compound

or a pharmaceutically acceptable salt thereof is dry-mixed with microcrystalline cellulose, polyvinylpyrrolidone and croscarmellose sodium in a ratio of 4:5:1:1 and compressed into tablets to obtain a unit dosage form, for example, 5, 20 or 40 mg of active substance per tablet.

Oral solid dosage form in the form of a capsule.

Formula Compound

or a pharmaceutically acceptable salt thereof is combined with microcrystalline cellulose, polyvinylpyrrolidone and croscarmellose sodium in a ratio of 4:5:1:1 by wet granulation and loaded into gelatin or hydroxypropyl methylcellulose capsules to obtain a unit dosage form, for example, 5, 20 or 40 mg of the active substance per capsule.

Oral solid dosage form in the form of a tablet.

Formula Compound

or a pharmaceutically acceptable salt thereof is dry or wet granulated with microcrystalline cellulose, lactose, hydroxypropyl methylcellulose, crospovidone and magnesium stearate. The composition includes, in % w/w, the compound of the present invention (4%), microcrystalline cellulose (45%), lactose (36%), hydroxypropyl methylcellulose (10%), crospovidone (3%) and magnesium stearate (2% ). Mixtures obtained by dry or wet granulation are compressed into tablets to obtain a unit dosage form of 10 mg of active substance per 250 mg tablet.

Oral solid dosage form in the form of a tablet.

Formula Compound

or a pharmaceutically acceptable salt thereof is dry or wet granulated with microcrystalline cellulose, hydroxypropyl methylcellulose, crospovidone and magnesium stearate

- 6 040544 niya.

The composition comprises, in % w/w, the compound of the present invention (40%), microcrystalline cellulose (45%), hydroxypropyl methylcellulose (10%), crospovidone (3%) and magnesium stearate (2%). Mixtures obtained by dry or wet granulation are compressed into tablets to obtain a unit dosage form of 100 mg of active substance per 250 mg tablet.

Liquid dosage form.

A liquid dosage form comprising a compound of the formula

(0.1%), water (98.9%) and ascorbic acid (1.0%) are obtained by adding the compound of the present invention to a mixture of water and ascorbic acid.

Enteric-coated oral dosage form.

Formula Compound

dissolved in an aqueous solution containing polyvinylpyrrolidone, and applied by spraying on granules of microcrystalline cellulose or sugar in a ratio of 1:5 wt./wt. active substance: granules and then enteric coating is applied, giving an increase in weight of about 5%, which includes an acrylic copolymer, for example a combination of acrylic copolymers available under the trademarks Eudragit-L® and Eudragit-S®, or hydroxypropyl methylcellulose acetate succinate. The enteric-coated beads are loaded into gelatin or hydroxypropyl methylcellulose capsules to form a unit dosage form, eg 30 mg active per capsule.

Enteric-coated oral dosage form.

An enteric coating comprising a combination of Eudragit-L® and Eudragit-S® or hydroxypropyl methylcellulose acetate succinate is applied to the oral tablet dosage form or the oral capsule dosage form described above.

Applicability.

It has been shown that the compound of formula

is a strong inhibitor of the JAK family of enzymes: JAK1, JAK2, JAK3 and TYK2.

Inhibition of the JAK family of enzymes can inhibit the signaling of many key pro-inflammatory cytokines. Thus, the JAK inhibitors of the present invention are expected to be useful in the treatment of inflammatory diseases such as ulcerative colitis, Crohn's disease, allergic rhinitis, asthma, and chronic obstructive pulmonary disease (COPD).

The compound of the invention is designed to have low absorption to minimize systemic exposure. As described in the experimental section below, the absorption and distribution of exemplary compounds have been extensively profiled in preclinical analyses.

Oxazolone-induced colitis is an experimental model that has histological similarity to human ulcerative colitis. As described below, the compound of Example 1, among other compounds of the invention, showed activity in a mouse model of oxazolone-induced colitis. In addition, when tested in a mouse immunosuppression model that examines systemic functional activity, the compound showed minimal immunosuppressive effect at the same dose required to demonstrate efficacy in the oxazolone model. Thus, the compound showed activity against colitis without systemic manifestations.

- 7 040544 effects in preclinical models.

The high colon:plasma ratio achieved by these compounds is expected to provide robust luminal-targeted anti-inflammatory activity, without associated systemic side effects.

The compounds are expected to be useful for various inflammatory indications of the gastrointestinal tract, which include, but are not limited to, ulcerative colitis (proctosigmoiditis, pancolitis, ulcerative proctitis, and left-sided colitis), Crohn's disease, collagenous colitis, lymphocytic colitis, Behcet's disease, celiac disease, colitis induced by cancer treatment (eg, CTLA-4 inhibitor-induced colitis), ileitis, eosinophilic esophagitis, graft-versus-host disease-associated colitis, and infectious colitis. Ulcerative colitis (Reimund et al., J. Clin. Immunology, 1996, 16, 144-150), Crohn's disease (Woywodt et al., Eur. J. Gastroenterology Hepatology, 1999, 11, 267-276), collagenous colitis ( Kumawat et al., Mol. Immunology, 2013, 55, 355-364), lymphocytic colitis (Kumawat et al., 2013), eosinophilic esophagitis (Weinbrand-Goichberg et al., Immunol. Res., 2013, 56, 249260) , graft-versus-host disease-associated colitis (Coghill et al., Blood, 2001, 117, 32683276), infectious colitis (Stallmach et al., Int. J. Colorectal. Dis., 2004, 19, 308-315 ), Behcet's disease (Zhou et al., Autoimmun Rev, 2012, 11, 699-704), celiac disease (de Nitto et al., World J. Gastroenterol., 2009, 15, 4609-4614), cancer treatment-induced colitis (e.g., CTLA-4 inhibitor-induced colitis (Yano et al., J. Translation. Med., 2014, 12, 191) and ileitis (Yamamoto et al., Dig. Liver. Dis., 2008, 40, 253 -259) are characterized by increased levels of certain pro-inflammatory cytokines. cytokines signal through JAK activation, the compounds described herein may alleviate inflammation and provide symptomatic relief.

In particular, it is expected that the compound of formula

would be useful for the induction and maintenance of remission of ulcerative colitis and for the treatment of Crohn's disease, CTLA-4 inhibitor-induced colitis, and gastrointestinal side effects in graft-versus-host disease.

In one aspect, the invention therefore provides a method of treating a gastrointestinal inflammatory disease in a mammal (e.g., a human) comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of formula

The invention also provides a method for treating ulcerative colitis in a mammal, comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the formula

When used to treat gastrointestinal inflammatory disease, a compound of formula

- 8 040544 is usually administered orally as a single daily dose or as multiple doses per day, although other forms of administration may be used.

The amount of active substance administered per dose, or the total amount administered per day, will generally be determined by the attending physician in light of the relevant circumstances, including the condition being treated, the mode of administration chosen, the particular compound administered and its relative potency, age, body weight, and response. a particular patient, the severity of the patient's symptoms, and the like.

It is expected that suitable doses for the treatment of ulcerative colitis and other inflammatory diseases of the gastrointestinal tract may range from about 1 to about 400 mg/day of the active substance, including from about 5 to about 300 mg/day and from about 20 to about 70 mg per day of the active substance for a person with an average body weight of 70 kg.

Examples

The following synthetic example and biological examples are provided to illustrate the invention. In the examples below, the following abbreviations have the following meanings, unless otherwise noted. Abbreviations not defined below have their conventional meanings.

ACN = acetonitrile

DCM = dichloromethane

DIPEA = Ν,Ν-diisopropylethylamine

DMF = N, W-dimethylformamide

DMSO = dimethyl sulfoxide

EtOAc = ethyl acetate h = hour(hours)

HATU= Ν,Ν,Ν',Ν'-tetramethyl-O-(Ίazabenzotriazol-1-yl)uronium hexafluorophosphate min = minute(s)

ΝΜΡ = M-methyl-2-pyrrolidone

Pd (dppf) C1 ₂ \u003d dichloro (1, l'-bis (diphenylphosphino) ferrocene) dipalladium (II)

Pd ₂ (dba) ₃ \u003d tris (dibenzylideneacetone) dipalladium (0)

PdXPhos = chloro(2-dicyclohexylphosphino-2',4',6' triisopropyl-1,1'-biphenyl)[2-(2'-amino-

1, 1'-biphenyl)]palladium(II)

RT = room temperature

Selectfluor = 1-chloromethyl-4-fluoro-1,4diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate)

TEA = triethylamine

TEA = trifluoroacetic acid

THF = tetrahydrofuran bis(pinacolato) 4,4,5, 5,4' , 4',5',5'-octamethyldiboron = [2.2']bi[[1.3.2]dioxaborolanyl]

Xantphos = 4,5-bis(diphenylphosphino)-9,9dimethylxanthene

Xphos = Dicyclohexylphosphino-2',4',6'trisopropylbiphenyl

Reagents and solvents were purchased from commercial suppliers (Aldrich, Fluka, Sigma, etc.) and used without further purification. The progress of the reaction was monitored using a thin-layer

- 9 040544 chromatography (TLC), analytical high performance liquid chromatography (anal. HPLC) and mass spectrometry. The reaction mixtures were processed as described specifically for each reaction; they have usually been purified by extraction and other purification methods such as temperature- and solvent-dependent crystallization and precipitation. In addition, the reaction mixtures were typically purified by column chromatography or preparative HPLC, typically using C18 or BDS packed columns and conventional eluents. Typical preparative HPLC conditions are described below.

Characterization of the reaction products was usually carried out by mass and 1H-NMR spectrometry. For NMR analysis, samples were dissolved in a deuterated solvent (such as CD3OD, CDCl3 or d ₆ -DMSO) and 1H-NMR spectra were obtained using a Varian Gemini 2000 (400 MHz) instrument under standard observation conditions. Mass spectrometric identification of compounds was performed by electrospray ionization (ESMS) using an Applied Biosystems (Foster City, CA) model API 150 EX or Waters (Milford, MA) 3100 device connected to auto-cleaning systems.

Example 1 3-((1R,3s,5S)-3-((7-((5-Methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino) -8azabicyclo[3.2.1]octan-8-yl)propanenitrile

N ⁵ -((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N ⁷ -(5-methyl-1H-pyrazol-3yl)-1, 6-naphthyridine-5,7-diamine, 2TFA (462.4 mg, 0.80 mmol), methanol (4 ml) and DIPEA (0.70 ml, 4.00 mmol). The reaction mixture was stirred at room temperature for 5 minutes and then acrylonitrile (0.058 ml, 0.88 mmol, 100 ml) was added slowly. The vessel was capped and the reaction mixture was stirred at room temperature for 20 h, concentrated, dissolved in 15% acetic acid in water and purified by preparative HPLC (method 1). Fractions were pooled, lyophilized, dissolved in 15% acetic acid in water and purified by reverse phase HPLC. Fractions were combined and lyophilized to give the title compound as a bright red solid (505 mg, 52% yield; 98% pure).

(m/z): [M+H] ⁺ calculated for C22H26N8 403.23 found 403.7.

Analysis 1. Biochemical analyzes of JAK and non-target kinases.

A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3, and Tyk2) were run in normal kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl ₂ and 1 mM EGTA). Recombinant GST-labeled JAK enzymes and GFP-labeled STAT1 peptide substrate were obtained from Life Technologies.

Serially diluted compounds were pre-incubated with each of the four JAK enzymes and substrate in white 384-well microplates (Corning) at ambient temperature for 1 h. ATP was then added to initiate the kinase reaction in a total volume of 10 ml, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3, and Tuk2 were 4.2, 0.1, 1, and 0.25 nM, respectively; the respective ATP Km concentrations used were 25, 3, 1.6 and 10 μM; while the substrate concentration was 200 nM for all four assays. Kinase reactions were allowed to run for 1 h at ambient temperature before adding 10 µl of EDTA preparation (final concentration 10 nM) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, final concentration 2 nM) in TR-FRET dilution buffer (Life Technologies). Plates were incubated at ambient temperature for 1 hour, then read on an Envision plate reader (Perkin Elmer). The ratio of emission signals (520 nm/495 nm) was recorded and used to calculate percent inhibition based on DMSO and background controls.

For dose-response analysis, percent inhibition was plotted against compound concentrations and IC ₅₀ values were determined using a 4-parameter good fit model and Prism (GraphPad Software). The results were expressed as pIC ₅₀ (negative logarithm of IC ₅₀ ) and then converted to pKi (negative logarithm of the dissociation constant, Ki) using the Cheng-Prusov equation.

Test compounds having a higher pKi value in each of the four JAK assays showed greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibit pKi values of about 7 to about 10.3.

A panel of non-target tyrosine kinase assays (Flt3, RET, FGFR2, TrkA and pDGFRP) was developed using the same methodology, with recombinant enzymes obtained from Life Technologies and biotinylated peptide substrates synthesized in AnaSpec. All assays were performed at ambient temperature with a final ATP concentration of 100 mM. Detection reagents including Eu-anti-phosphotyrosine (pY20) antibody and SureLight APC-SA were purchased from

- 10 040544

Perkin Elmer. The ratio of emission signals (665 nm/615 nm) was recorded and used for data analysis and the final results were expressed as pIC ₅₀ .

Assay 2 Cellular JAK activity assay.

Cellular analysis of JAKI AlphaScreen activity was performed by measuring interleukin-13 (IL-13, R&D Systems)-induced STAT6 phosphorylation in BEAS-2B human lung epithelial cells (ATCC). Anti-STAT6 antibody (Cell Signaling Technologies) was conjugated to AlphaScreen acceptor beads (Perkin Elmer), while anti-pSTAT6 (pTyr641) antibody (Cell Signaling Technologies) was biotinylated using EZ-Link Sulfo-NHS-Biotin (Thermo Scientific).

BEAS-2B cells were grown at 37° C. in a 5% CO ₂ humidified incubator in 50% DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS (Hyclone), 100 U/ml penicillin, 100 μg/ ml streptomycin (Life Technologies) and 2 mM GlutaMAX (Life Technologies). On day 1 of the assay, cells were seeded at a density of 7500 cells/well in white poly-D-lysine coated 384-well plates (Corning) with 25 μl of medium and allowed to adhere overnight in the incubator. On day 2 of the assay, media was removed and replaced with 12 ml of assay buffer (Hank's balanced salt solution/HBSS, 25 mM HEPES and 1 mg/ml bovine serum albumin/BSA) containing dose-response test compounds. Compounds were serially diluted in DMSO and then further diluted 1000-fold in medium to bring the final DMSO concentration to 0.1%. Cells were incubated with test compounds at 37° C. for 1 hour followed by the addition of 12 μl of prewarmed IL-13 (80 ng/ml in assay buffer) for stimulation. After incubation at 37° C. for 30 min, the assay buffer (containing the compound and IL-13) was removed and 10 μl of cell lysis buffer (25 mM HEPES, 0.1% SDS, 1% NP-40, 5 mM MgCl _{2 )} was added. , 1.3 mM EDTA, 1 mM EGTA and supplemented with a cocktail of protease inhibitors Complete Ultra mini and PhosSTOP from Roche Diagnostics). The plates were shaken at ambient temperature for 30 minutes before adding detection reagents. First, a mixture of biotin-anti-pSTAT6 and anti-STAT6-conjugated acceptor beads was added and incubated at ambient temperature for 2 h, then streptavidin-conjugated donor beads (Perkin Elmer) were added. After a minimum of 2 hours of incubation, the assay plates were read on an Envision plate reader. AlphaScreen luminescent signals were recorded and used to calculate percent inhibition based on DMSO and background controls.

For dose-response analysis, percent inhibition was plotted against compound concentrations and IC ₅₀ values were determined using a 4-parameter good fit model and Prism software. The results were expressed as the negative logarithm of the IC ₅₀ value, pIC ₅₀ .

Test compounds having a higher PIC ₅₀ value in this assay showed greater inhibition of IL-13-induced STAT6 phosphorylation. Compounds of the invention tested in this assay typically exhibited pIC ₅₀ values of about 6.8 to about 8.5.

Interleukin-4 (IL-4, R&D Systems)-induced STAT6 phosphorylation assay was also performed in THP-1 human monocytes (ATCC) using the same assay formats and detection reagents. IL-4 stimulation was performed for 30 min at a final concentration of 30 ng/ml. Data collection and analysis was also carried out in a similar manner. Compounds of the invention tested in this assay typically exhibited pIC ₅₀ values of about 6.8 to about 8.5.

Assay 3 Cytotoxicity assay.

The CellTiter-Glo luminescent cell survival/cytotoxicity assay was performed in BEAS-2B human lung epithelial cells (ATCC) under normal growth conditions.

Cells were grown at 37°C in a 5% CO ₂ humidified incubator in 50% DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS (Hyclone), 100 U/ml penicillin, 100 μg/ml streptomycin ( Life Technologies) and 2 mM GlutaMAX (Life Technologies). On day 1 of analysis, cells were seeded at a density of 500 cells/well in white 384-well tissue culture plates (Corning) with 25 μl of medium and left to adhere overnight in an incubator. On day 2 of the assay, 5 µl of medium containing the dose-response test compounds was added and incubated at 37°C for 48 h. Then 30 ml of CellTiter-Glo detection solution (Promega) was added, mixed on an orbital shaker for 5 min and incubated for an additional 10 min, then read on an Envision plate reader. Luminescence signals were recorded and the percentage of the DMSO control was calculated.

For dose-response analysis, the percentage of the DMSO control was plotted against compound concentrations to obtain dose-response curves along the line connecting each data point. The concentration at which each curve crosses the 15% inhibition threshold is defined as CC ₁₅ . The results were expressed as the negative logarithm of the CC ₁₅ value, pCC ₁₅ .

Test compounds that have a lower pCC ₁₅ value in this assay are expected to be less likely to cause cytotoxicity. Compounds of the invention tested in this assay typically exhibit pCC ₁₅ values of less than 5 to about 6.

Assay 4 Determination of absorbance in cannulated rats.

Oral bioavailability (F%), absorbed fraction (F _a %) and hepatic clearance escaping fraction (F _h %) were determined in Sprague Dawley rats in the following two tests:

- 11 040544 (1) Pharmacokinetics in rats after i.v. dose of test compound: After i.v. dosing, plasma samples were usually collected between 0-6 hours. Drug levels were determined using LC-MS-MS method. The resulting drug levels were used to calculate IV pharmacokinetic parameters: AUC i.v. and dose i.v.

(2) Rats cannulated into their portal vein (PV) as well as their jugular vein (JV) were administered the test compound orally. After oral dosing, plasma samples were typically collected between 0-6 hours from both the portal vein and the jugular vein. Drug levels were determined using the LC-MS-MS method. The resulting drug levels were used to calculate the following pharmacokinetic parameters: AUC p.o. PV, AUC p.o. JV and dose p.o.

Using the data obtained in the trials described above, the oral bioavailability F% and the amounts of F _a % and Fh% were calculated from the following formulas:

F% \u003d (AUC p.o. JV / AUC i.v.) x (Dose i.v. / Dose p.o.) x100

F _a % \u003d (AUC p.o. PV / AUC i.v.) x (Dose i.v. / Dose p.o.) x100

F _h %= AUC p.o. JV/AUC p.o. PV where AUC p.o. JV = area under the curve after oral dose and plasma collected from the jugular vein;

AUC p.o. PV = area under the curve after oral dose and plasma collected from the portal vein;

AUC v.v. = area under the curve after intravenous dose;

Dose i.v. = intravenous dose in mg/kg;

Dose p.o. = oral dose in mg/kg.

3-((1R,3s,5S)-3-((7-((5-Methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)αmino)-8-azαbicyclo [3.2.1]octan-8-yl)propanenitrile typically exhibited oral bioavailability (F%) of less than about 5% and Fa% values of less than about 10%.

Analysis 5. Pharmacokinetics in the colon of rats.

3-((1R,3s,5S)-3-((7-((5-Methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo [3.2.1]octan-8-yl)propanenitrile was individually formulated in 0.5% methylcellulose in water and administered by oral gavage at 5 mg/kg to Sprague Dawley rats. At various time points (typically 1, 2, 4, 6, 24 hours) after dosing, blood samples were taken by cardiac puncture and intact colons were excised from rats. Blood samples were centrifuged at 1500xg for 15 min to collect plasma. The colons were washed with ice-cold phosphate-buffered saline (PBS), weighed and homogenized at a 1:10 dilution in PBS. Plasma and colon test compound levels were determined by LC-MS analysis against analytical standards embedded in a standard curve in the test matrix. Colon to plasma ratio was defined as the ratio of colon AUC to plasma AUC in μg h/g. For example, 3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrαzol-3-yl)αmino)-1,6-nαphthyridin-5-yl)amino)-8 -azabicyclo[3.2.1]octan-8-yl)propanenitrile exhibited a colon:plasma ratio greater than about 450.

Analysis 6. Plasma and gastrointestinal pharmacokinetics in rats and dogs.

3-((1R,3s,5S)-3-((7-((5-Methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)αmino)-8-azαbicyclo [3.2.1]octan-8-yl)propanenitrile was administered to male Sprague Dawley rats (n=3) as described in analysis 5. Plasma samples were taken at each time point (0.5, 1, 3, 6, and 24 h). by cardiac puncture and immediately thereafter, the gastrointestinal tract was removed and the following segments were excised: duodenum, proximal colon, and distal colon. Plasma and segment test compound levels were determined as described in Assay 5. At all time points, the plasma concentration was below the quantitation limit of 0.001 μg/mL. For 3-((1R,3s,5S)-3-((7((5-methyl-1H-pyrαzol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo [3.2.1]octan-8yl)propanenitrile, for each segment, the tissue-to-plasma ratio was greater than about 14,000.

A similar experiment was carried out in dogs. Male beagle dogs (n=2) were administered by oral gavage at 5 mg/kg of test compound as above. From dog #1, plasma samples were taken at time points 0.25, 1, 2, 4, 6 and 24 hours post dosing. In dog #2, plasma samples were taken at the 6 hour time point. In both dog #1 (at 24 hours) and dog #2 (at 6 hours), the gastrointestinal tract was removed and segmented as follows: duodenum, ileum , the caecum and the colon, divided into three equal parts (proximal, middle and distal colon). For each of the segments of the gastrointestinal tract, a piece of approximately 2 cm was cut out in the middle of the segment. Each of them was thoroughly washed in ice-cold PBS buffer and then homogenized in 5 volumes of PBS buffer and analyzed as above. For the compound of Example 1, the ratio of compound concentration in gastrointestinal tissue to compound in plasma for samples taken 6 hours after an oral dose ranged from about 9 to about 165, with the concentration in the colon taken as the sum of three segments of the colon . The ratio of concentration in the gastrointestinal tract to plasma concentration ranged from 7 to 30 for the measurement

- 12 040544 ions, which were carried out 24 hours after the oral dose.

Analysis 7. Mouse model of oxazolone-induced colitis.

Oxazolone-induced colitis is an experimental model that has histological similarity to human ulcerative colitis (Heller et al., Immunology, 2002, 17, 629-638). Adult Balb/C mice from Harlan were used in the assay. On day 1, animals were lightly anesthetized with isoflurane and hairs between the shoulders were carefully removed before oxazolone (4%, 150 μl, in 4:1 acetone:olive oil) or vehicle solution was slowly applied to skin sensitization. Seven days after skin sensitization, the mice were fasted overnight, anesthetized with isoflurane inhalation, and a 1 ml syringe equipped with a 3.5-F catheter filled with oxazolone solution was gently inserted about 4 cm into the colon of the mouse. After injection, 50 μl of a solution of oxazolone (1%, in a mixture of 1:1 ethanol:water) was injected very slowly (over 30 seconds using an injection pump) into the colon. The catheter was removed and the mice were held upright (head down) for 2 min to ensure that all of the oxazolone solution remained inside the colon. Treatment with drug (p.o., twice a day (BID) or three times a day (TID)) or vehicle was started the day before intrarectal (i.r.) administration of oxazolone. Two days after intrarectal administration of oxazolone, the disease activity index (DAI) was assessed by treatment-unaware experimenters for each mouse according to the criteria:

assessment of stool consistency (0 normal, 2 - liquid, 4 - diarrhea);

overall assessment of bleeding (0 - no, 2 - shade of blood, 4 - presence);

assessment of body weight loss (0 - no, 1 - from 1 to 5%, 2 - from 5 to 10%, 3 - from 10 to 20%, 4 - more than 20%);

DAI = mean of (stool consistency score + total bleeding score + weight loss score).

Efficacy in this model is supported by a reduction in the DAI score compared to that of vehicle-treated animals. Compound of Example 1 (3-((1R,3s,5S)-3-((7-((5-methyl-1Hpyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8 -azabicyclo[3.2.1]octan-8-yl)propanenitrile) showed a statistically significant reduction in DAI score compared to vehicle-treated animals in the oxazolone model at 1, 3 and/or 10 mg/kg BID.

Analysis 8 Effects of Immunosuppression in Natural Killer (NK) Mouse Spleen Cells.

Depletion of mouse spleen cells is an experimental model for immunosuppression (Kudlacz et al., Am. J. of Transplantation, 2004, 4, 51-57). Compound of Example 1 (3-((1R,3s,5S)-3-((7-((5methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8 -azabicyclo[3.2.1]octan-8-yl)propanenitrile) was evaluated in a mouse spleen cell model following the same treatment regimen as in an oxazolone-induced colitis model (Analysis 7).

Adult male Balb/C mice (12-14 weeks old) from Harlan were used for the study. Compound (1, 10 and 100 mg/kg, BID) and tofacitinib (30 mg/kg, BID) were dosed orally for three days to previously naive mice as positive controls. Spleens were harvested 1 or 2 hours after the last dose and immediately minced for cell subtype staining. Prior to fixation, fluorophore-labeled antibodies for CD19 (FITC; B cells), CD3e (PE, pan T cells), and DX5 (APC; NK cells) were incubated with splenocyte samples from each animal to enable simultaneous analysis of % of multiple subtypes per flow cytometer. The number of all spleen cells for each animal was measured using a pocket automated cell counter Scepter™ 2.0.

The absolute population count of lymphocyte subtypes (eg, splenic B, T, and NK cells) was calculated as the percentage of each subtype per total spleen cell count for each animal. One-way ANOVA with Dunnett's post hoc test was used to compare the number of splenic lymphocytes in the vehicle and test compound groups. The α level was set at p<0.05. Data were presented as mean ± SEM for each group.

The positive control tofacitinib (30 mg/kg, po, BID) was dose dependent and significantly reduced spleen NK cells. In the same study, the compound of Example 1 (3-((1R,3s,5S)-3-((7((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl )amino)-8-azabicyclo[3.2.1]octan-8yl)propanenitrile) did not affect the number of splenic NK cells at p.o. doses. (BID) up to 100 mg/kg (tested maximum dose). No treatment effect was observed for the B and T cell populations with any compound.

These data, combined with the minimum dose of 1 mg/kg, which produced a significant anti-colytic effect in a mouse model of oxazolone-induced colitis (Test 7), allow a functional therapeutic index of >100 to be calculated for the compound of Example 1.

Assay 9. First in a human trial to assess safety, tolerability and pharmacokinetics in healthy subjects.

Compound of Example 1 (3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5yl)amino)-8 -azabicyclo[3.2.1]octan-8-yl)propanenitrile) was evaluated in a double-blind, randomized, placebo-controlled, single dose escalation (SAD) and multiple escalation

-

Claims (14)

dose (MAD) study of safety, tolerability and pharmacokinetics in healthy subjects. PK samples were collected after the final dose up to 72 hours for both the SAD test (after the first dose) and the MAD test (after 14 days of single daily dosing). There were 5 groups in the SAD trial and 4 groups in the MAD trial with a total of 72 subjects, of which 71 completed the full dosing period. Plasma pharmacokinetic (PK) parameters were determined by non-compartmental analysis using WinNonLin Version 6.4.0 (Pharsight, St Louis, MO). The PK parameter in plasma presented here is as follows. Cmax: maximum plasma concentration. After a single dose up to 1000 mg, the mean plasma concentration of the compound Cmax was less than 50 ng/mL, with no individual subject achieving Cmax greater than 100 ng/mL. After 14 days of administration of the compound up to 300 mg, the mean plasma concentration of the compound Cmax was less than 15 ng/ml, with no individual subject achieving Cmax greater than 30 ng/ml. Comparison of these data with other orally administered compounds suggests that the compound of Example 1 has a very low oral bioavailability. In addition, a high concentration of the drug was observed in stool samples, indicating a significant effect in the gastrointestinal tract. While the present invention has been described with reference to specific embodiments thereof, those skilled in the art will appreciate that various variations and equivalents are possible without departing from the true spirit and scope of the invention. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, method, method step or steps to the objectives, spirit and scope of the present invention. All such modifications are intended to be covered by the scope of the appended claims. In addition, all publications, patents and patent documents referred to above are incorporated herein by reference in their entirety as if they were incorporated individually by reference. CLAIM 1. Pharmaceutical composition for the treatment of a gastrointestinal inflammatory disease, comprising a compound of the formula or a pharmaceutically acceptable salt thereof; microcrystalline cellulose; lactose; magnesium stearate. 2. The composition of claim 1 wherein the compound is in free base form. 3. Composition according to claim 1 or 2, wherein the compound is present in an amount of 1 to 400 mg. 4. Composition according to claim 1 or 2, wherein the compound is present in an amount of 5 to 300 mg. 5. Composition according to claim 1 or 2, wherein the compound is present in an amount of 20 to 70 mg. 6. Composition according to claim 1 or 2, wherein the compound is present in an amount of 10 mg. 7. Composition according to claim 1 or 2, wherein the compound is present in an amount of 20 mg. 8. Composition according to claim 1 or 2, wherein the compound is present in an amount of 40 mg. 9. Composition according to claim 1 or 2, wherein the compound is present in an amount of 100 mg. 10. A method of treating a gastrointestinal inflammatory disease in a human, comprising administering to a human a pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, wherein the subject's C _max is less than 100 ng/mL after administration to a human of a single dose containing up to 1000 mg of the compound, or less than 30 ng/mL after administration to a human of a dose containing up to 300 mg compound per day for 14 days. - 14 040544 11. The method of claim 10, wherein the subject's C _max is less than 50 ng/mL following administration of a single dose containing up to 1000 mg of the compound to the human. 12. The method of claim 10 wherein the subject is administered a single dose containing 1000 mg of the compound. 13. The method of claim 10, wherein the subject is administered 300 mg of the compound per day for 14 days, wherein the C _max is less than 30 ng/ml. 14. The method of claim 10, wherein C _max is less than 15 ng/mL after administration to a human of a dose containing up to 300 mg of the compound per day for 14 days. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2