WO2007042885A2 - Therapeutic combination comprising methotrexate and a specified inhibitor of mek1 and mek2 - Google Patents

Abstract

The present invention relates to a method of treating rheumatoid arthritis with a combination, consisting essentially of N-[4-[[(2,4-diamino-6- pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid, or a pharmaceutically acceptable salt thereof, and N-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide, and related combinations and pharmaceutical compositions.

Description

THERAPEUTIC COMBINATION

FIELD OF THE INVENTION

The present invention generally relates to a therapeutic combination of compounds and the use of the combination to treat rheumatoid arthritis.

BACKGROUND

Sodium N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L- glutamate, which has the generic name methotrexate sodium, is indicated by the U.S. Food and Drug Administration for treating certain neoplastic diseases, psoriasis, and rheumatoid arthritis in human patients.

The RAF-MEK-ERK pathway mediates immunomodulation, inflammation, and proliferative and anti-apoptotic signaling from growth factors and oncogenic factors such as Ras and Raf mutant phenotypes. Mitogen-activated protein kinase (MAPK) and extracellular regulated kinase (ERK, e.g., ERK-j and ERK2) are enzymes involved in controlling certain cellular processes. Each MAPK consists of a series of three cytoplasmic kinases: a MAPK, a mitogen-activated protein kinase kinase (MAPKK), and a mitogen-activated protein kinase kinase kinase (MAPKKK). MAPK/ERK Kinase (MEK) enzymes are dual specificity kinases that activate ERK. MEK enzymes (e;g.; MEK-| and MEK2) are involved in, for example, rheumatoid arthritis, cancer, and restenosis.

BRIEF SUMMARY OF THE INVENTION

An aspect of the present invention is a method of treating rheumatoid arthritis in a patient, the method comprising administering to a patient in need of rheumatoid arthritis treatment a therapeutically effective amount of a combination, consisting essentially of a first amount of N-[4-[[(2,4-diamino-6- pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid, or a pharmaceutically acceptable salt thereof, or a mixture thereof, and a second amount of N-[(R)-2,3- dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide. Preferred is the method, wherein the patient is a human and the first amount is from 1 mg to 30 mg and the second amount is from 1 mg to 30 mg. More preferred is any one of the above aspects of the method, wherein the patient is a human and the N-[4- [[(2,4-diamino-6-pteridinyi)methyl]methylamino]benzoyl]-L-glutamic acid, or the pharmaceutically acceptable salt thereof, is sodium N-[4-[[(2,4-diamino-6- pteridinyl)methyl]methylamino]benzoyl]-L-glutamate and the first amount is from 1 mg to 20 mg or from 1 mg to 25 mg. Still more preferred is any one of the above aspects of the method, wherein the patient is a human and the second amount is from 1 mg to 15 mg; still more preferred is wherein the patient is a human and the second amount is from 1 mg to 8 mg. Still more preferred is any one of the above aspects of the method, wherein the patient is a human and the second amount is 2 mg, 4 mg, or 8 mg; still more preferred is wherein the patient is a human and the second amount is 2 mg, 4 mg, or 8 mg, which is administered once per day, or 1 mg, 2 mg, or 4 mg, which is administered twice per day. Still more preferred is any one of the above aspects of the method, wherein the N-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2- fluoro-4-iodo-phenylamino)-benzamide is polymorph Form IV of N-[(R)-2,3-dihydroxy- propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 to 4 graphically display changes in paw edema versus experiment day from a mouse collagen-induced arthritis experiment with Compound (I) alone, methotrexate alone, combinations of Compound (I) and methotrexate, and vehicle and normal controls, and recite AUC % lnh of paw edema.

Figures 5 to 8 graphically display changes in average total clinical score versus experiment day from the mouse collagen-induced arthritis experiment with Compound (I) alone,. methotrexate alone, combinations of Compound (I) and - - methotrexate, and vehicle and normal controls, and recite AUC % lnh of a decrease in average total clinical score.

Figures 9 to 12 graphically display changes in body weight for each treatment and control group of animals from the mouse collagen-induced arthritis experiment.

DETAILED DESCRIPTION The structure of N-[4-[[(2,4-diamino-6- pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid is:

This compound is also known by the names: methotrexate; (S)-2-{4-[(2,4-diamino- pteridin-6-ylmethyl)-methyl-amino]-benzoylamino}-pentanedioic acid; N-[p-[[(2,4- diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-(+)-glutamic acid; (+)- amethopterin; L-amethopterin; amethopterin; amethopterine; 4-amino-10-methylfolic acid; 4-amino-N¹⁰-methylfolic acid; 4-amino-N¹⁰-methylpteroylglutamic acid; antifolan; CL-14377; EMT-25299; emtexate; L-methotrexate; ledertrexate; MTX; metatrexan; methotrexat-ebewe; methylaminopterin; mexate; and NSC-740;R-9985. RHEUMATREX^® (American Cyanamid Company Corp., Wayne, New Jersey) is a brand name for methotrexate sodium tablets.

The structure of N-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo- phenylamino)-benzamide (Compound (I)) is:

(Compound (I))

This compound also has the name, (R)-N-(2,3-dihydroxy-propoxy)-3,4-difluoro-2-(2- fluoro-4-iodo-phenylamino)-benzamide, and the Chemical Abstracts Registry No. [391210-10-9]. Compound (I) may be amorphous or crystalline. Crystalline forms of Compound (I) include polymorph Forms I, II, and IV. Compound (I) is a highly specific, non-ATP-competitive inhibitor of the enzymes MEK1 and MEK2.

Definitions of some acronyms used herein are: AE - adverse event ^{. . . . .}

AR - accumulation ratio

AUC - area under the plasma drug concentration-time curve AUC₍o-₂₄₎ - area under the plasma drug concentration-time curve from 0 to 24 hours post dose AUC₍o-_ti_ast) - area under the plasma drug concentration-time curve from Time

0 to the last recorded observation

AUC % lnh - area under the curve-derived percent inhibition ATP - adenosine triphosphate BID - bis in die (i.e., twice per day) C₀ - drug concentration at Time 0

Cm_ax- maximum plasma drug concentration Css, avg - steady-state average plasma drug concentrations C_tr_oug_h - pre-dose plasma drug concentration CV% - coefficient of variation in percentile DLT - dose-limiting toxicity

ECG - electrocardiogram EDTA - ethylenediaminetetraacetic acid FDA - United States Food and Drug Administration HERG - human ether-a-go-go related gene

HPMC - hydroxypropylmethylcellulose

IP - intraperitoneal

IV - intravenous Ki^app - apparent dissociation constant of the enzyme-inhibitor complex

LC/MS - liquid chromatography-mass spectrometry

LC/MS/MS - liquid chromatography-mass spectrometry-mass spectrometry

LPS - lipopolysaccharide

MAD - maximum administered dose MAP - mitogen-activated protein

MTD - maximum tolerated dose

PD - pharmacodynamics

PK - pharmacokinetic

PO - peroral (i.e., oral) pTsOH - para-toluenesulfonic acid

QD - quaque die (i.e., once per day)

QTc - corrected QT-interval

T_max - time of occurrence of C_max

USP - United States Pharmacopeia . .v/v -..volume/Volume _ ..

Another aspect of the present invention is a combination, consisting essentially of N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid, or a pharmaceutically acceptable salt thereof, or a mixture thereof, and N-[(R)- 2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide. Preferred is the combination consisting essentially of N-[4-[[(2,4-diamino-6- pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid and N-[(R)-2,3-dihydroxy- propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide. Also preferred is the combination consisting essentially of sodium N-[4-[[(2,4-diamino-6- pteridinyl)methyl]methylamino]benzoyl]-L-glutamate and N-[(R)-2,3-dihydroxy- propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide. More preferred is any one of the combinations, wherein the N-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro- 2-(2-fluoro-4-iodo-phenylamino)-benzamide is polymorph Form IV of N-[(R)-2,3- dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide.

A "combination" includes admixed components, packages containing each component, and combinations prepared in vivo in a patient by administration of the combination's components to the patient. Preferred is a combination of admixed components. The phrase "consisting essentially of," when referring to a combination of the present invention, means the combination may contain, in addition to the methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, and the Compound (I), pharmacologically-inactive components, but the combination may not contain additional components that are alleged in the prior art to have pharmacological activity.

The phrase "or a mixture thereof" means the combination of the present invention optionally may consist essentially of Compound (I) and: (i) a mixture of methotrexate and one or more salts thereof, preferably no more than two salts thereof; or (ii) a mixture of two or more salts of methotrexate, preferably no more than two salts thereof. The invention combination includes heterogeneous and homogenous mixtures of its components. The combination includes mixtures of amorphous forms, crystalline forms, and an amorphous form and a crystalline form of Compound (I). The term "treating" means prophylactic treatment, i.e., delaying the onset of a disease, or palliative treatment, which palliative treatment alleviates, inhibits the progress of, prevents further progress of, or reverses progression of, in part or in whole, any one or more pathologies or symptoms of the disease being treated. Preferred is palliative treatment. The effect of clinical treatment of rheumatoid arthritis in human patients may be assessed by a physician of ordinary skill in-the art using a primary outcome measure for regulatory approval of anti-rheumatoid arthritis drugs. The American College of Rheumatology (ACR)/World Health Organization (WHO) responder index, ACR-20, standard is a 20% improvement in multiple measures, expressed as a composite score; see, for example, Felson, David T., et al., ACR Preliminary Definition of Improvement in Rheumatoid Arthritis, Arthritis &

Rheumatism, June, 1995;38(6):727-735. Other standards include ACR-50% and ACR-70%. The physician may diagnose rheumatoid arthritis in a human patient upon clinical evaluation of the patient's signs and symptoms using ACR-20 criteria or the like. Clinical signs and symptoms of rheumatoid arthritis include pain, swelling, stiffness, and loss of function in a joint such as knuckle, knee, and the like. For the present purposes, the ACR-20% standard is the preferred primary outcome measure. A veterinarian of ordinary skill in the art may diagnose rheumatoid arthritis in non- human patients. A human patient in need of prophylactic treatment of rheumatoid arthritis may be identified by the physician as someone having a family history of, a genetic marker for developing, or premature clinical signs and symptoms of rheumatoid arthritis. The veterinarian may identify non-human patients in need of prophylactic treatment of rheumatoid arthritis similarly. The term "administering," as applied to the combination of the present invention, includes simultaneous or substantially simultaneous administration of different pharmacologically-active components, and sequential administration, wherein a pharmacologically-active component is administered, and then, after a waiting period, another pharmacologically-active compound is administered. The waiting period may be readily determined by the physician or veterinarian. The pharmaceutically-active components of the combination may be administered in any order. For example, a first amount of one pharmaceutically-active component may be administered before, at the same time as, or after administration of a second amount of another pharmaceutically-active component.

Use of the terms "first" and "second" when referring to an amount, a pharmaceutical composition, etc. is done herein for convenience and the terms do not refer to an order or sequence of using the amounts, pharmaceutical compositions, etc. in a method of the present invention. A first and a second amount may be the same or different.

The term "patient" means a mammal and includes humans, companion animals such as cats, dogs, and the like, primates such as monkeys, chimpanzees, and the like, livestock animals such as horses, cows, pigs, sheep, and the like, and laboratory animals such as cats, dogs, rats, mice, guinea pigs, hamsters, rabbits, monkeys, pigs, and the like. A preferred patient is a human. In another aspect of the present invention, the patient is a non-human mammal. Preferred non-human mammals are cats, dogs, and horses. More preferred non-human mammals are cats and dogs.

A "patient in need of rheumatoid arthritis treatment" means a patient who is in need of prophylactic treatment of rheumatoid arthritis, i.e., in need of delaying the onset of rheumatoid arthritis, or in need of palliative treatment of rheumatoid arthritis, which palliative treatment alleviates, inhibits the progress of, prevents further progress of, or reverses progression of, in part or in whole, any one or more pathologies or symptoms of rheumatoid arthritis in the patient. A "therapeutically effective amount of a combination" is an amount of the combination that is effective for treating the disease being treated. A therapeutically effective amount for the treatment of rheumatoid arthritis includes an amount sufficient to effect, even temporarily, an improvement in one of a patient's clinical signs or symptoms or ACR-20 evaluation. This amount may comprise an amount of an individual component of the combination that is less than the amount required for monotherapeutic treatment of the disease with the individual component.

Methotrexate is capable of forming pharmaceutically acceptable salts, including acid addition and base addition salts. All pharmaceutically acceptable salt forms of methotrexate are included within the scope of the present invention. Scientific literature describes types of pharmaceutically acceptable salts and their general preparations; see, for example, Berge S.M. et al., "Pharmaceutical Salts," J. of Pharma. Sci., 1977;66:1. Pharmaceutically acceptable acid addition salts of methotrexate include salts derived from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, hydrofluoric, phosphorous, and the like, as well salts derived from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Such salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, malate, tartrate,- methanesulfonate, and the like. Also contemplated are methotrexate salts with amino acids such as arginate and the like and gluconate, galacturonate.

An acid addition salt of methotrexate may be prepared by contacting methotrexate with a sufficientamount of a desired acid to produce-the saltin a conventional manner. The acid addition salt may be converted back to methotrexate by contacting the acid addition salt with a base, and isolating methotrexate in a conventional manner.

Pharmaceutically acceptable base addition salts of methotrexate include sodium cation (Na⁺), potassium cation (K⁺), magnesium cation (Mg^⁺), calcium cation (Ca^⁺), and the like salts and salts with suitable amines such as N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine. Monosodium and disodium salts, and the like of methotrexate, and divalent metal cation salts of methotrexate that have 1 :1 or 1 :2 ratios of the divalent metal cation to methotrexate, are contemplated.

A base addition salt may be prepared in a conventional manner by contacting methotrexate with a sufficient amount of a desired base such as a metal cation hydroxide, e.g., a hydroxide of an alkali or alkaline earth metal cation, to give a metal cation salt, or the amine, especially an organic amine, to give an amine salt. A base addition salt of methotrexate may be converted back to methotrexate by contacting the base addition salt with an acid, and isolating methotrexate in a conventional manner. Preferred is methotrexate. More preferred is methotrexate sodium. Methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, and Compound (1) can exist in unsolvated forms as well as solvated forms, including hydrated forms and partially solvated forms (i.e., forms wherein the molar ratio of compound to solvent is not 1 :1 , i.e., is greater than or less than 1 ). Preferred solvates are those wherein the molar ratio of compound to solvent is greater than or equal to 1. Unsolvated and hydrated forms are preferred. The solvated forms and unsolvated forms are all encompassed within the scope of, and useful in, the present invention. The present invention includes the use of isotopically-labeled methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, and of isotopically- labeled Compound (1), which are identical to "unlabeled" methotrexate, a salt thereof, or Compound (I) recited above, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (i.e., different from the naturally abundant atomic mass or mass number). Examples of contemplated isotopes include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸0, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶CI, respectively. The isotopically-labeled compounds, for example those into which radioactive isotopes such as ³H and ¹⁴C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., ³H and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. An isotopically-labeled compound can generally be prepared by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent in a conventional method of preparing the compound.

Pharmaceutical compositions of the present invention include homogeneous and heterogeneous mixtures. The methods of the present invention may employ any dosage form of methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, or Compound (I), or both. Preferred is an oral dosage form of Compound (I); more preferred is a tablet or capsule oral dosage form. Preferred is an oral or injectable dosage form of methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof; more preferred is an oral dosage form; and still more preferred is a tablet or capsule oral dosage form.

Pharmaceutically acceptable excipients include pharmaceutically acceptable diluents, carriers, stabilizers, and other components such as capsule shells, for example gelatin capsule shells. Examples of pharmaceutically acceptable excipients include sugars such as lactose and sucrose; starches such as corn starch and potato starch; cellulose derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, methyl cellulose, and cellulose acetate phthalate; gelatin; talc; stearic acid; magnesium stearate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and oil of theobroma; propylene glycol, glycerin; sorbitol; polyethylene glycol; water; agar; alginic acid; isotonic saline, and phosphate buffer solutions; as well as other compatible substances normally used in pharmaceutical formulations. The compositions to be employed in the present invention may also contain other components such as coloring agents, flavoring agents, and/or preservatives. These other components, if present, are usually used in relatively small amounts. Suitable pharmaceutically acceptable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.

Another aspect of the present invention is a package, comprising (i) a first pharmaceutical composition containing methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, and a first pharmaceutically acceptable excipient, (ii) a second pharmaceutical composition containing Compound (I) and a second pharmaceutically acceptable excipient, and (iii) instructions for using-the first and second pharmaceutical compositions to treat rheumatoid arthritis in a patient in need of rheumatoid arthritis treatment, wherein the first pharmaceutical composition and the second pharmaceutical composition do not contain any other pharmacologically-active component. In the package, the first pharmaceutically acceptable excipient and the second pharmaceutically acceptable excipient may be the same or different, and the first pharmaceutical composition and the second pharmaceutical composition may be suitable for routes of administration that are the same or different.

Another aspect of the present invention is a pharmaceutical composition, comprising methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, Compound (I), and a pharmaceutically acceptable excipient, wherein the pharmaceutical composition does not contain any other pharmacologically-active component.

Another aspect of the present invention is a method of treating rheumatoid arthritis in a patient, the method comprising administering to a patient in need of rheumatoid arthritis treatment a therapeutically effective amount of a pharmaceutical composition, comprising a first amount of methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, a second amount of Compound (I), and a pharmaceutically acceptable excipient. A preferred aspect of the present invention is the method of treating rheumatoid arthritis, wherein the methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, and Compound (I) are as described herein in any one of the preferred aspects of the combination or method of the present invention.

Another aspect of the present invention is a method of treating rheumatoid arthritis in a patient, the method comprising separately administering to a patient in need of rheumatoid arthritis treatment a first pharmaceutical composition, comprising a first pharmaceutically acceptable excipient and a first therapeutically effective amount of methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, and a second pharmaceutical composition, comprising a second pharmaceutically acceptable excipient and a second therapeutically effective amount of Compound (I). In this method, the first pharmaceutically acceptable excipient and the second pharmaceutically acceptable excipient may be the same or different, and the first pharmaceutical composition and the second pharmaceutical composition may be administered to the patient at the same time or atdifferent times and by the same or different routes. A preferred aspect of the present invention is the method of treating rheumatoid arthritis, wherein the methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, and Compound (I) are as described herein in any one of tha preferred aspects of the combination or method of the - - present invention.

Another aspect of the present invention is a use of a combination consisting essentially of a first amount of N-[4-[[(2,4-diamino-6- pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid, or a pharmaceutically acceptable salt thereof, or a mixture thereof, and a second amount of N-[(R)-2,3- dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide in the manufacture of a medicament for treating rheumatoid arthritis in a patient. A preferred use is wherein the patient is a human.

A therapeutically effective amount of the methotrexate sodium in a method of treating rheumatoid arthritis in a human patient according to a method of the present invention is typically from 1 mg to 30 mg in a human patient of average weight. A preferred amount of methotrexate sodium for such treatment is 2.5 mg, 5.0 mg, 7.5 mg, 10 mg, 15 mg, 20 mg, or 30 mg; also preferred is from 1 mg to 25 mg or from 1 mg to 20 mg. A preferred therapeutically effective amount of methotrexate, or a non- sodium pharmaceutically acceptable salt thereof, or a mixture thereof, for treating human rheumatoid arthritis is an amount that will provide 0.0050 mol, 0.010 mol, 0.015 mol, 0.020 mol, 0.030 mol, 0.040 mol, or 0.060 mol of methotrexate, or the salt thereof, or the mixture thereof. Such therapeutically effective amounts are preferably for weekly administration. The weekly amounts may be divided and administered in portions throughout a seven day period. For example, a 17.5 mg per week dosage regimen may be divided into seven 2.5 mg dosages, which are administered daily for the week. Divided weekly amounts may further be subdivided for administration of sub-portions throughout any 24-hour period. For example, a weekly 15 mg dosage may be divided into two 7.5 mg portions for administration 3 days apart, and each 7.5 mg portion may be subdivided into three 2.5 mg portions for administration every 12 hours such that each 7.5 mg portion is administered within its own 24 hour period, which 24 hour periods are three days apart. Drug holidays from methotrexate are included within the present invention. In treating non-human patients, a starting weekly-administered therapeutically effective amount of the methotrexate sodium is from 0.1 mg/kg to 1 mg/kg. Thereafter, a veterinarian may optionally increase the amount of methotrexate sodium to find an optimal amount for the particular non- human patient being treated, which optimal amount may be up to 10 mg/kg/week. The molar equivalent starting therapeutically effective amount of methotrexate, or another pharmaceutically acceptable salt thereof, may optionally be used by the veterinarian in treating non-human patients.

A therapeutically effective amount of Compound (I) in a method of treating rheumatoid arthritis in a human patient according to a method of the present invention is-typically from 1 mg to 30 mg. A preferred therapeutically effective amount of

Compound (I) is from 1 mg to 20 mg, from 1 mg to 15 mg, or from 1 mg to 8 mg. A more preferred therapeutically effective amount of Compound (I) is 2 mg, 4 mg, or 8 mg. Such therapeutically effective amounts are preferably for daily administration. The daily amounts may be divided and administered in portions throughout a 24-hour period. Preferred is BID administration; more preferred is QD administration, although more or less frequent administration is within the scope of the present invention. Drug holidays from Compound (I) are included within the present invention. For example, the dosing regimen for Compound (I) may be BID dosing for 21 days, followed by a 7 day drug holiday, to give a 28-day regimen that can be repeated for as long as a physician determines to be appropriate. Alternatively, Compound (I) can be administered continuously without interruption until treatment is discontinued. Compound (I) can be administered to a fasted patient (for example, no food or beverage within 2 hours before and after administration) or, preferably, to a non- fasted (i.e., with food) patient. Administration of a therapeutically effective amount of Compound (I) once per day refers to QD dosing, while administration of a therapeutically effective amount of Compound (I) twice per day refers to BID dosing. In treating non-human patients, a starting therapeutically effective amount of Compound (I) is from 0.01 mg/kg to 0.1 mg/kg. Thereafter, a veterinarian may optionally increase the amount of Compound (I) to find an optimal amount for the particular non-human patient being treated, which optimal amount may be up to 1 mg/kg/day.

Drug holidays from both methotrexate and Compound (I) are included within the scope of the present invention. For example, the dosing regimen may be BID dosing of Compound (I) and twice weekly dosing of methotrexate sodium for 21 days, followed by a 7 day drug holiday from both Compound (I) and methotrexate sodium, to give a 28-day regimen that can be repeated for as long as a physician determines to be appropriate. In determining what constitutes a therapeutically effective amount in a method of the present invention, a number of factors will generally be considered by a physician or veterinarian in view of his or her experience. These factors include, for example, regulatory guidelines, including FDA guidelines, the results of published clinical studies, the particular patient being treated, the patient's age, sex, weight and general health condition, as well as the type and extent of the disease being treated, the use of other medications, if any,, by the patient, the particular formulation being employed, and the route of administration. Accordingly, the administered dose may fall within the ranges recited herein, or may be either below or above those ranges. Determination of a proper dose for a particular situation is routine and within - the ordinary skill of the physician or- veterinarian. Generally, treatment may-be initiated using smaller dosages of a pharmacologically-active component that are less than optimal for a particular patient. Thereafter, the dosage can be increased by small increments until an acceptable effect under the circumstance is reached.

Methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, and Compound (I) may be formulated in bulk form or dosage unit form. Preferably the pharmaceutical compositions of the present invention are in unit dosage form. In unit dosage form, a pharmaceutical preparation is subdivided into unit doses containing an appropriate quantity of the pharmacologically-active component(s). The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparations such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. A unit dosage formulation may contain less than a therapeutically effective amount of a pharmacologically-active component for the disease or disorder being treated, and thus require administration of more than one unit dosage to the patient to treat a disease. Some examples of dosage unit forms are tablets, capsules, pills, powders, aqueous and nonaqueous oral solutions and suspensions, and parenteral solutions packaged in containers containing either one or some larger number of dosage units and capable of being subdivided into individual doses.

In the pharmaceutical composition of the present invention, the total percentage of the pharmacologically-active components can be varied within wide limits, but for practical purposes it is preferably at least 5% by weight in a solid composition and at least 2% by weight in a primary liquid composition. The most satisfactory compositions are those in which a much higher proportion of the pharmacologically-active components is present, for example, up to about 95% by weight. Different routes of administration may require different amounts, dosages, or percentages.

A pharmaceutical composition of the present invention that is suitable for oral administration includes a tablet, capsule, powder, pill, lozenge, aqueous suspension or solution, melt formulation, and the like; preferred is a tablet or capsule. The pharmaceutical composition includes the formulation of the pharmacologically-active component(s) with encapsulating material such as a capsule shell, providing a capsule in which the pharmacologically-active component(s), with or without other carriers, is surrounded by the capsule shell. Solid form preparations are preferred.

A pharmaceutical composition that is suitable for administration by injection includes an aqueous solution, a lyophilized powder capable of being dissolved in sterile water or saline, and the like. In veterinary use_; a preferred therapeutic composition for dogs comprises an ingestible liquid peroral dosage form selected from the group consisting of a solution, suspension, emulsion, inverse emulsion, elixir, extract, tincture and concentrate, optionally to be added to the food or drinking water of the dog being treated. A concentrate liquid form may be formulated for dissolution in a given amount of water, from which solution an aliquot amount may be withdrawn for administration directly to the dog or addition to the dog's food or drinking water.

Other preferred pharmaceutical compositions provide delayed-, sustained- and/or controlled-release of a pharmacologically-active ingredient. Controlled-release forms are particularly useful when formulating an invention combination having pharmacologically-active components with different half-lives. Controlled-release forms can be prepared that have different release times for each of the pharmacologically-active components, which forms allow relatively uniform dosing.

In the case of non-human patients, pharmaceutical compositions also include a medicated feed dosage form in which the pharmacologically-active components are present together in a feed composition.

Methods for preparing the pharmaceutical compositions are known, or will be apparent and routinely determined in light of this disclosure, to those skilled in the art. For example, methods for preparing pharmaceutical compositions of the present invention may be adapted from those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 19^th edition (1995) and in the six-volume series, Handbook of Pharmaceutical Manufacturing Formulations, CRC Press, Boca Raton, Florida (2004).

Solid form preparations such as powders, tablets, pills, capsules, cachets, suppositories, lozenges, and dispersible granules can be formulated with a solid carrier. A solid carrier can be one or more substances that may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In tablets, the pharmacologically-active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. In powders, the carrier is a finely divided solid that is in a mixture with the finely divided pharmacologically-active component. Aqueous solutions suitable for oral use can be prepared by dissolving the pharmacologically-active component in water and adding suitable colorants, flavors, stabilizing, and thickening agents as desired.

Aqueous suspensions suitable for oral use can be made by dispersing the finely divided pharmacologically-active component in water with viscous material, such as natural-or-synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

Liquid form preparations include solutions, suspensions, and emulsions, for example, water, saline, or water propylene glycol solutions. For parenteral injection, liquid preparations can be formulated using solvents such as saline and aqueous polyethylene glycol solution. Powders suitable for intravenous administration or administration by injection may be lyophilized. Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations for injection or oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the pharmacologically-active component(s), colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

Methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, and Compound (1) can be administered by different routes such as oral ingestion of a tablet, capsule, powder, solution and the like or by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally. Continuous administration such as by intra-arterial infusion from a surgically implanted pump is also contemplated. Preferred routes of administration of methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, are oral or by injection; more preferred is oral. A preferred route of administration of Compound (I) is oral. However, another route of administration of the compounds may be preferred depending upon the particular condition being treated. Topical administration by transdermal patch may be preferred where, for example, it is desirable to effect sustained systemic dosing.

There is further provided in accordance with the present invention substantially simultaneous administration or sequential administration of methotrexate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, and Compound (I). The sequential administration may be performed in any order and in accordance with different but regular and continuous dosing schedules, whereby therapeutically effective plasma levels of these pharmacologically-active components are maintained in the patient being treated, even though the individual components are not administered to the patient simultaneously. Methotrexate is commercially available from Sigma-Aldrich Company, Sigma

Catalog item no. M 9929. (USP grade, anhydrous) and item no. A 6770 ((+)- amethopterin trihydrate).

Preparation of Compound (I) is described in U.S. Patent Application Publication No. 2004/0054172 and its corresponding U.S. Patent Application No. -10/333,399, now allowed. Preparation of Compound (I) is also described-in U:S. Provisional Patent Application No. 60/690,620, filed June 14, 2005.

Preparations of polymorph Forms I, II, and IV of Compound (I) are described below and in U.S. Patent Application No. 10/969,681 and its corresponding PCT International Patent Application Publication No. WO 2005/040098.

Preparation 1 : Λ/-[(R)-2,3-Dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo- phenylamino)-benzamide (polymorph Form I)

Step A: To a solution of 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzoic acid (39.3 g, 100.0 mmol) in dry tetrahydrof uran (500 mL, 0.2 M), under nitrogen atmosphere, was added ('f?_/)-O-(2,2-dimethyl-[1 ,3]dioxolan-4-ylmethyl)-hydroxylamine (14.7 g, 100.0 mmol), followed by Λ/-methylmorpholine (27.5 mL, 0.25 mole). The orange-colored solution was cooled with an ice-water bath. Diphenylphosphinic chloride (22.9 mL, 0.12 mol) was added dropwise. Some solid formed. The mixture was warmed to ambient temperature and stirred for 18 hours. Water was added to quench the reaction, and the tetrahydrofuran was rotary evaporated in vacuo. The remaining oil was dissolved in ethyl acetate (500 mL) and washed with a mixture of saturated brine and saturated sodium bicarbonate (1 :1) two times. The ethyl acetate was removed and the crude oily solid was purified by flash chromatography (silica gel, hexane-acetone / 2 : 1) to give N-((R)-2,2-dimethyl-[1 ,3]dioxolan-4-ylmethoxy)- 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide as an off-white solid after drying in a vacuum oven at 40 ⁰C for 20 hours: 41.7 g (79.8%), m.p. 124-125 ⁰C. The impure fractions were combined and purified by a second column chromatography using the same conditions to give a second batch of 6.4 g (12.3%), mp 124-125 ⁰C, total yield 48.1 g (92.1%). ¹H NMR (d⁶-DMSO): δ 11.9 (s, br, 1 H), 8.7 (s, br, 1 H), 7.6 (d, 1 H, J=10.99 Hz), 7.4 (m, 2H), 7.2 (m, 1 H), 6.7 (m, 1 H)₁ 4.2 (m, 1 H), 4.0 (t, 1 H, J1=8.3 Hz, J2=6.8 Hz), 3.8 (m, 2H), 3.7 (m, 1 H), 1.3 (s, 3H), 1.2 (s, 3H); ¹⁹F NMR (d⁶-DMSO): δ -128.0, -133.1 , -144.3; MS: 523 (M⁺+1). Step B: N-((R)-2,2-Dimethyl-[1 ,3]dioxolan-4-ylmethoxy)-3,4-difluoro-2-(2- fluoro-4-iodo-phenylamino)-benzamide (22.3 g, 42.7 mmol) was suspended in methanol (223 mL, 10 mL/g), and a solution of pTsOH-H₂O (4.1 g, 21.35 mmol) in water (22.3 mL) was added. The mixture was stirred at ambient temperature for 18 hours, during which all solids dissolved to give a colorless, clear solution. The solution was concentrated and extracted with ethyl acetate (2 x 300 mL). The organic solution was washed with sodium bicarbonate, dried over MgSO₄. After filtration, the filtrate was concentrated, and co-evaporated with heptane to give a foaming solid. To this solid was added hexane-CH₂CI₂ (1 : 1 , 100 mL) and the mixture was stirred for 30 min. A white solid formed, which was filtered, washed with hexane. The solid was recrystallized from hexane-AcOEt to give N-((R)-2,3-dihydroxy-propoxy)-3,4-difluoro- 2-(2-fluoro-4-iodo-phenylamino)-benzamide as white crystals, 13.57 g (65.9%), after drying at 60 °C vacuum oven for 3 days. A second crop of 5.05 g was obtained from the mother liquor, after recrystallization from the same solvent system. The total yield was 18.62 g (90.4%): m.p. 89-90 °C (polymorph Form I). The combined crystals were ground with a set of mortar and pestle to fine powder, and dried at 60 ⁰C in a vacuum oven for 3 days: m.p. 117-118 ⁰C (Form I); [α]= -2.05° (c=1.12, methanol); Analysis: Calcd. For: C₁₆H₁₄F₃I₁N₂O₄: C, 39.85; H, 2.93; N, 5.81 ; F, 11.82, 1, 26.32. Found: C, 39.95; H, 2.76; N, 5.72; F, 11.71 ; I, 26.53. ¹NMR (400MHz, DMSO-d₆) δ 11.87 (s, 1 H), 8.69 (s, 1 H), 7.54 (dd, 1 H₁ J=10.9, 1.5), 7.32-7.38 (m, 2H), 7.17 (dd, 1 H, J=16.8, 9.0), 6.61 -6.66 (cm, 1 H), 4.82 (bs, 1 H), 4.58 (bs, 1 H), 3.84-3.85 (m, 1 H), 3.71-3.64 (cm, 2H), 3.33 (2H, partially hidden by HDO).

Preparation 2: Λ/-[(R)-2,3-Dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo- phenylamino)-benzamide (polymorph Form II) Step A: To a solution of 3,4-difluoro-2-(2-f!uoro-4-iodo-phenylamino)-benzoic acid (2.25 g, 5.10 mmol) in dry tetrahydrofuran under nitrogen atmosphere, at -15⁰C was added diphenylphosphinic chloride (1.26 mL, 6.63 mole) dropwise. After stirring 20 min., N-methyl morpholine (0.70 mL, 6.375 mmol) was added and the reaction stirred an additional 20 minutes. (fl>O-(2,2-dimethyl-[1 ,3]dioxolan-4-ylmethyl)- hydroxylamine (0.748 g, 5.1 mmol) was added and the reaction stirred for 1 hour, at which point Λ/-methylmorpholine (0.7 ml_, 6.37 mmol) was added. The mixture was warmed to ambient temperature and stirred for 12 hours. The reaction was concentrated in vacuo and then diluted with EtOAc. The organic layer was washed with saturated NaHCO₃ (2 times), brine (1 time), dried over Na₂SO₄, filtered and concentrated. The crude product was purified on silica gel using 4:1 hexane/EtOAc as eluant to provide 1.82g (68%) of N-((R)-2,2-Dimethyl-[1 ,3]dioxolan-4-ylmethoxy)- 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide as a brownish red solid. Step B: N-((R)-2,2-Dimethyl-[1 ,3]dioxolan-4-ylmethoxy)-3,4-difluoro-2-(2- fluoro-4-iodo-phenylamino)-benzamide (0.210 g, 0.40 mmol) was suspended in 10:1 methanol/H₂O and pTsOHΗ₂O (0.008 g, 0.04 mmol) was added. The mixture was stirred at ambient temperature for 18 hours, during which all solids dissolved to give a colorless, clear solution. The solution was diluted with EtOAc. The organic solution was washed with sodium bicarbonate (2 times), brine (1 time) and dried over Na₂SO₄. After filtration, the filtrate wasxoncentrated, and recrystallized from EtOAc and heptane. This solid was washed with heptane-CH₂CI₂ (1 :1 and dried in vacuo at 6OC to give N-((R)-2,3-Dihydroxy-propoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide (polymorph Form II) as a white solid, (0.136g, 70%). Product shrinks at 90.8C, melts at 115-1 -17⁰C. Analysis shows C 40,92, H 3.16, N 5.41 , F 11.30, I 23.92 (6.75% EtOAc, 0.96 % heptane).

Preparation s: Λ/-[(R)-2,3-Dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo- phenylamino)-benzamide (polymorph Form IV ) To a flask containing 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzoic acid (2.6 kg, 6.6 mol) and N, N'-carbonyldiimidazole (1.1 kg, 6.8 mol) under nitrogen atmosphere, was added 12 L of dry acetonitrile. After stirring at 22° +5⁰C for about 90 minutes, a solution of (fiJ-O-(2,2-dimethyl-[1 ,3]dioxolan-4-yImethyl)-hydroxylamine in toluene was added ( 8.5 L total volume, about 8 moles of amine). The solution was stirred for at least 6 hours at 22° ±5 C. Aqueous hydrochloric acid (9 L, 1.5 molar) was added, and after stirring for about 5 minutes, the layers were separated. Aqueous hydrochloric acid (9 L, 1.5 molar) was added to the remaining top layer, and after stirring for about 20 hours, the layers were separated. The remaining top layer was concentrated by vacuum distillation, and then diluted with 15 L of toluene and 2 L of ethanol. The mixture was warmed to 35 - 45°C and diluted with 20 L of warm water, then cooled to 0 - 5°C. The product was collected by filtration and washed with 2 L of toluene. The product was recrystallized by dissolving in 12 L of toluene and 2 L of ethanol (50° ±5 C), adding 10 L of water and cooling to 0 - 5⁰C. After collecting the product by filtration and washing with toluene, the product was dried in a vacuum oven resulting in 2.6 kg of Λ/-[(R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2- fluoro-4-iodo-phenylamino)-benzamide.

2.4 kg of this compound, which was a mixture of different crystalline forms, was stirred in a mixture of 10 L of water and 1 L of ethanol at 35+ 5⁰C for 20-30 hours, then cooled to 25+ 5°C. The product was collected by filtration and washed with 1 L of water, then dried in a vacuum oven at 65°C. This resulted in 2.3 kg of material which was greater than 90% polymorph Form IV. Note : DSC analysis shows an onset of melting at 110°C with only a small amount of the peak with an onset of melting at 117⁰C.

An X-ray diffraction pattern of polymorph Form IV of Compound (I) was measured on a Rigaku Ultima + diffractometer with CuK₀ radiation. Rigaku Ultima + Diffractometer with an IBM-compatible interface equipped with 6 position autosampler, software = RigMeas v2.0 (Rigaku, December 1995) and JADE 3.1 (Materials Data, Inc.). CuK_α radiation (40 mA, 40 kV, λ = 1.5419 A). Slits I and Il at 0.5°, slit III at 0.3°. Continuous Θ/2Θ coupled scan: 3.00° to 45.00° in 2Θ, scan rate of 0.2°/min: 15.0 sec/0.05^o step. Sample tapped out of vial and pressed onto zero- background silicon in aluminum holder. Sample width 5 mm. Samples were stored and run at room temperature. Samples were spun at 40 rpm around vertical axis during data collection.

Table 1 lists the X-ray powder diffraction pattern for polymorph Form IV of Compound (I), expressed in terms of the 2-theta ("2Θ"), d-spacings or d(A), and relative intensities by peak area with a relative intensity of >10% measured on a Rigaku Ultima + diffractometer with CuK_α radiation. Table 1

Experiment 1 : Inhibition of activated MEK1 and MEK2, and Enzyme Specificity

Compound (I) was shown to be a selective inhibitor of MEK1 and MEK2 in vitro. The Ki^app of Compound (I) for an activated form of MEK1 , MEK1-S218D/S222D, was 1.1+0.2 nM, while the Ki^app of Compound (I) for an activated form of MEK2, MEK2-S222D/S226D was 0.79±0.2 nM. Compound (I) was also tested in a "cascade" assay in which activated B-Raf, unactivated MEK1 , and ERK1 were present. In this cascade assay, activated B-Raf phosphorylates and activates MEK1 , and activated MEK1 in turn phosphorylates ERK1. The readout is phosphorylation of ERK1. Inhibitors that bind to either the unactivated or the activated form of MEK1 can inhibit this assay. The Ki^app for Compound (I) was 0.90+0.09 nM in the cascade assay. The kinase specificity of Compound (I) was evaluated against a panel of 27 kinase enzymes. This panel, which was comprised of tyrosine kinases as well as a multitude of serine/threonine kinases, was refractory to inhibition by a 10 //M concentration of Compound (I). Therefore, Compound (I) appears to be a highly specific inhibitor of MEK1 and MEK2 versus other kinases. Example 2: Genetic Toxicology and Safety Pharmacology Studies of Compound (I) Compound (I) has been examined in various genetic toxicology and safety pharmacology (cardiovascular, central nervous system (CNS), and pulmonary) studies. General toxicology studies have been conducted in rats, dogs, and monkeys. Most studjes used QD oral dosing by gavage, although a limited number of studies used IV administration. Definitive toxicology studies were conducted in rats and dogs, in which Compound (I) was administered orally by gavage QD for 1 -month, followed by a 1 -month reversal period.

Time- and dose-dependent dysregulation in calcium and phosphorus homeostasis with subsequent mineralization of vasculature and various soft tissues (i.e., systemic mineralization) was observed in rat toxicology studies. This toxicity was not observed in dogs or monkeys despite administration of lethal doses, exposures in excess of those in rats that produced the toxicity, and >70% suppression in tissue pERK levels (the substrate of MEK). The dysregulation of serum calcium and phosphorus metabolism observed in rats treated with Compound (I) occurs prior to the onset of systemiαmineralization and appears to correlate with significant elevations in plasma concentration of 1 ,25-dihydroxyvitamin D. Based upon the toxicology data, rats appear to be uniquely sensitive to this toxicity. Gastrointestinal tract toxicity is dose-limiting in dogs and monkeys and is anticipated to be the dose-limiting toxicity of Compound (I) in the human patients. To ensure patient safety in a Phase I clinical trial with Compound (I), protocol incorporated a plan for intensive monitoring focused on detecting early abnormalities in calcium and phosphorus metabolism, and no significant abnormalities in serum phosphorus and calcium regulation were seen. The primary Compound (I) toxicities in preclinical studies were to the gastrointestinal tract (rat, dog, and monkey), skin (rat, dog, and monkey), liver (rat), bone (rat), and systemic mineralization (rat only). Injury to the mucosa of the gastrointestinal tract was the dose-limiting toxicity in non-rodents. Results of the safety pharmacology studies were generally unremarkable. Compound (I) indicated a potential for clastogenicity in the in vivo micronucleus study in rats.

The preclinical studies of Compound (I) (Purkinje fiber and HERG in vitro assays, and ECG assessment of dogs and monkeys receiving Compound (I)) did not indicate a risk of QT/QTc prolongation or arrhythmia. A systematic ECG evaluation incorporated into the Phase I study demonstrated no evidence of significant QT/QTc effects. All patients enrolled in the trial were required to have ECGs performed. A small number of patients enrolled in trials of MEK inhibitors have developed noticeable decreases in ejection fraction of unknown relation to the study drug. Monitoring of ejection fraction with multiple gate acquisition scans was conducted in this trial.

During the Phase I portion of this study, 40 patients were treated with Compound (I). Dose escalation was conducted from 1 mg QD through 30 mg BID days 1-21 in 28 day cycles. Continuous dosing for 56 days was subsequently tested. Drug related toxicities that were seen included acneiform rash, mucositis, blurry vision, confusion, edema, diarrhea, and fatigue. Grade 1-3 acneiform skin rash occurred in a dose related pattern, and was a DLT at 30 mg BID. The rash was managed with minocycline hydrochloride, 50-100 mg per day. Two episodes of syncope occurred, possibly drug related, and were considered DLT at 30 mg BID. Grade 1-2 transient and reversible visual effects including blurred vision and halos were seen without a discernable pattern relevant to dose level or patient characteristics. These typically were less severe later in a drug cycle, during intercycle drug holidays, and during subsequent cycles. One patient developed unilateral visual decrements, and was found to have ipsilateral optic disc edema and intraretina] hemorrhages in one eye, whereas opthamologic exam of the contralateral eye revealed no abnormalities. Grade 1-2 dependent and facial edema was seen intermittently with all dose levels. Grade 1-2 diarrhea was seen; the diarrhea responded well to loperamide hydrochloride treatment. Grade 1-2 confusion and ataxia were seen in two patients at-doses of 30 mg BID, but given the patients'- concomitant illnesses, it is unclear if the confusion and ataxia were drug related. One case of worsening of a pre-existing congestive heart failure at 1 mg BID and one case of new onset minimally symptomatic congestive heart failure at 20 mg BID were seen, and were of unclear relation to Compound (I). The maximum administered dose (MAD) was 30 mg BID, and the MTD was determined to be 20 mg BID, secondary to 1/6 cases of DLT Grade 3 acneiform skin rash and 2/6 cases of DLT syncope (Grade 3 by CTC AE 3.0) at 30 mg BID on 21 day dosing in 28 day cycles. A cohort of 6 additional patients was entered onto a cohort of 20 mg BID continuous dosing in two 28 day cycles (56 days total). In this cohort, one patient developed a DLT of grade 3 acneiform rash and four patients demonstrated upwards trends in liver function tests during the second cycle. Thus, 20 mg BID was considered above the MTD in a continuous dosing regimen and subsequent patients were treated at 15 mg BID continuous dosing.

Experiment 3: Phase I Clinical Study of Compound (I) in Human Patients with Advanced Cancer

During a Phase I/Phase Il study, 42 cancer patients have been treated with Compound (I). Oral dose escalation was conducted from 1 mg QD through 30 mg BID days 1-21 of 28-day cycles in patients with breast, colon, nonsmall cell lung cancer (NSCLC) or melanoma. Continuous dosing in 28-day cycles was subsequently tested.

Tumor tissue was assessed by immunohistochemistry (IHC) for pERK at baseline (BL) and Day 15 of Cycle 1 as described in Experiment 4 and for the amount of Ki67 as described below in Experiment 5. Pharmacokinetic (PK) samples were obtained on Days 1 and 21 in all patients and also on Day 1 of Cycle 2 in patients participating in a 2-way crossover food effect component. Due to elevated serum phosphorus with corresponding soft tissue mineralization observed in rats, serum calcium, phosphorous and (Ca x P) product were monitored closely.

Drug related toxicities seen included acneiform rash, mucositis, blurry vision, confusion, edema, diarrhea, and fatigue. Grade 1-2 acneiform skin rash occurred in a significant portion of patients, and Grade 3 rashes were seen at doses above 15 mg BID. This rash has been managed with minocycline, given 50 to 100 mg per day. Two episodes of syncope occurred, possibly drug related, and considered to be DLT at 30 mg BID. Grade 1-2 transient and reversible- visual effects including blurred vision and halos have been reported. These typically were less severe later in a drug cycle, during intercycle drug holidays, and during subsequent cycles. Grade 1-2 dependent and facial edema was seen intermittently with all dose levels. Grade 1 -2 diarrhea was seen and responded well to loperamide hydrochloride. - - -

The MAD was 30 mg BID, and the MTD when dosing 21 days of a 28-day cycle was determined to be 20 mg BID, secondary to 1/6 cases of DLT Grade 3 acneiform skin rash and 2/6 cases of DLT syncope (Grade 3 by CTC AE 3.0) at 30 mg BID on 21 day dosing in 28-day cycles. Cohorts of 6 patients each were subsequently treated at 20 mg bid and 15 mg bid with dosing continuously over 28- day cycles. A DLT was seen in one patient in each cohort (grade 3 acneiform skin rash).

Of 42 patients treated, 2 partial responses (melanoma) and 10 patients with stable disease have been reported. There was no notable effect on serum (Ca x P) product.

Experiment 4: lmmunohistochemical Analysis of Phosphorylated ERK Protein (p- ERK) in Paired Serial Biopsy Specimens from Patients Treated with Compound (I) The ERK protein (also known as MAP kinase, or MAPK) is a substrate for MEK kinase activity, and thus reduction in the phosphorylation of the ERK protein is indicative of a reduction in MEK activity. The phosphorylation status of ERK can be assessed through antibody-based detection methods, utilizing phosphorylation site- specific antibodies. In a clinical study of cancer patients treated with Compound (I), biopsy specimens were collected immediately before the onset of treatment and on the 14^th day of daily dosing with Compound (I). The biopsies were collected from tumor lesions amenable to either excisional or core needle biopsy procedures; the tissue specimens were immediately placed into 10% neutral-buffered formalin solution for fixation (nominally 6-8 hours, but no more than 24 hours in formalin). The fixed tissues were then transferred to 70% ethanol solution and submitted to the histopathology assay lab for immunohistochemistry (IHC) analysis of both p-ERK and the proliferation marker Ki67 (Experiment 5). The biopsy tissues were paraffin- embedded following standard tissue processing. The antibody used for detection of p-ERK was the mouse monoclonal anti-MAP kinase (activated) antibody, manufactured by Sigma-Aldrich Co. (catalog number: M8159). This antibody is reacts specifically with the di-phosphorylated form of MAPK (both the ERK-1 and ERK-2 forms). Tissue sections prepared from the paraffin blocks were pretreated with Heat-induced Epitope Retrieval conditions (3 minutes at 120⁰C) prior to the detection procedure. A DAKO Envision plus kit was used for visualization of the antibody bound to sections, with DAB Chromogen as the chromogen.- A Biogenex Autostainer system was used in the staining procedure (with hematoxylin counterstaining). The extent of staining in individual sections was assessed by pathologist review, with scoring based on the relative staining color intensities in tumorcells in each section (prior to biomarker evaluation, a hematoxylin-eosin - stained slide from each biopsy tissue was used for evaluation of whether and to what extent tumor cells were present in a given biopsy specimen; tissues in which little or no tumor cell content was observed were not included in the analysis). Staining intensity was grouped in categories from 0 to 3+, with 0 being little or no staining, and 3 being the most intense staining. The final score for each section was given in the form of an H-score, which was calculated as follows: 3 X (% of cells with 3+ staining) + 2 X (% of cells with 2+ staining) + 1 X (% of cells with 1+ staining). For each biopsy specimen, 2 sections were analyzed and the average of these was taken for the p- ERK H-score. In addition to H-scores, the total % of positive cells was also recorded. Comparison of pre-treatment biopsies to the post-treatment biopsy from the same patient was calculated based on H-scores using the following formula: the negative of ((pre-post)/pre) X 100; to give the percentage difference in p-ERK associated with treatment with Compound (I).

Experiment 5: lmmunohistochemical Analysis of Cell Proliferation Marker Ki67 in Paired Serial Biopsy Specimens from Human Cancer Patients Treated with Compound (I) The same biopsy specimens as those analyzed for p-ERK staining described in Experiment 4 were also assessed for the amount of Ki67 detectable by immunohistochemistry. The Ki67 marker is considered an indicator of cell proliferation and thus reduction in Ki67 staining levels may be correlated with anti- tumor activity of anticancer agents. Tissue sections from the paraffin-embedded biopsy tissues were pretreated with Heat-induced Epitope Retrieval (in this case, microwaving for 3 minutes) prior to the Ki67 detection procedure. The antibody used was the mouse monoclonal Ki67/MM1 manufactured by Novocastra Laboratories, Ltd. (catalog number: NCL-Ki67-MM1). A Biogenex Supersensitive DAB/HRP detection kit was used for visualization of the antibody bound to sections, with DAB Chromogen as the chromogen. A Biogenex Autostainer system was used in the staining procedure (with hematoxylin counterstaining). The extent of staining in individual sections was assessed by pathologist review, with scoring based on the relative staining color intensities in tumor cells in each section. Scoring of staining intensity was performed in same manner as for p-ERK assay, with H-scores generated for each section. For Ki67, only one section was assayed for each biopsy specimen. The percentage difference in Ki67 scores associated with Compound (I) was calculated in the same manner as for p-ERK.

Experiment 6: Pharmacokinetics (PK), Pharmacodynamics (PD) and Product

Metabolism of Compound (I) Following Multiple Oral Doses to Advanced Cancer Patients

A first-in-human Phase I/Phase Il trial employed an open-label, dose- escalating design where patients with various advanced solid tumors were treated orally with Compound (I) (QD or BID) for 21 or 28 days in 28-day cycles.

Pharmacokinetic data are presented for the first 38 subjects. Dose escalation was conducted from 1 mg QD, and 1 mg BID through 30 mg BID. Continuous dosing at 20 mg BID for 56 days was subsequently tested.

Serial blood samples were collected on Days 1 and 21 of Cycle 1 , and Day 1 of Cycle 2 for pharmacokinetic evaluation of the parent Compound (I), its major metabolite Compound (II)

and the S-enantiomer of Compound (I), which S-enantiomer is Λ/-[(S)-2,3-dihydroxy- propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide (Compound (III)) (Compound (III))

The effect of food on the PK of Compound (I) was evaluated in 16 patients (Day 1 of Cycles 1 & 2).

A liquid chromatographic method with tandem mass spectrometry detection was used to quantitate Compound (I), Compound (III), and Compound (II) in human plasma with EDTA as an anticoagulant. Compound (I) and its stereoisomer Compound (III) along with the metabolite Compound (II) were isolated from EDTA- treated human plasma by liquid/liquid extraction. Plasma samples were stored at - 2O⁰C until analysis. Analysis is by LC/MS/MS in the negative ion mode using multiple reaction monitoring. The range of quantitation for Compound (I), Compound (III), and Compound (II) was 0.100 to 100 ng/mL This assay required a 0.200 ml_ aliquot of plasma. Samples were extracted by using the following procedure. Sample Extraction:

Added 0.650 mL 95:5 methyl-t-butyl ether/ethyl alcohol, v/v. Vortexed using a multi-tube vortex machine for 1 minute. Repeated this step three more times.

After vortexing step, centrifuged at 4°C for 20 minutes at 4000 rpm. After centrifugation, transferred approximately 0.550 mL to a 1-mL 96-well polypropylene plate. Evaporated to dryness under nitrogen at 3O⁰C to 35°C.

Reconstituted sample with 0.075 mL 70:30 methanol/water containing 0.1% acetic acid.

Vortexed using the multi-tube vortex machine for 1 minute. Centrifuged for 5 minutes at 4000 rpm. Injected 1 to 10 μL of sample extract into liquid chromatography instrument.

Chromatography Conditions:

Analytical Column: CHIRALPAK AD-RH, 0.46 x 15 cm, 5 ^

Guard Column: MetaGuard 2.0 mm Polaris C18-A, 5μ

Mobile Phase: Mobile Phase A: 0.1% acetic acid in water, v/v

Mobile Phase B: 90:10 acetonitrile/isopropanol, v/v 17:83 Mobile Phase A/Mobile Phase B Needle Flush Solvent: 300:300:400:1

Acetonitrile/isopropanol/water/acetic acid, v/v/v/v

Injector Loop: 50 juL

Pump Program: lsocratic

Flow Rate: 0.750 mL/min

Column Temperature: 35⁰C

Injection Volume: 1-10 //L

Approximate Retention Times: Compound (I) 2.6 to 3.2 minutes

Compound (III) 3.2 to 3.8 minutes

Compound (II) 2.7 to 3.3 minutes

Mass Spectrometry:

Mass Spectrometer: Sciex API 4000

Ionization Mode: Turbo ion spray/negative ion mode

Acquisition Mode: MRM

Pause Time: 5 ms

Masses of Interest: .

Compound Q1 m/z Q3 m/z Dwell³ (ms) CE^a (V) C (V) CXP^a (V) (I) 481.0 (±0.5) 389.0 (±0.5) 125 -25 -55 -15 (III) 481.0 (±0.5) 389.0 (±0.5) 125 -25 -55 ^"-15^" (II) 392.0 (±0.5) 328.0 (±0.5) 125 -21 -42 -15 instrument dependent, may be adjusted to optimize performance.

Run Time: 6.0 or 12.0 minutes, depending on column condition Quantitation:

PE Sciex Analyst software (Version 1.2) was used to measure peak areas. Watson LIMS (Version 6.4.0.04, CPL #63) was used for data reduction. Samples are quantified by applying a 1 /(concentration)² weighted quadratic regression analysis.

Pharmacokinetic parameters were determined by the non-compartmental method using WINNONLIN Professional Edition (Version 4.1). The liner trapezoidal method was used to calculate area under the concentration-time curve from 0 to 24 hours (AUCo-₂₄). The half-life of the terminal phase (t_1/2) in the plasma concentration- time curve was calculated from t_1/2 = 0.693/λ_z, where λ_z was estimated by linear regression using the last three concentration data points (r² > 0.9). The maximum observed concentration (C_max) was obtained by inspection of the concentration data. The time to reach the C_max was the first time at which C_max is observed and obtained by inspection of the data. The pre-dose concentration (C_trough) was obtained by inspection of the concentration data. The average concentration at steady state (C_ss, _avg) was calculated from AUC₀.₂₄ on Day 21 divided by the daily dosing interval (24 hours).

The following results are based on preliminary pharmacokinetic parameters from the 38 subjects, estimated using nominal collection times and quality-controlled, non quality-assured bioanalytical data.

Preliminary plasma pharmacokinetic parameters of Compound (I) are presented in Table 2. Compound (I) administered in the fasted state was absorbed rapidly, with peak plasma concentrations occurring within 1 to 2 hours after dosing. Under fasting conditions, both peak plasma levels (C_max) and area under the curve (AUC) were roughly dose proportional in a range of 1 mg QD or BID - 30 mg BID. Plasma concentrations declined with an elimination plasma half-life generally ranging between 5 to 14 hours. After 21 -day multiple oral BID dosing, AUC of Compound (I) increased slightly with an accumulation ratio of 1.1-2.2. The inter-subject variability in Compound (I) pharmacokinetics was evaluated in this subject population. The overall coefficients of. variation (CV) for AUC₍₀-₂₄₎ in the fasted state was 39%. Table 2: Plasma Pharmacokinetic Parameters of Compound (I) (Mean Estimates with Coefficient of Variation, CV%) on Day 21 of Cycle 1 (C1 D21) under

Fasting Conditions

After oral administration, the major circulating metabolite of Compound (I) was Compound (II). The AUC of Compound (II) was approximately 66% and 120% (medians) of the parent following single and multiple dosing of Compound (I) in human plasma, respectively. The preliminary plasma pharmacokinetic parameters of Compound (II) are summarized in Table 3. Following oral administration of Compound (I) under fasting conditions, both AUC and C_maxof Compound (II) were generally increased with doses ranging from 1 mg QD or BID - 30 mg BID. The terminal plasma half-life of Compound (II) was longer than that of the parent drug. After 21 - day multiple BID dosing, AUC of Compound (II) increased 2.0 to 5.0 folds. Table 3: Plasma Pharmacokinetic Parameters of Compound (II), the major metabolite (Mean Estimates With Coefficient of Variation, CV%) on Day

21 of Cycle 1 (C1 D21) under Fasting Conditions

A pilot food effect evaluation was conducted on Day 1 of Cycle 1 (C1 D1) and Day 1 of Cycle 2 (C1 D2). Sixteen subjects completed the pilot food evaluation and their data are presented in Table 4. When Compound (I) was taken with a high fat meal, peak plasma levels were delayed for about 2 to 7 hours with a decrease in Cm_ax. suggesting that the absorption rate of Compound (I) was reduced by food ingestion. However, the effect of food on Compound (I) AUC was variable. The coefficients of variation (CV) for AUC₍₀-₂₄₎ in the fed state was 78%, about twice of that in the fasted state, indicating that food ingestion increased the inter-subject pharmacokinetics variability.

Table 4: Pilot Food Effect Assessment: Comparison of Plasma Pharmacokinetics of Compound (I)Jn the Fed versus Fasted State

Compound (I) was dosed as purified (R)-enantiomer. The in vivo interconversion from (R)- (i.e., Compound (I)) to S-enantiomer (i.e., Compound (III)) was evaluated in twenty-four subjects across eight different dosing regimens. The average (S)-to-(R) ratio for AUC on Day 21 of cycle 1 was low, 0.03 (CV 38%); this excludes two outlier subjects with ratios of 12 and 21% each from the 1 mg BID cohort.

In summary, the plasma pharmacokinetics of Compound (I) in cancer patients is characterized by rapid absorption, with peak concentrations occurring within 1 to 2 hours of dosing, generally dose-proportional changes in exposures, and an elimination half-life of 5 to 16 hours. Food appeared to reduce Compound (I) peak plasma concentrations, but the effect on AUC was variable. Plasma pharmacokinetics for the major circulation metabolite Compound (I) was characterized by a longer half-life and up to 120% higher plasma exposures than the parent.

PD markers of MEK1/MEK2 activity (pERK) and cell proliferation (Ki67) were assessed by quantitative immunohistochemistry in tumor biopsies obtained at baseline and on Day 15 (2 to 4 hours after dosing). Strong tumor pERK suppression was observed across different dose regimens ranging from 1 mg QD or BID to 30 mg BID.; in results being gathered from the ongoing study, the average pERK decline was 68% over all dose (N = 23), and 75% from 2 mg BID to 30 mg BID (N = 20), relative to baseline. When only those cases with baseline pERK score above 20 are considered, the average pERK decline was 63 % over all dose groups from 1 mg QD to 30 mg BID (N=19), and 74% from 2 mg BID to 30 mg BID (N=14). Ki67 was also affected over this dose range; the average Ki67 decline was 31% over dose ranges from 1 mg QD to 30 mg BID (N = 22) and 46% from 2 mg BID to 30 mg BID (N = 19). When only those cases with baseline Ki67 scores above 20 are considered, the average Ki67 decline was 39% across all dose groups (N=18), and 49 % over the 2 mg BID to the 30 mg BID groups (N=13). As a result of these investigations of pERK in tumor biopsies, it has been shown that Compound (I) led to suppression of pERK

10 levels in tumors at all doses tested, and particularly at doses of 2 mg or greater. The proliferation marker Ki67 also showed a decline in post-treatment tumors and a similar dose effect. Table 5 represents data for the Phase I/Phase Il study. Table 5: Data for the Phase I/Phase Il Study in Human Cancer Patients

ND - not determined.

Example 1 : In Vivo Treatment of Rheumatoid Arthritis and Protection from Death 5

In an experiment using a mouse model of collagen-induced arthritis, a combination of methotrexate, USP (Sigma catalog number M-9929) and Compound (I) effectively inhibited edema and improved clinical scores. Compound (I) protected the combination-treated mice from death observed for some mice treated with

10 methotrexate alone.

In this experiment, bovine type Il collagen (University of Utah) was diluted with 0.01 N acetic acid to a concentration of 2 mg/mL, and the mixture was emulsified with an equal volume of Freund's complete adjuvant (Difco, Detroit, Michigan) supplemented with 1 mg/mL of Mycobacterium tuberculosis Hra37. Age-matched (8

15 to 12 weeks) female DBA/1 mice (Harlan, UK) were immunized on Day 0 with 100 μl_ of emulsion (100 μg collagen) intradermal^ at the base of the tail. Mice were weighed on Day 27. Oral dosing with methotrexate and Compound (I) began on Day 27 and continued through Day 41 or Day 42; methotrexate was administered on a schedule of three times per week, Mondays, Wednesdays, and Fridays, and Compound (I) was

20 administered QD during the dosing period. Vehicle control mice were administered vehicle (HPMC/Tween-80) only and normal control mice were not administered anything. On Day 28 after immunization, the mice were given 50 μg LPS (Sigma Aldrich, St Louis, Missouri) intraperitoneal^ (IP) in 100 μL saline. The development of arthritis was assessed on Days 27, 31 , 34, 37, and 41 by giving a clinical score to

25 each limb of each animal using the following scale: (0) normal, (1) erythema (i.e., abnormal redness of skin) and edema (i.e., swelling due to abnormal collection of fluid), (2) joint distortion, or (3) joint ankylosis (i.e., stiffness or fixation of joint). Edema measurements on front and rear paws were taken using constant tension calipers (Dyer, Lancaster, Pennsylvania). Mice were weighed again on Day 42. On Day 42 in

30 all groups but the 10 mg methotrexate group (i.e., Group no. 5), plasma samples were collected at 1 hour (from 3 animals), 3 hours (from 2 animals), 7 hours (from 2 animals) post Day 42 dose, and 24 hours (from 2 animals) post-Day 41 dose. By Day 42 in the 10 mg/kg methotrexate group, three animals had died so plasma samples were collected at 1 hour (2 animals), 3 hours (2 animals), 7 hours (1 animal) post Day 42 dose and 24 hours (1 animal) post Day 41 dose. The animals providing the 24 hour plasma samples were not dosed on Day 42. Each remaining live mouse was euthanized, its liver was collected, and one front joint and one rear joint were fixed in 10% buffered formalin and one front joint and one rear joint were flash frozen in liquid nitrogen. The mice were grouped as shown in Table 6, which also reports the number of deaths observed per group. Table 6: Mouse collagen-induced arthritis

The data from the experiment are graphically displayed in Figures 1 to 12 and numerically in Table 6. Figures 1 to 4 also recite AUC % lnh of paw edema, and Figures 5 to 8 recite AUC % lnh of a decrease in average total clinical score, for Day 27 to Day 41. Statistical significance was determined using a Student t-test. The data show that a combination of Compound (I) and methotrexate is efficacious in treating rheumatoid arthritis in mice and indicates that Compound (I) protects mice treated with methotrexate from death and inhibits methotrexate related body weight loss.

Claims

CLAIMS What is claimed is:

1. A method of treating rheumatoid arthritis in a patient, the method comprising administering to a patient in need of rheumatoid arthritis treatment a therapeutically effective amount of a combination consisting essentially of a first amount of N-[4- [[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid, or a pharmaceutically acceptable salt thereof, or a mixture thereof, and a second amount of N-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide.

2. The method according to claim 1 , wherein the patient is a human and the first amount is from 1 mg to 30 mg and the second amount is from 1 mg to 30 mg.

3. The method according to claim 2, wherein the N-[4-[[(2,4-diamino-6- pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid, or the pharmaceutically acceptable salt thereof, is sodium N-[4-[[(2,4-diamino-6- pteridinyl)methyl]methylamino]benzoyl]-L-glutamate and the first amount is from 1 mg to 20 mg or from 1 mg to 25 mg.

4. The method according to claim 3, wherein the second amount is from 1 mg to 15 mg.

5. The method according to claim 4, wherein the second amount is from 1 mg to 8 mg.

6. The method according to claim 5, wherein the second amount is 2 mg, 4 mg, or 8 mg.

7. The method according to claim 6, wherein the second amount is 2 mg, 4 mg, or 8 mg, which is administered once per day, or 1 mg, 2 mg, or 4 mg, which is administered twice per day.

8. The method according to any one of claims 1 to 7, wherein the N-[(R)-2,3- dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide is polymorph Form IV of N-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo- phenylamino)-benzamide.