TREATMENT OF INFLAMMATORY BOWEL DISEASE

^' PRIORITY

|000I J The present application claims the benefit of Indian Provisional Patent Application No. 2Q62/CHE/2012 filed on 23-May-20l2, the entire disclosure of which is relied on for all purposes and is incorporated into this application by reference.

FIELD OF THE INVENTION 0002] This disclosure generally relates to compounds and compositions for the treatmen of Inflammatory Bowel Disease More particularly, this invention relates to treating subjects with a pharmaceutically acceptable dose of compounds, stereoisomers, enantiomers, crystals, esters, salts, hydrates, prodrugs, or mixtures thereof.

BACKGROUND OF THE INVENTION

[0003] Inflammatory bowel disease (IBD) encompasses several chronic inflammatory conditions, most significantly ulcerative colitis (UC) and Crohn's disease (CD). While these two conditions share many commo features - diarrhea, bloody stools, weight loss, abdominal pain, fever, and fatigue - each has unique features, A complete discussion of Crohn's disease will be addressed in a future article. Ulcerative colitis and associated risk factors, pathogenesis, nutrient deficiencies, conventional treatment approaches, natural treatment approaches, and extra-intestinal mani estations of the disease.

|O0O | Ulcerative colitis affects the colon and rectum and typically involves onl the innermost lining or mucosa, manifesting as continuous areas of inflammation and ulceration, with no segments of normal tissue. The Crohn's and Colitis Foundation of America defines several varieties of UC. Disease involving only the most distal part of the colon and the rectum is termed ulcerative proctitis; disease from the descending colon down is referred to as limited or distal colitis; and disease involving the entire colon is called pancolitis. UC may be insidious, with gradual onset of symptoms, or the first attack may be acute and fulminate.

{0005} More mild symptoms include a progressive loosening of the stool, abdominal cramping, and diarrhea, As the disease progresses from mild to more severe, the patient may also experience weight loss, fatigue, loss of appetite that may result in nutrient deticiencies, mucus in the stool, severe recta! bleeding, fever, and anemia. It is estimated that 1 -2 million Americans suffer from IBD; approximately half of these have ulcerative colitis. UC can occur anytime in life, but is usually diagnosed prior to age 30. The disease appears to affect men and women equally. Approximately 20 percent of people with UC have a close relati ve with IBD. Caucasians have a higher incidence of UC, with Jewish people of European descent 3-6 times more likely to develop the disease. Regions with a low incidence of UC include Asia, Japan, Africa, and Sout America,

|0006] Differing cytokine and other inflammatory-mediator profiles have been identified for UC and CD. The classic lesions of UC, involving the mucosal layer with extensive epithelial damage, abundant neutrophils, and crypt abscesses have led to a search for an immune mechanism to explain the epithelial damage. Signs of increased oxidative stress are in evidence in the intestinal mucosa of patients with ulcerative colitis and may be secondary to inflammation. One study examined signs of oxidative stress and plasma antioxidant levels in controls compared to patients with UC and CD. Oxidative DNA damage was noted in both IBD groups compared to controls, measured by production of 8-hydroxy-deoxyguanosine (S-OHdG). UC patients were found to have significantly lower plasma levels of vitamins A and E and several carotenoids compared to controls; there were no differences between UC and CD groups,

[0007J Mucosal biopsies of UC patients were analyzed and shown to have increased reactive oxygen intermediates, DNA oxidation products (8-OHdG). and iron in inflamed tissue compared to controls. Decreased levels of copper and zinc, cofactors for the endogenous antioxidant superoxide dismutase, were also observed.

JOOOS] Managing acute pathology of often relies on the addressing underlying pathology and symptoms of the disease. There is currently a need in the art for new compositions t treatment or delay of the onset of Inflammatory' Bowel Disease and its associated complications progression .

SUMMARY OF THE INVENTION

[0009J The present invention provides compounds, compositions containing these compounds and methods fo using the same to treat prevent and/or ameliorate the effects of the conditions such as Inflammatory Bowel Disease.

[001.0J The invention herein provides compositions comprising of formula Ϊ or pharmaceutical acceptable salts thereof The invention also provides pharmaceutical compositions comprising one or more compounds of formula 1 or intermediates thereof and one or more of pharmaceutically acceptable carriers, vehicles or diluents. These compositions may be used in the treatment of Inflammatory Bowel Disease and its associated complications.

Formula Ϊ

[0 11] In certain embodiments, the present invention relates to the compounds and compositions of formula i, or pharmaceutically acceptable salts thereof.







a is independentiv 2,3 or 7;

each b is independently 3, 5 or 6;

e is independently 1 , 2 or 6;

c and d are each independently H, D, -OH, -OD, C C«-alky1, ~N¾ or ~COC¾. jOO j The inveotion herein aiso provides compositions comprising of formuia II or pharmaceutical acceptable salts thereof. The invention also provides pharmaceutical compositions comprising one or more compounds of formula 11 or intermediates thereof arid one or more of pharmaceutically acceptable carriers, vehicles or diluents. These compositions may be used in the treatment of inflammatory Bowel Disease and its associ ated com pli caii oris .

Formula II

|00I.4J Accordingly, in certain embodiments, the present invention relates to the compounds and compositions of formula II, or pharmaceutically acceptable salts thereof,

Formula II

Wherein

R ^'¾ each independently represents H, D,

11









a is independently 2,3 or 7;

each b is i ndependently 3, 5 or 6;

e is independently 1, 2 or 6;

c and d are each independently H, D, -OH, -GD, Ct-CValkyi, - B ₂ or -COCH ₃

[0016] In the iiiusirative embodiments, examples of compounds of formula 1 are as set forth below:

(1-1)

(1-2) 0017 j in the illustrative embodi ments, examples of compounds of formula 11 are as set forth below:

j ^' 001.8] Herein the application also provides kit comprising any of the pharmaceutical compositions disclosed herein. The kit may comprise instructions for use in the treatment of Inflammatory Bowel Di sease or its rel ated complications.

[0019] The application also discloses a pharmaceutical composition comprising a pharmaceutically acceptable carrier and any of the compositions herein, in some aspects, the pharmaceutical com position is formulated for systemic administration, oral administration, sustained release, parenteral administration, injection, subdermal administration, or transdermal administration.

[0020] Herein, the application additionally provides kits comprising the pharmaceutical compositions described herein. The kits may further comprise instructions for use in the treatment of Inflammatory Bowel Disease or its related complications.

[0021] The compositions described herein have several uses. The present application provides, for example, methods of treating a patient suffering from Inflammatory Bowel Disease or its related complications manifested from Inflammation, metabolic conditions or disorders, metabolic syndrome, chronic diseases or disorders; Hyperinsulinemia, Insulin resistance. Glucose intolerance, Hepatology, Cancer, Respiratory, Hematological, Orthopedic, Cardiovascular, Renal, Skin, Nephrological, or Ocular complicatio . DETAILED DESCRIPTION OF THE INVENTION

Definitions

{0022} As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.

10023} The compounds of the present invention ca be present in the form of pharmaceutically acceptable salts. The compounds of the present invention can also be present in the form of pharmaceutically acceptable esters (I.e., the methyl and ethyl esters of the acids of formula 1 and formula 11 to be used as prodrugs). The compounds of the present invention can also be solvated, i.e. hydrated. The solvation can be affected in the course of the manufacturing process or can take place i.e. as a consequence of hygroscopic properties of an initially anhydrous compound of formula Ϊ and formula Π (hydrati n),

{0024} Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed "isomers " Isomers that diffe in the arrangement of their atoms in space are termed "stereoisomers." Diastereomers are stereoisomers with opposite configuration at one or more chiral centers which are not enantiomers. Stereoisomers bearing one or more asymmetric centers that are tion- superimposable mirror images of each other are termed ''enantiomers." When a compound has an asymmetric center, for example, if a carbon atom is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center or centers and is described by the R- and S-sequencing rules of Cahn, Ingold a d Preiog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+} or (~)~isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a "racemic mixture" .

[0025J As used herein, the term "metabolic condition" refers to an inborn errors of metabolism (or genetic metabolic conditions) are genetic disorders that result from a defect in one or more metabolic pathways; specifically, the function of an enzyme is affected and is either deficient or completely absent. 0 26J The term "polymorph* as used herein is art-recognized and refers to one crystal structure of a given compound.

[0027] The phrases "parenteral administration" and "administered parenteral iy" as used herein refer to modes of administration other than enteral and topical administration, such as injections, and include w thout limitation intravenous, i tramuscular, intrapleural, intravascular, intrapericardiat, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemat injection and infusion,

{00281 A "patient," "subject," or "host" to be treated by the subject method may mean either a human or non-human animal, such as primates, mammals, and vertebrates.

J0029J The phrase "pharmaceutically acceptable" is art-recognized. In certain embodiments, the term includes compositions, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals, human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0030] The phrase "pharmaceutically acceptable carrier" is art-recognized, and includes, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, solvent or encapsulating material involved in carrying or transporting any subject composition, from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable ' in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient. In certain embodiments, a pharmaceutically acceptable carrier is non-pyrogenic. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1.) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate, (4) powdered tragacanth; (5) malt; (6) gelatin; (?) talc; (8) cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, com oil and soybean oil; ( 10) glycols, such as propylene glycol; (11 ) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; ( 14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer ^' s solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21 ) other non-toxic compatible substances employed in pharmaceutical formulat ons. 0031 J The term "prodrug" is intended to encompass compounds that, under physiological conditions, are converted into the therapeutically active agents of the present invention. A common method for making a prodrug is to include selected moieties that are hydrolyzed under physiological conditions to reveal the desired molecule, in other embodiments, the prodrug is converted by an enzymatic activity of the host animal,

[0032] The term "prophylactic or therapeutic" treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i .e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0033] The term "predicting" as used herein refers to assessing the probability related diseases patient will suffer from abnormalities or complication and'Or terminal platelet aggregation or failure and/or death (i.e. mortality) within a defined time window (predictive window) in the future. The mortality may be caused by the central nervous system or complication. The predictive window is an interval in which the subject will develop one or more of the said complications according to the predicted probability. The predictive window may be the entire remaining lifespan of the subject upon analysis by the method of the present invention.

[0034] The term "treating" is art -recognized and includes preventing a disease, disorder or conditio from occurring in an animal which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, such as treating the gastrointestinal disorders such as inflammatory bowel disease, ulcerative colitis and mild-to-moderate Crohn's disease of a subject by administration of an agent even though such agent does not treat the cause of the conditio The term "treating", "treat" or "treatment" as used herein includes curative, preventative (e.g., prophylactic), adjunct and palliative treatment.

[0035] The phrase "therapeutically effective amount" is an art-recognized term, in certain embodiments, the term refers to an amount of a salt or composition disclosed herein that produces some desired effect at a reasonable benefit risk ratio applicable to any medical treatment, la certain embodiments, the term refers to that amount necessary or sufficient to eliminate or reduce medical symptoms for a period of time. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art may enipirically determine the effective amount of a particular composition without necessitating undue experimentation.

[0036] In certain embodiments, the pharmaceutical compositions described herein are formulated in a manner such that said compositions will be delivered to a patient in a therapeutically effective amount, as part of a prophylactic or therapeutic treatment. The desired amount of the composition to be administered to a patient will depend on absorption, inactivation, and excretion rates of the drug as well as the delivery rate of the salts and compositions from the subject compositions. It is to be noted thai dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Typically, dosing wi ll be determined using techniques known to one skilled i the an. 0037] Additionally, the optimal concentration and/or quantities or amounts of any particular salt or composition may be adjusted to accommodate variations in the treatment parameters. Such treatment parameters include the clinical use to which the preparation is put, e.g., the site treated, the type of patient, e.g., human or non-human, adult or child, and the nature of the disease or condition.

[0038] in certain embodiments, the dosage of the subject compositions provided herein may be determined by reference to the plasma concentrations of the therapeutic composition or other encapsulated materials. For example, the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve from time 0 to infinity may be used.

[0039) When used with respect to a pharmaceutical composition or other material, the term "sustained release" is art-recognized. For example, a subject, composition which releases a substance over time ma exhibit sustained release characteristics, in contrast to a bolus type administration, in which the entire amount of the substance is made biologically available at one time. For example, in particular embodiments, upon contact with body fluids including blood, spinal fluid, mucus secretions, lymph or the like, one or more of the pharmaceutically acceptable exci ients may undergo gradual or delayed degradation (e.g., through hydrolysis) with concomitant release of an material incorporated therein, e.g., an therapeutic and/or biologically active salt and/or composition, for a sustained or extended period (as compared to the elease from a bolus). This release may result in prolonged delivery of therapeutically effective amounts of any of the therapeutic agents disclosed herein. j 004 J The phrases "systemic administration," "administered systemically," "'peripheral administration" and "administered peripherally" are art-recognized, and include the administration of a subject composition, therapeutic or other material at a site remote from the disease being treated. Administration of an agent for the disease being treated, even if the agent is subsequently distributed systemically, may be termed "local" or "topical" or "regional" administration, other than directly into the central nervous system, e.g., by subcutaneous administration, such that it enters the patient's system and, thus, is subject to metabolism and other like processes.

£004 J J The phrase ''therapeutically effective amount'' is an art-recognized term. In certain embodiments, the term refers to an amount of a salt or composition disclosed herein that produces some desired effect at a reasonable benefit/risk ratio applicable to an medical treatment, in certain embodiments, the term refers to that amount necessary or sufficient to eliminate or reduce medical symptoms for a period of time. The effective amount may vary depending on such factors as the disease or conditio being treated, the particular targeted constructs being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular composition without necessitating undue experimentation, f(MM2j The present disclosure also contemplates prodrugs of the compositions disclosed herein, as well as pharmaceutically acceptable salts of said prodrugs,

|0043j This application also discloses a pharmaceutical composition comprising a pharmaceutically accepiable carrier and the composition of a compound of Formula ί and formula 11 may be formulated for systemic or topical or oral administration. The pharmaceutical composition may be also formulated for oral administration, oral solution, injection, subdermal administration, or transdermal administration. The pharmaceutical composition may further comprise at least one of a pharmaceutically acceptable stabilizer, diluent, surfactant, filler, binder, and lubricant.

|0044] In many embodiments, the pharmaceutical compositions described herein will incorporate the disclosed compounds and compositions (Formula I and Formula O) to be delivered in an amount sufficient to deliver to a patient a therapeutically effective amount of a compound of formula 1 and formula 11 or composition as part of a prophylactic or therapeutic treatment. The desired concentration of formula 1 and formula 11 or its pharmaceutical acceptable salts will depend on absorption, inactivation, and excretion rates of the drug as well as the delivery rate of the salts and compositions from the subject compositions. It is to be noted that dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Typically, dosing will be determined using techniques known to one skilled in the art. |0045J Additionally, the optimal concentration and/or quantities or amounts of any particular compound of formula 1 and formula 0 ma be adjusted to accommodate variations in the treatment parameters. Such treatment parameters include the clinical use to which the preparation is put, e.g., the site treated, the type of patient., e.g., human or non-human, adult or child, and the nature of the disease or condition.

[0046] The concentration and/or amount of any compound of formula I and formula 11 may be readily identified by routine screening in animals, e.g., rats, by screening range of concentration and/or amounts of the material in question using appropriate assays. Known .methods are also available to assay local tissue concentrations, diffusion rates of the salts or compositions, and local blood flow before and after administration of therapeutic formulations disclosed herein. One such method is microdialysis, as reviewed by T. E. Robinson et a ., 1991 , microdialysis in the neurosciences. Techniques, volume 7, Chapter 1. The methods reviewed by Robinson .may be applied, in brief as follows. A microdialysis loop is placed in situ in a test animal. Dialysis fluid is pumped through the loop. When compounds with formula 1 and formula 0 such as those disclosed herein are injected adjacent to the loop, released drugs are collected in the dialysate in proportion to their local tissue concentrations. The progress of diffusion of the salts or compositions may be determined thereby with suitable calibration procedures using known concentrations of salts or compositions.

[0047] In certain embodiments, the dosage of the subject compounds of formula I and formula H provided herein may be determined by reference to the plasma concentrations of the therapeutic composition or other encapsulated materials. For example, the maximum plasma concentrati n (Cmas) and the area under the plasma concentration- time curve from time 0 to infinity may be used.

[0048] Generally, in carrying out the methods detailed in this application, an effective dosage for the compounds of Formulas I is in the range of about 0,01 mg/kg/da.y to about 100 mg kg/day in single or divided doses, for instance 0.01 mg/kg/day to about 50 mg kg/day in single or divided doses. The compounds of Formulas Ϊ may be administered at a dose of, for example, less than 0.2 mg/kg/day, 0.5 mg/kg/day, 1.0 mg/kg day, 5 mg/kg day, 10 mg kg day, 20 mg/kg/day, 30 mg/kg/day, or 40 mg kg day. Compounds of Formula I and formula 11 m y also be administered to a human patient at a dose of, for example, between 0.1 mg and 1000 mg, between 5 mg and 80 mg, or less than 1 .0, 9.0, 12.0, 20.0, 50.0, 75.0, 100, 300, 400, 500, 800, 1000, 2000, 5000 mg per day. In certain embodiments, the compositions herein are administered ai an amount, that is less than 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the compound of formula 1 and formula II required for the same therapeutic benefit.

|0049| An effective amount of the compounds of formula Ϊ and formula Π described herein refers to the amount of one of said salts or compositions which is capable of inhibiting or preventing a disease.

[0050] An effective amount may be sufficient to prohibit, treat, alleviate, ameliorate, halt, restrain, slow or reverse the progression, or reduce the severity of a complication resulting from nerve damage or demyelizatlon and/or elevated reactive oxidative- nitrosative species and/or abnormalities in neurotransmitter homeostasis' s, in patients who are at risk for such complications. As such, these methods include both medical therapeutic (acute) and/or prophylactic (prevention) administration as appropriate. ^' The amount and timing of compositions administered will, of course, be dependent on the subject being treated, on the severity of the affliction, on the manner of administration and on the judgment of the prescribing physician. Thus, because of pattent-to-paiient variability, the dosages given above are a guideline and the physician may titrate doses of the drug to achieve the treatment that the physician considers appropriate for the patient, in considering the degree of treatment desired, the physician must balance variety of factors such as age of the patient, presence of preexisting disease, as well as presence of other diseases. {0051] The compositions provided by this application may be administered to a subject in need of treatment fay a variety of conventional routes of administration, including orally, topically, parenteral.}', e.g., intravenously, suhcutaneously or intramedullary. Further, the compositions may be administered intranasal.}', as a rectal suppository, or using a "flash" formulation, i.e., allowing the medication to dissolve in the mouth without the need to use water. Furthermore, the compositions may be administered to a subject in need of treatment by controlled release dosage forms, site specific drug delivery, transdermal drug delivery, patch (active/passive) mediated drug delivery, by stereotactic injection, or in nanoparticles.

|0052] The compositions may be administered alone or in combination with pharmaceutically acceptable carriers, vehicles or diluents, in either single or multiple doses. Suitable pharmaceutical carriers, vehicles and diluents include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. The pharmaceutical compositions formed by combining the compositions and the pharmaceutically acceptable carriers, vehicles or diluents are then readily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, injectable solutions and the like. These pharmaceutical compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients and the like. Thus, for purposes of oral administration, tablets containing various excipients such as L-arginine, sodium citrate, calcium carbonate and calcium phospiiate may be employed along with various disintegrates such as starch, alginic acid and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium siearate, sodium lauryl sulfate and talc are often useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules. Appropriate materials for this include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the essential active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if desired, emulsifying or suspending agents, together with diluents such, as water, et anol, propylene glycol, glycerin and combinations thereof. The compounds of formula 1 and formula II ma also comprise enterieally coated comprising of various excipients, as is well known in the pharmaceutical art. 0053J For parenteral administration, solutions of the compositions may be prepared in (for example) sesame or peanut oil, aqueous propylene glycol, or in sterile aqueous solutions may be employed. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration, in this connection, the sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art. 0054] The formulations, for instance tablets, may contain e.g. 10 to 100, 50 to 250, 150 to 500 mg, or 350 to 800 mg e.g. 10, 50, 100, 300, 500, 700, 800 nig of the compounds of formula ! and formula li disclosed herein, for instance, compounds of formula I and formula Π or pharmaceutical acceptable salts of a compounds of Formula I. 0055] Generally, a composition as described herein may be administered orally, or parenterally (e.g., intravenous, intramuscular, subcutaneous or intramedullary). Topical administratio may also be indicated, for example, where the patient is suffering from gastrointestinal disorder that prevent oral administration, or whenever the medication is best applied to the surface of a tissue or organ as determined by the attending physician. Localized administration may also be indicated, for example, when a high dose is desired at the target tissue or organ. For buccal administration the active composition may take the form of tablets or lozenges formulated in a conventional manner. {0056 j The dosage administered wiil be dependent upon the identity of the inflammatory bowel disease: the type of host involved, including its age, health and weight; the kind of concurrent treatment, if any; the frequency of treatment and therapeutic ratio,

|0057J Illustratively, dosage levels of the administered active ingredients are: intravenous, 0.1 to about 200 mg/kg; intramuscul r., 1 to about 500 nig/kg; orally, 5 to about 1000 mg/kg; intranasal instillation, 5 to about 1000 mg/kg; and aerosol, 5 to about 1000 mg/kg of host body weight.

(0058) Expressed in terms of concentration, an active ingredient can be present in the compositions of the present invention for localized use about the cutis, intranasal!y, pharyngolaryngeally, hronchially, intravaginaJly, rectally, or ocularly in a concentration of from about 0.0.1 to about 50% w/ ^' of the composition, preferably about .1 to about 20% w/w of the composition; and for parenteral use in a concentration of from about 0.0S to about 50% /v of the composition and preferably from about 5 to about 20% ^" w/v,

|0059| The compositions of the present invention are preferabl presented for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, suppositories, sterile parenteral solutions or suspensions, sterile non-parenteral solutions of suspensions, and oral solutions or suspensions and the like, containing suitable quantities of an active ingredient. For oral administration either solid or fluid unit dosage forms can be prepared.

{006CIJ As discussed above, the tablet core contains one or more hydrophilic polymers. Suitable hydrophilic polymers include, but are not limited to, water sweliable cellulose derivatives, polyalkylene glycols, thermoplastic polyalkylene oxides, acrylic polymers, hydrocolloids, clays, gelling starches, swelling cross-linked polymers, and mixtures thereof. Examples of suitable water sweliable cellulose derivatives include, but are not limited to, sodium carboxymethylcelluiose, cross-linked hydroxypropylcellulose, hydroxy propyl cellulose (UPC), hydroxypropylmethylcellulose {HPMC), hydroxyisopropyiceilulose. hydroxybutylcellul ose, hydroxyphenylcellulose, h droxy ethyl cell ulose (HEC), hydroxypentylcellulose, hydroxypropylethylcell ulose, hydraxypropylbutylcellulose, and hydroxypropylethylcellulose, and mixtures thereof. Examples of suitable polyalkylene glycols include, but are not limited to, polyethylene glycol. Examples of suitable thermoplastic polyalkylene oxides include, but are not. limited to, poly (ethylene oxide). Examples of suitable acrylic polymers include, but are not limited to., potassium methaerylaiedivinySbenzene copolymer, polymethylmethacrylate, high-molecular weight crosslinked acrylic acid homopolymers and copolymers suc as those commercially availabie from Noveon Chemicals under the tradename CARBOPOL ^r¾i . Examples of suitable hydrocolloids include, but are not limited to, alginates, agar, guar gum, locust bean guru, kappa earrageenan, iota earrageenan, tara, gum arabic, tragacanth, pectin, xanthan gum, gel I an gum, maltodextrin, galactomannan, pusstulan, laminarin, sclerogluean, gum arabic, inulin, pectin, gelatin, whelan, rhamsan, zooglan, methylan, chitin, cyclodextrin, chitosan, and mixtures thereof. Examples of suitable clays include, but are not limited to, smectites such as bentonite, kaolin, and laponite; magnesium trisihcate; magnesium aluminum silicate; and mixtures thereof. Examples of suitable gelling starches include, but are not limited to, acid hydrolyzed starches, swelling starches such as sodium starch glycolate and derivatives thereof and mixtures thereof. Examples of suitable swelling cross-linked polymers include, but are not limited to, cross-linked polyvinyl pyrrolidorie, cross-linked agar, and cross-linked carboxymethyl cellulose sodium, and mixtures thereof

|0061] The carrier may contain one or more suitable excipients for the formulation of tablets. Examples of suitable excipients include, but are not limited to, fillers, adsorbents, binders, disintegrarits, lubricants, glidants, release-modifying excipients, supefdisiiitegrants, antioxidants, and mixtures thereof.

{0062] Suitable binders include, but are not limited to, dry binders such as polyvinyl pyrrolidorie and hydroxypropyhnethylcelluiose; wet binders such as water-soluble polymers, including hydrocolloids such as acacia, alginates, agar, guar gum, locust bean, carrageenan, oarboxymetliylcelhilose, tara, gum arable, tragacanth, pectin, xanthan, gelian, gelatin, maltodextrin, galactomannan, pusstulan, laminarin, scierogl cati, inulin, vvhelan, rhamsan, zooglan, methyl an, chitin, cyelodextrin, chitosan, polyvinyl pyrrol i done, cellulosics, sucrose, and starches; and mixtures thereof Suitable disinte&rants include, but are not limited to, sodium starch alveolate, cross-linked polyvinylpyrrolidone, cross-linked carboxymethylceHulose, starches, microcrystai!iiie cellulose, and mixtures thereof.

[00631 Suitable lubricants inciude, but are not limited to, long chain fatty acids and their salts, such as magnesium stearate and stearic acid, talc, glyeerides waxes, and mixtures thereof. Suitable glidants include, but are not limited to, colloidal silicon dioxide. Suitable release-modifying excipients include, but are not limited to, insoluble edible materiais, pH-dependent polymers, and mixtures thereof.

|006 | Suitable insoluble edible materiais for use as release-modifying excipients include, but are not limited to, water-insoluble polymers and low-melting hydrophobic materials, copolymers thereof, and mixtures thereof. Examples of suitable water- insoluble polymers inciude, but are not limited to, ethylcelluiose, polyvinyl alcohols, polyvinyl acetate, polycaprolaetones, cellulose acetate and its derivatives, acrylates, methaery!ates, acrylic acid copolymers, copolymers thereof and mixtures thereof. Suitable low-melting hydrophobic materials inciude, but are not limited to, fats, fatty acid esters, phospholipids, waxes, and mixtures thereof. Examples of suitable fats include, but are not limited to, hydrogenated vegetable oils such as for example cocoa butter, hydrogenated palm kernel oil, hydrogenated cottonseed oil, hydrogenated sunflower oil, and hydrogenated soybean oil, free fatty acids and their salts, and mixtures thereof Examples of suitable fatty acid esters inciude, but are not limited to, sucrose fatty acid esters, mono-, di-, and triglycerides, glyceryl behenate, glyceryl palmitosiearate, glyceryl monostearate, glyceryl tiistearate, glyceryl tri!aurylate, glyceryl myri state, GlycoWax- 932, lauroyl macrogol-32 glyeerides, stearoyl macrogoi-32 glyeerides, and mixtures thereof. Examples of suitable phospholipids include phosphoiidyi choline, phosphotidyl serene, phosphotidyl enositoL phosphotidic acid, and mixtures thereof Examples of suitable waxes include, but are not limited to, canrauba wax, spermaceti wax, beeswax, candelilla wax, shellac wax., nucrociystalline wax, and paraffin wax; fat-containing mixtures such as chocolate, and mixtures thereof. Examples of super disintegrants include, but are not limited to, croscarmeilose sodium, sodium starch gl col ate and cross- linked povidone (crospovidone). In one embodiment the tablet core contains up to about 5 percent by weight of such super disintegrant.

{006 1 Examples of antioxidants include, but are not limited to, tocopherols, ascorbic acid, sodium pyrosulfHe, but lh droxy toluene, butylated hydroxyanisole, edetic acid, and edetale salts., and mixtures thereof. Examples of preservatives include, but are not limited to, citric acid, tartaric acid, lactic acid, malic acid, acetic acid, benzoic acid, and sorbic acid, and mixtures thereof.

| 066| in one embodiment, the immediate release coating has an average thickness of at least 50 microns, such as from about 50 microns to about 2500 microns; e.g., from, about 250 microns to about 1000 microns. In embodiment, the immediate release coating is typically compressed at a density of more than about 0.9 g/cc, as measured by the weight and volume of that specific layer. 0067] In one embodiment, the immediate release coating contains a first portion and a second portion, wherein at least one of the portions contains the second pharmaceutically active agent, in one embodiment, the portions contact each other at a center axis of the tablet In one embodiment, the first portion includes the first pharmaceutically active agent, and the second portion includes the second pharmaceutically active agent.

[0068] In one embodiment, the first portion contains the first pharmaceutically active agent, and the second portio contains the second pharmaceutically active agent. In one embodiment, one of the portions contains a third pharmaceutically active agent. In one embodiment one of the portions contains a second immediate release portion of the same pharmaceutically active agent as that contained in the tablet core,

[0069) in one embodiment, the outer coating portion is prepared as a dry blend of materials prior to addition to the coated tablet core, in another embodiment the outer coating portion is included of a dried granulation including the pharmaceutically active agent. 0070] Formulations with different drug release mechanisms described above could be combined in a final dosage form containing single or multiple units. Examples of multiple units include multilayer tablets, capsules containing tablets, beads, or granules in a solid or liquid form. Typical, immediate release formulations include compressed tablets, gels, films, coalings, liquids and particles that can be encapsulated, for example, in a gelatin capsule. Many methods for preparing coatings, covering or incorporating drugs, are known in the art.

[0071] The immediate release dosage, unit of the dosage form, i.e., a tablet, a plurality of drug-containing beads, granules or particles, or an outer layer of a coated core dosage form, contains a therapeutically effective quantity of the active agent with conventional pharmaceutical excipients. The immediate release dosage unit may or may not be coated, and may or may not be admixed with the delayed release dosage unit or units (as in an encapsulated mixture of immediate release drug-containing granules, particles or beads and delayed release drug-containing granules or beads),

[0072] Extended release formulations are generally prepared as diffusion or osmotic systems, for example, as described in "Remington— The Science and Practice of Pharmacy", 20th, Ed., Lippincott. Williams & Wilkins, Baltimore, d., 2000), A diffusion system typically consists of one of two types of devices, reservoir and matrix, which are weiiknown and described in die art. The matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form. 0073] An immediate release portion can foe added to the extended release system by means of either applying an immediate release layer on top of the extended release core; using coating or compression processes or in a .multiple unit system such as a capsule containing extended and immediate release beads. 0O74] Delayed release dosage formulations are created by coating a solid dosage form with a film of a polymer which is insoluble in the acid environment of the stomach, but soluble in the neutral environment of small intestines. The delayed release dosage units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material. The drug-containing composition may be a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coated core" dosage form, or a plurality of drug-containing beads, particles or granules, for incorporation into either a tablet or capsule. j ^' 0075| A pulsed release dosage form is one that mimics a multiple dosing profile without repeated dosing and typically allows at least a twofold reduction in dosing frequency as compared to the drug presented as a conventional dosage form (e.g., as a solution or prompt drug-releasing, conventional solid dosage form), A pulsed release profile is characterized by a time period of no release (lag time) or reduced release followed by rapid drug release.

|0076| Each dosage form contains a therapeutically effective amount of active agent. In one embodiment of dosage forms that mimic a twice daily dosing profile, approximately 30 wt. % to 70 wt. %, preferably 40 wt. % to 60 wt. %, of the total amount of active agent in the dosage form is released in the initial pulse, and, correspondingly approximately 70 wt. % to 3.0 wt. %, preferably 60 wt. % to 40 wt. %, of the total amount of active agent in the dosage form is released in the second pulse. For dosage forms mimicking the twice daily dosing profile, the second pulse is preferably released approximately 3 hours to less than 14 hours, and more preferably approximately 5 hours to 1.2 hours, following administration.

{0077) Another dosage form contains a compressed tablet or a capsule having a drug- containing immediate release dosage unit, a delayed release dosage unit and an optional second delayed release dosage unit, in this dosage form, the immediate release dosage unit contains a plurality of beads, granules particles that release drug substantially immediately following oral administration to provide an initial dose. The delayed release dosage unit contains a plurality of coated beads or granules, which release drug approximately 3 hours to 14 hours following oral administration to provide a second dose.

[0078] For purposes of transdermal (e.g., topical) administration, dilute sterile, aqueous or partially aqueous solutions (usually in about 0.1% to 5% concentration), otherwise similar to the above parenteral solutions, may be prepared,

|0O79] Methods of preparing various pharmaceutical compositions with a certain amount of one or more compounds of formula i and formula II or other active agents are known, or will be apparent in light of this disclosure, to those skilled in this art. For examples of methods of preparing pharmaceutical compositions, see Remington' Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th Edition (1 95).

|0O8O] In addition, in certain embodiments, subject compositions of the present application maybe lyophilized or subjected to another appropriate drying technique such as spray drying. The subject compositions may be administered once, or may be di ided into a number of smaller doses to be administered at varying intervals of time, depending in part on the release rate of the compositions and the desired dosage.

{ ^' 0081 ) Formulations useful in the methods provided herein include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared fay any methods well known in the art of pharmacy. The amount of a subject composition which may be combined with a carrier material to produce a single dose may vary depending upon the subject being treated, and the particular mode of administration.

{0082] Methods of preparing these formulations or compositions include the step of bringing into association subject compositions with the carrier and, optionally, one or more accessory ingredients. In general , the formulations are prepared by uniformly and intimately bringing into association a subject composition with liquid carriers, or finely di vided solid carriers, or both, and then, if necessary, shaping the product. 00S3] The compounds of formula ί and formula II described herein may be administered in inhalant or aerosol formulations. The inhalant or aerosol formulations may comprise one or more agents, such as adjuvants, diagnostic agents, imaging agents, or therapeutic agents useful in inhalation therapy. The final aerosol formulation ma for example contain 0.005-90% w/w, for instance 0.005-50%, 0.005-5% w/w, or 0.01 -1 .0% w/w, of medicament relative to the total weight of the formulation. 0084] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the subject composition is mixed with one or more pharmaceutically acceptable carriers and/or any of the following: (I) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymetrryiceliulose, alginates, gelatin, polyvinyl pyrroiidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical composi tions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. 00S5J Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the subject compositions, the liquid dosage forms may contain inert diluents commonly used in the art, 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 (in particular, cottonseed, corn, peanut, sunflower, soybean, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan.. and mixtures thereof. 0086] Suspensions, in addition to the subject compositions, may contain suspending agents such as, for example, ethoxyiated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide. bentonite, agar- agar and tragacanth, and mixtures thereof.

[0087] Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing a subject composition with one or more suitable non-irritating carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax, or a salicylate, and which is solid at. room tem erature., but liquid at body temperature and, therefore, will melt in the appropriate body cavity and release the encapsulated compound(s) and composition^). Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing such, carriers as are known in the art to be appropriate.

[0088) Dosage forms for transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. A subject composition may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required. For transdennai administration, the complexes may include lipophilic and h drophilic groups to achieve the desired water solubility and transport properties.

[0089] The ointments, pastes, creams and gels may contain, in addition to subject compositions, other carriers, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof Powders and sprays may contain, in addition to a subject composition, exci ients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and poly amide powder, or mixtures of such substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0090] Methods of delivering a composition or compositions via a transdermal patch are known in the art. Exemplar)' patches and methods of patch deliver are described in US Patent Nos. 6,974,588, 6,564,093, 6,312,716, 6,440,454, 6,267,983, 6,239, 180, and 6,103,275.

[0091 ] In another embodiment, a transdennai patch may comprise: a substrate sheet comprising a composite film formed of a resin composition comprising 100 parts by weight of a polyvinyl chloride-polyurethane composite and 2-10 parts by weight of a styrene-ethylene-butylene-styrene copolymer, a first adhesive layer on the one side of the composite film, and a polyaikylene terephthaiate film adhered to the one side of the composite film by means of the first adhesive layer, a primer layer which comprises a saturated polyester resin and is formed on the surface of the polyaikylene terephthaiate film; and a second adhesive layer comprising a styrene-diene-styrene block copolymer containing a pharmaceutical agent layered on the primer layer. A method for the manufacture of the above-mentioned substrate sheet comprises preparing the above resin composition molding the resin composition into a composite film by a calendar process, and then adhering a polyaikylene terephthaiate film on one side of the composite fi lm by means of an adhesive layer thereby forming the substrate sheet, and forming a primer layer comprising a saturated polyester resin on the outer surface of the polyaikylene terephthaiate film,

|0092] Another type of patch comprises incorporating the drug directly in a pharmaceutically acceptable adhesive and laminating the drug-containing adhesive onto a suitable backing member, e.g. a polyester backing membrane. The drug should be present at a concentration which will not affect the adhesive properties, and at the same time deliver the required clinical dose. 0093] Transdermal patches may be passive or active. Passive transdermal drag delivery systems currently available, such as the nicotine, estrogen and nitroglycerine patches, deli ver small-molecule drugs. Many of the newly developed proteins and peptide drugs are too large to be delivered through passive transdermal patches and may be delivered using technology such as electrical assist (iontophoresis) for large-molecule drugs.

[0094J Iontophoresis is a technique employed for enhancing the flux of ionized substances through membranes by application of electric current. One example of an iontophoretic membrane is given in U. S. Pat. No. 5,080,646 to Theeuwes. The principal mechanisms by which iontophoresis enhances molecular transport across the skin are (a) repelling a charged ion from an electrode of the same charge, (b) electroosmosis, the convective movement of solvent that, occurs through a charged pore in response the preferential passage of counter-ions when an electric field is applied or (c) increase skin permeability due to application of electrical current.

|0095] In some cases, it may be desirable to administer in the form of a kit. it may comprise a container for containing the separate compositions such as a divided bottle or a divided foil packet. Typically the kit comprises directions for the administration of the separate components. The kit .form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals., or when titration of the individual components of the combination is desired by the prescribing physician.

100961 An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a plastic material that may be transparent.

{ ^' 0097| Methods and compositions for the treatment of Inflammatory Bowel Disease. Among other things, herein is provided a method of treating Inflammatory Bowel Disease, comprising administering to a patient in need thereof a therapeutically effective amount of compound of Formula 1:

Formula I

8] Wherein,

R ¹ , * each independently represents -C¾CO-, acetyl, D, H, CD3CO-,

42

43



a is independentiv 2,3 or 7;

each b is independently 3, 5 or 6;

e is independently 1 , 2 or 6;

c and d are each independently H, D, -OH, -OD, C C«-alky1, ~N¾ or ~COC¾; 100 9) Accordingly, in another aspect the methods and compositions for the treatment of Inflammatory Bowel Disease. Among other things, herein is provided a method of treating Inflammatory Bowel Disease, comprising administering to a patient in need thereof a therapeutically effective amount of compound of Formula II:





51

52

a is independently 2,3 or 7,

each b is independently 3, 5 or 6;

e is independently 1, 2 or 6;

c and d are each independently H, D, -OH, -OD, Ci-Gs-alky!, -N¾ or -COCH ₃ .

Methods for using compounds of formula I and formula 11:

[OOl OOJ The invention also includes methods for treating gastrointestinal diseases and inflammation such as inflammatory bowel disease, ulcerative colitis, mild-to-moderate Crohn's disease, rheumatoid arthritis, inflammatory arthritis, psoriatic arthritis, liver cirrhosis and idiopathic urticaria,

METHODS OF MAKING

[00101J Examples of synthetic pathways useful for making compounds of formula ί and formula 11 are set forth in example below and generalized in scheme I through scheme 2;

Scheme- 1 :

■10 °G

{0

1 2

{00103} To a stirred solutioii of potassium hydroxide (70kg, 1250 moi) and water (250 L) was added 2 (50 kg, 248 mo!) over a period of 20 minutes between 25 and 30°C in an autoclave. The reaction mixture was heated to 125-130*0, stirred at the same temperature for 5 hours under 2.5 kg/cm2 . The reaction mass was cooled to 25°C and acidified to pH 1.0-2.0 using hydrochloric acid (85 L), stirred for 1 hour. The precipitated soiid was filtered and washed with water (150 L), and the cake was slurried in water (300 L) at 30*C for I hour, filtered, washed with water (150 L) and dried at 65°C for 10 hours to afford 3 43.5 kg Yield: 43.5 kg (96 %) (Purity: 99.87 %). 00104| St.ep-2: Synthesis of compound 3:

2 3

1001.05) Compound 2 (40 kg, 218 mo!) was added slowly to a sti rred mixture of water (320 L) and sodium carbonate (16.3 kg, 153 mol), stirred for 30 minutes and the mass pH maintained between 8.0 and 9.5. 10% Raney Nickel (8 L) along with water (80 L) was added to the above solution. The resultant mixture was hydrogenated at atmospheric pressure at 60° C for 6 hours. 5% sodium hydroxide (130 L) was added to the mass at. 25°C, stirred for 30 minutes. The catalyst was filtered through celite, and washed with water (80 L). The solution was acidified to pH 2.5-3.0 with hydrochloric acid (3 I.) and stirred for 1 hour. The obtained compound was filtered, washed with water (40L). To the solution of wet cake and water (560 L) and hydrochloric acid (41 L), active charcoal (2.8 kg) was added, and the contents were heated to 70 ^f> C, maintained at the same temperature for 2 hours. Filtered the total solution through celite, washed with water (28 L). Active charcoal (2,8 kg) was added to the filtrate, stirred at a temperature of 70 C for 2 hours, filtered through celite and washed with water (28 L). To the obtained filtrate, pH was adjusted to 3.0-3.5 with aq, sodium bicarbonate solution (15 L), stirred for 1 hour, filtered the solid and washed with water (42 L), dried the solid at 80°C for 9 hours to give the title compound 3 as a oft ^" white colored powder. Yield: 27,5 kg (82 %) (Purity: 99.9 %).

[00106] Step-3: Synthesi s of compound 4:

3 4 00107| Compound 3 ( 1.0 mmol) was kept in a B, added thionyi chloride 10 ml and refluxed the reaction mixture. After compietion of the reaction, excess thionyi chloride was distilled off and the crude acid chloride (4) was used for next step without further purification.

{00108} Step-4: Sy nthesis of compound 6.

{00109} To a solution of compound 5 { 1 .0 mmol) in dry DCM ( 1.8 ml) was added N, N- diisopropylethylamine (2.0 mmol) at - 10°C, followed by drop wise addition of 4 (1.2 mmol) for 30 min at the same temperature and the reaction mixture was allowed to stir for 1 h at 0°C. On compietion of the reaction (monitored by TLC), the reaction mixture the solvent was evaporated and the crude was purified through column to obtain compound 6. -5: Synthesis of compound 8:

[001.1 if A 50 ml, vial was charged with a magnetic spin bar, compound 6 (0.38 mmol), acid 7 ( 0.57 mmol ), DMF ( 1 .912 ml), and di I sopropyl eth l amine (0.334 ml, 1 .91 mmol). With stirring, HATU (0.291 g.. 0.76 mmol) was added and the reaction was warmed to 50°C for 3 h. The reaction was then diluted with water and exti'acted with ethyl acetate (3X50 mL). The combined organic extract was dried with MgS04, filtered through a bed of Celite, and cone, in vacuo to yield the product which was purified via silica gel to get compound 8. M.F: C35H49N05 ; Mol. Wt : 563.36 ; Elemental Analysis: C, 74.57; H, 8.76; N, 2.48; O. 14. 19.

Scheme-2:

{00112} Step- 1 : Synthesis of compound 2:

1 2

{00! 13} Compound 1 (10 mmol) was kept in a RB, added thionyl chloride 10 ml and refluxed the reaction mixture. After completion of the reaction, excess thionyl chloride 2 was distilled off and the crude acid chloride was used for next step without further purification.

14} Step-2: Sy nthesis of compound 4.

{00115} To a solution of compound 3 { 1 0 mmol) in dry DCM (1.8 ml) was added , N- diisopropyiethylamine (2,0 mmol) at -10°C, followed by drop wise addition of 2 (1.2 mmol) for 30 min ai the same temperature and the reaction mixture was allowed to stir for 1 li at OoC, On completion of the reaction (monitored by TLC), tine reaction mixture the solvent was evaporated and the crude was purified through column to get compound 4,

|00116| Step-3 : Synthesi s of compound 6:

S 6

[001.171 Compound 5 (10 mmol) was kepi in a RB, added thionyl chloride 10 ml and refluxed the reaction mixture. After completion of the reaction, excess thionyl chloride 6 was distilled off and the crude acid chloride was used for next step without further purification.

[001 -4: Synthesis of compound 8:

{00119} To a solution of compound 7 ( 1.0 mraol) in dry DCM ( 1 .8 mi ) was added N, - diisopropylethylamine (2.0 mmol) at - 10°C, followed by drop wise addition of 6 (1,2 mmol) for 30 mm at the same temperature arid the reaction mixture was allowed to stir for 1 h at OoC. On completion of the reactio (monitored by TLC), the reaction mixture the solvent was evaporated and the crude was purified through column to get compound 8.

{00120! Step-5: Synthesis of compound 9:

jOOi ilJ NaH (15 mniol) was taken in a RB, added DMF, cooled to 0 ^1> C and added Compound 8 in DMF (10 mniol) dropwise with stirring. The stirring was continued for 20 min and then added CH3I, stirred the reaction mixture at room temperature for 4h. After com letion of the reaction the mixture was cooled to OoC and added ice cold water dropwise. The reaction mixture was extracted with diethyl ether and the organic layer was dried over Na2S0 and evaporated to get the crude product which was purified through column to get compound 9, 0 1221 Step-6: Synthesis of compound 10:

H ₂ 0 001231 Compound 9 (0.56 mmol) was dissolved in a mixture of 5 mL of concentrated HCi and 2.5 mL of water and diazoti ed with a solution of sodium nitrite (0.56 mmol). In the meantime compound 4 (0.56 mmol), potassium hydroxide (1.12 irtmoi) and sodium carbonate are dissolved in water. The diazo suspension is added in portions to the alkaline solution of salicylic acid and the alkalinity maintained at a sufficiently high level during the whole reaction by means of addition of further quantities of potassium hydroxide solution. After 2 days the reaction mixture is heated for 30 min at 50°C. The azo compound 10 was precipitated by means of HCI and filtered off (yield 85%), to yield the compound 10. 001241 Step-7: Synthesi of compound I I

!00125| Compound 10 (I mrool) was dissolved in dry DCM (5 ml) arid added boron tri bromide (BB.r3) (1.0 M, 2mmoi) at 0 oC and stirred the reaction mixture at rt for 5h. After completion of reaction the mixture wa cooled to 0 oC and added saturated aqueos aBCO?) dropwise to neutralize the reaction mixture. The two layers were separated and the organic layer was dried over and evaporated to get the crude material which on purification through column to yielded compound 1 1. M.F: C42H52N208 ; Mo\. Wt: 712.37 ; Elemental Analysis: C, 70.76; H, 7.35; N, 3.93, O, 17,95. j00126| The term "sample" refers to a sample of a body fluid, to a sample of separated cells or to a sample from a tissue or an organ. Samples of bod fluids can be obtained by well known techniques and include, preferably, samples of blood, plasma, serum, or urine, more preferably, samples of blood, plasma or serum. Tissue or organ samples may be obtained from any tissue or organ by, e.g., biopsy. Separated cells may be obtained from the body fluids or the tissues or organs by separating techniques such as centrifugation or cell sorting Preferably, ceil-, tissue- or organ samples are obtained from those cells, tissues or organs which express or produce the peptides referred to herein.

EQUIVALENTS jOO l 27| The present disclosure provides among other things compositions and methods for treating epilepsy and their complications. While specific embodiments of the subject disclosure have been discussed, the above speciiication is illustrative and not restrictive. Many variations of the systems and methods herein wilt become apparent to those skilled in the art upon review of this specification. The full scope of the claimed systems and methods should be detemiined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

INCORPORATION BY REFERENCE

[001281 AH publications and patents mentioned herein, including those items listed above, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control .