NZ714098B2 - Use of a thiazolo pyrimidinone for the treatment of inflammatory bowel disease - Google Patents

Abstract

Present invention relates to a low dose pharmaceutical composition, preferably oral composition comprising therapeutically effective amount of [(2-hydroxy-4-oxo-6,7,8,9- tetrahydro-4H,5H-10-thia-1,4a-diaza-benzo[a]azulene-3-carbonyl)-amino]-acetic acid (compound A) in the range of 2.5 mg to 60 mg. Present invention also relates to a method of treating inflammatory bowel disease in a mammal by administrating said low dose pharmaceutical composition. Further, present invention relates to a use of the compound A for the preparation of low dose pharmaceutical composition for the treatment of inflammatory bowel disease in a mammal. resent invention also relates to a method of treating inflammatory bowel disease in a mammal by administrating said low dose pharmaceutical composition. Further, present invention relates to a use of the compound A for the preparation of low dose pharmaceutical composition for the treatment of inflammatory bowel disease in a mammal.

Description

USE OF A THIAZOLO PYRIMIDINONE FOR THE TREATMENT OF INFLAMMATORY BOWEL DISEASE Field of the Invention: Present invention relates to a low dose pharmaceutical composition, preferably oral composition sing therapeutically ive amount of [(2-hydroxyoxo-6,7,8,9- tetrahydro-4H,5H-1 0-thia-l,4a—diaza—benzo[a]azulene-3 —carbonyl)-amino]—acetic acid (compound A) in the range of 2.5 mg to 60 mg. Present ion also relates to a method 'of treating inflammatory bowel disease in a mammal by administrating said low dose pharmaceutical composition. Further, present invention s to a use of the compound A for the preparation of low dose pharmaceutical composition for the treatment of inflammatory bowel e in a mammal.

Background of the invention: Inflammatory bowel disease (IBD) is the name given to a group of diseases causing chronic inflammation of the gastrointestinal tract. It is an idiopathic disease caused by a dysregulated immune se to host intestinal microflora. The course of IBD varies widely, with intermittent periods of remission followed by periods of acute illness. The 2 major types of IBD are ulcerative colitis and Crohn’s disease.

Ulcerative’ colitis affects the colon and rectum and typically involves only the innermost lining or , manifesting as uous areas of inflammation and ulceration, with no segments of normal tissue. The disease involving only the most distal part of the colon and the rectum, termed as ulcerative proctitis; disease from the desCending colon down is referred to as limited or distal colitis; s e involving the entire colon is called pancolitis (Kathleen Head \et al; Altern Med Rev. 2003;8(3):247—83).

Crohn's disease is transmural (affecting all layers of the intestine) inflammation that can affect any portion of the digestive tract from mouth to anus, but is predominantly seen in the terminal ileum and/or colon. Intestinal inflammation and ulceration in Crohn's disease is asymmetrical [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp ation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp and occurs in “patches,” with areas of healthy tissue persed, and s deeply into the intestinal wall, forming omatous lesions. Several categories of Crohn's disease have been described, defined by the portion of the digestive tract involved and the presenting symptomatology (Kathleen Head et al; Altern Med Rev. 2004; 9(4):360-401). The symptoms are often more variable than tive colitis depending on which part of the bowel is involved.

Clinical symptoms of IBD include abdominal cramps and pain, bloody diarrhea, severe urgency to have a bowel movement, sensation of incomplete evacuation, fever, loss of appetite, weight loss and anemia. Available therapeutic gies for management of IBD include 5-aminosalicylate (mesalamine), corticosteroids, immunomodulators, antibiotics and anti-tumor necrosis factor (TNF) agents. Most commonly used medication for IBD is mesalamine, which is available in the dose strengths range 250mg to 2g, which are recommended for dose of at least 1g per day' or even higher by oral route of stration, Though mentioned therapeutic gies are found useful, non-responsiveness of patients to 5- aminosalicylate, side-effects associated with high-level and prolonged corticosteroid usage and non- response/loss of response, high cost and increased risk of infection and malignancy with the use of biologic agents, especially when combined with immunomodulators are limitinngummins et al; Lab Invest, 2013; 93: 378-383). This indicates at existence of gap in the available therapeutics and re-inforces need for the development of newer therapeutic ches for the treatment of IBD. Also, as mentioned, IBD is a chronic disease, the tion is ed for longer duration of time, for example, mesalamine is recommended generally for 6 to 8 weeks for the total daily dose of 4g by oral route. Therefore, high dose medication for longer duration may reduce patient compliance.

Although exact etiology for IBD is not yet established but it has been found by researchers that during IBD, increased tissue metabolism and itis s the chronically inflamed mucosa and particularly the epithelium hypoxic, giving rise to the activation of the hypoxia- responsive transcription factor hypoxia-inducible factor (HIF).

[Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp The protective role of HIF—l in murine colitis was first investigated by Karhausen'et al (J Clin Invest. 2004; 114: 1098-1106). Conditional on of HIF-lu in c epithelium increased the severity of colitis and r dysfunction in both the oxazolone- and trinitrobenzene sulfonic acid (TNBS)- induced model of acute colitis. HIF activation is also known for up- regulation of set of target genes linked with maintenance of intestinal barrier function such as HSP 70 and anti-inflammatory cytokines interleukin (IL)-10 (Braai et a]; al . Gastroenterology-and Hepatology 2006;4: 754 —759) Intestinal epithelial HSP70 plays an ant role in protecting mucosal integrity and function by stabilizing the tight junctions between intestinal epithelial cells. Such intestinal epithelial protection is associated with cted ial translocation-and a reduction in inflammation (Liedel JL et al;, Pediatr Res. 2011;69:395-400). Also IL-10 expression is directly linked with inflammatory disorders. It was shown that IL-lO—deficient mice develop a chronic bowel disease resembling Crohn’s disease in human beings and intragastric administration of recombinant lactococcus lactis strain secreting murine IL-lO, prevented onset of colitis in IL- knockout mice, and caused a 50% reduction of the inflammation in dextran sulfate - induced c colitis. (Braat et al; Clinical Gastroenterology and Hepatology 2006;4: 754 — 759) W02009002533 discloses. method for treating IBD (by administering an agent that inhibits HIF hydroxylase activity. It discloses pyridine-Z-carboxamide, quinolinecarboxamide and isoquinolinecarboxamide as HIF hydroxylase inhibitors for treatment of IBD. It discloses that compound was stered daily as intraperitoneal dose in TNBS induced animal model of colitis at the dose of 20mg/kg and 40mg/kg, and higher dose was found more ive.

Above data shows that disclosed HlF ylase inhibitors may have potential in treatment of IBD in animals. However there is no conclusive ce available yet that HIF hydroxylase inhibitor can be effective for the treatment of IBD, particularly by oral administration and there is no HIF hydroxylase inhibitor drug yet ble in the market which could be used for treatment of IBD effectively.

[Annotation] jennyp None set by jennyp ation] jennyp MigrationNone set by jennyp [Annotation] jennyp ed set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp W02011045811 discloses oxazolo and thiazolo derivatives as HIF hydroxylase inhibitors for treatment of anemia, ia or tissue damage caused by ischemic disorders. It discloses that compound 10 was effective for chronic kidney disorder (CKD) at higher doses such as at the dose of 20mg/kg bid (approx 450 mg/day equivalent human dose), when administered through intraperitoneal route to diseased animals.

Jamadarkhana et a1 discloses in Am J. of nephrology (2012, 36: 8) that compound [(2- hydroxyoxo-6,7,8,9-tetrahydro-4H,5 1 1 0-thia-1,4a¥diaza-benzo [a]azulene—3-carbonyl)— amino]-acetic acid, a novel HIF hydroxylase inhibitor, when administered intraperitoneally it was found effective in prevention and for treatment of ischemic acute kidney injury (AKI).

Both Jamadarkhana et a1 and W02011045811 teach to use HIF hydroxylase inhibitor, [(2- hydroxyoxo-6,7,8,9-tetrahydro-4H,SH-1_0-thia-l,4a-diaza-benzo [a]azulenecarbonyl)- amino]-acetic acid, for the ent of anemia, ischemia or tissue damage caused by ischemic disorder such as CKD or AKI, cally by eral route.

Present invention es ‘ a low dose pharmaceutical composition for non-parenteral administratirin, preferably'for oral administration, comprising HIF hydroxylase inhibitor, [(2- hydroxyoxo-6,7,8,9-tetrahydro-4H,5Hthia-l,4a-diaza-benzo[a]azulene-i3-carbony1)-' amino]—acetic acid (hereinafier mentioned as compound A) which is useful in the dosage range of 2.5 mg to 60 mg per day for treating inflammatory bowel disease (herein after referred as IBD). Present invention also provides a method of treating inflammatory bowel disease in a mammal by administering low dose ceutical compositions comprising therapeutically effective amount of compound A, which is effective in low dosage range of 2.5 mg to 60 mg per day.

[Annotation] jennyp None set by jennyp ation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp Summary of the invention: One aspect of the present invention is to e a low dose ceutical composition comprising compound A and at least one pharmaceutically acceptable carrier selected from diluent, binder, disintegrant, pH adjusting agent-and lubricant, wherein said composition is effective in the dosage range of 2.5 mg to 60 mg per day for the treatment of IBD in a Another aspect of the present invention is to provide a low dose pharmaceutical composition for oral stration comprising compound A and at least one pharmaceutically acceptable carrier selected from diluent, binder, disintegrant, pH adjusting agent and lubricant, wherein said composition is effective in the dosage range of 2.5 mg to 60 mg per day for the treatment of IBD in a .

Another aspect of the present invention is to provide a low dose pharmaceutical composition comprising compound A in an amount of 2.5 mg to 60 mg and at least one pharmaceutically acceptable carrier selected from diluent, binder, disintegrant, pH adjusting agent and lubricant, wherein the said composition is effective in treating IBD in a mammal. r aspect of the present invention is to provide a low dose pharmaceutical ition for oral administration comprising compound A in an amount of 2.5 mg to 60 mg and at least one pharmaceutically acceptable carrier selected from diluent, binder, disintegrant, pH adjusting agent and lubricant, wherein the said composition is effective in treating IBD in a mammal.

Another aspect of the t invention is to provide a low dose ceutical composition comprising compound A and at least one ceutically able carrier selected from diluent, binder, disintegrant, pH adjusting agent and lubricant for the treatment of IBD in a mammal, wherein the said composition provides at least 50% local exposure of the total amount of said compound adminiStered.

Another aspect of the present ion is to provide a method of treating IBD in a mammal, by administering a low dose pharmaceutical composition comprising eutically effective amount of nd A.

Another aspect of the present invention is to provide use of compound A for preparation of the low dose pharmaceutical composition for the treatment of IBD in a mammal.

The present invention as claimed herein is described in the following items 1 to 4: 1. Use of [(2-hydroxyoxo-6,7,8,9-tetrahydro-4H,5Hthia-1,4a-diazabenzo [a]azulenecarbonyl)-amino]-acetic acid or its pharmaceutically acceptable salt for preparation of a pharmaceutical composition for the ent of inflammatory bowel disease in a human being wherein said compound is for non-parenteral administration. 2. Use according to item 1, n droxyoxo-6,7,8,9-tetrahydro-4H,5Hthia- 1,4a-diaza-benzo[a]azulenecarbonyl)-amino]-acetic acid or its pharmaceutically acceptable salt is in the range of 2.5 mg to 60 mg. 3. Use according to item 1, wherein [(2-hydroxyoxo-6,7,8,9-tetrahydro-4H,5Hthia- 1,4a-diaza-benzo[a]azulenecarbonyl)-amino]-acetic acid or its pharmaceutically acceptable salt is effective in the range of 2.5 mg to 60 mg per day. 4. Use according to item 1 wherein said pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier selected from diluent, binder, disintegrant, pH adjusting agent and lubricant.

Figures: Fig 1: Mean DAI score (Fig 1a), mean macroscopy score (Fig 1b) mean percent change in body weight (Fig 1c) and survival rate (Fig 1d) of animal model of colitis (TNBS induced colitis in male BALB/c mice) on per-oral administration of pharmaceutical composition comprising compound A or placebo (Composition without compound A).

Fig 2: Mean DAI score (Fig 2a), mean percent change in body weight (Fig 2b) and mean s score (Fig 2c) of animal model of colitis (DSS induced colitis in female BALB/c mice) or healthy animals and HSP70 n expression in colon tissue (Fig 2d) of animal model of colitis (DSS d colitis in female BALB/c mice) on per-oral administration of 17078093_1 ters) P101481.NZ pharmaceutical composition comprising nd A or placebo (Composition without compound A).

Fig 3: Tissue distribution of compound A in ileum, caecum and colon after administration of pharmaceutical composition comprising compound A by oral and intraperitoneal route in animal model of colitis (DSS induced colitis in female BALB/c mice).

Fig 4: Crypt architecture and inflammatory cells in colon of healthy s on l administration of placebo (composition without compound A) (a); and on per-oral administration of placebo (composition without compound A) (b), 1mg/kg bid of compound A 17078093_1 (GHMatters) P101481.NZ [Annotation] jennyp None set by jennyp ation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp ation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp composition (c) and 2.5 mg/kg bid of compound A composition ((1) to animal model of colitis (DSS induced s in female BALB/c mice) upon 10 days of treatment.

Fig 5: Colon images of animal model of colitis (TNBS induced colitis in male BALB/c mice) on per-oral administration of ceutical composition comprising compound A at the dose of 1mg/kg bid'or Vehicle (Placebo-composition t compound A). - Fig 6: Mean percent improvement in DAI scores (Fig 6a) and macroscopy score (Fig 6b) of animal model of colitis (TNBS induced colitis in male BALB/c mice) on eritoneal and per-oral administration of pharmaceutical composition comprising compound A.

Fig 7: mRNA expression of IL-10, TNF-a, and INF-y in colon tissue of animal model of colitis (DSS induced colitis in female BALB/c mice) on 2.5mg/kg bid g/day) al administration of pharmaceutical ition comprising compound A or placebo (composition without compound A).

Fig 8: Crypt architecture and inflammatory cells on per-oral administration of placebo (composition without compound A) (a), 1mg/kg bid of compound A (b) to animal model of colitis (TNBS induced colitis male BALB/c mice) upon 6 days of treatment Detailed description of the invention: The following paragraphs detail various embodiments of the invention. For the avoidance of doubt, it is specifically intended that any particular feature(s) described individually in any one of these paragraphs (or part thereof) may be combined with one or more other features described in one or more of the remaining paragraphs (or part thereof). In other words, it is explicitly intended‘that the features described below dually in each paragraph (or part thereof) represent important aspects of the invention that maybe taken in isolation and also combined with other important aspects'of the invention described elsewhere within this specification as a whole, and including the examples and figures. The skilled person will [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp appreciate that the ion extends to such combinations of es and that these have not been recited in detail here in the sts of brevity.

The term “[(2-hydroxyoxo-6,7,8,9-tetrahydro-4H,5H-lO-thia-l,4a-diaza-benzo[a]azulene carbonyl)-amino]-acetic acid” or “compound A” as used herein is defined to mean [(2- hydroxyoxo-6,7,8,9-tetrahydro-4H,5H-10—thia-l,4a-diaza—benzo[a]azulenecarbonyl)- amino]—acetic acid as its base or pharmaceutically acceptable salts thereof or rph thereof or ester thereof. Any of these said forms can be lline or amorphous.

The term “Inflammatory bowel disease or IBD” as used herein is the condition which results because of inflammation of epithelium, particularly gastrointestinal epithelium. It includes conditions like ulcerative colitis, Crohn’s disease, collagenous s, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet‘s syndrome, and indeterminate s.

The term “effective amount”, “therapeutically effective amount” or effective dose” as used herein meaning the amount or dose of the compound A, that is sufficient to initiate therapeutic response in a mammal including human being.

The term “mammal” means a human- being or an animal including monkey, es, dogs, cats, horses or cows etc, preferably human being.

The term “dosage range” or “range” as described herein means the theraPeutically effective range of the compound A, which is le for administration to a mammal.

The term “low dose” as described herein means the total daily dose for a mammal, preferably human being, which is less than 100 mg per day, preferably less than 60mg per day, more preferably less than 50mg per day. Dose conversion for different types of mammals, including human being, can be done as per FDA guidelines ble on www.fda.gov. For human being, 70 kg has been considered as average weight.

[Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp ation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 1010 The term antially insoluble” as used herein means at least 75% of the composition is not soluble at pH below 5. Preferably at least 80% ofthe composition is not soluble at pH below 5.

More preferably at least 90% of the composition is not soluble at pH below 5.

The term “systemic exposure” as used herein means availability of the compound A including its active metabolites, if any, in the systemic circulation of a mammal: The term “local exposure” as used herein means the availability of the compound A including its active metabolites, if any, at the affected site or diseased area or in its vicinity.

The term “upper GI tract” means the part of GI tract comprising esophagus, stomach and duodenum. The term “lower GI tract” means ing part of GI tract.

The use of the terms “a” and “an” and “the” and similar nces in the context of describing the invention (especially in the context of the ing ) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by Present ion provides that a low dose pharmaceutical composition of compound A is effective in a dosage range of 2.5mg to 60mg per day, for the treatment of ”IBD. Present invention also provides a method of treating IBD in a mammal by administering a low dose pharmaceutical composition comprising therapeutically effective amount of compound A.

Compound A, which is a_HIF hydroxylase tor disclosed in WO201145811 for the treatment of anemia, ia or tissue damage caused by ischemic disorders when administered parenterally. Patent application discloses that compound 10 was found effective for chronic kidney disorder,(CKD), at the dose of 20mg/kg bid (approx 450 mg/day human dose), when administered thrOugh intraperitoneal route to diseased animals.

[Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 1111 Inventors of present ion have found that the low dosage range of 2.5 mg/day to 60 mg/day as an optimal dosage range which achieve desired therapeutic effect, when given through non-parenteral route, preferably through oral route, for the treatment of IBD.

IBD which can be treated by administering therapeutically effective amount of nd A according to present invention includes conditions like ulcerative s, Crohn’s disease, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's syndrome, . and indeterminate colitis. Any other disease which includes inflammation of epithelium is also within the scope of present invention.

None of the prior art tried per-oral administration and mainly discloses parenteral route as preferred route of administration for HIF hydroxylase inhibitors for treatment of IBD. It is general art known to person having Ordinary skilled that effective amount of any compound would mainly be dependent on the circulating concentration/plasma levels of the active moiety.

,Inventors of present invention have found dose 2.5 mg to 60 mg of compound A as effective dose for the treatment of IBD, when administered orally. singly, when ceutical composition comprising compound A was given orally as well as intra peritoneal (IP), it was found that oral dose was more efficacious and provided more therapeutic benefit than intra' peritoneal dose, with very less ic exposure (circulating concentration/plasma levels) and high targettissue exposure as compared to IP for treatment of IBD. Hence present ion provides better therapeutic efficacy with improved safety margin.

Thus, one embodiment of the present invention provides a low dose pharmaceutical composition comprising nd A' and at least one pharmaceutically acceptable carrier ed from diluent, , disintegrant, pH adjusting agent and lubricant, n said composition is ive in the dosage range of 2.5 mg to 60 mg per day for the treatment of [BD in a mammal.

Another embodiment of the present invention provides a low dose pharmaceutical composition sinf compound A and at least one ceutically acceptable carrier selected from [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp ed set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 1212 diluent, binder, disintegrant, pH adjusting agent and lubricant, wherein said composition is ive for the treatment of [BD in_a mammal when stered in a dosage range of 2.5 mg to 60 mg per day.

Another embodiment of the present invention provides a low dose pharmaceutical composition comprising compound A in an amount of 2.5 mg to 60 mg and at least one ceutically acceptable carrier selected from diluent, binder, disintegrant, pH adjusting agent and lubricant, n the said composition is effective in ng inflammatory bowel disease in a mammal.

Another embodiment of the present ion provides a method of treating IBD in a mammal by administering a low dose pharmaceutical composition sing therapeutically effective amount of compound A.' A preferred embodiment of present invention provides a method of treating IBD in a mammal by administering a low dose pharmaceutical composition comprising therapeutically effective amount of' compound A, wherein said composition is effective in the dosage range of 2.5 mg to 60 mg per day.

Another preferred embodiment of present ion provides a method of ng IBD in a mammal by administering a low dose pharmaceutical composition comprising compound A in a range of 2.5mg to 60mg. Said low dose pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier selected from diluent, binder, disintegrant, pH adjusting agent and lubricant.

Another embodiment of the t invention provides use of compound A for preparation of a low dose pharmaceutical composition for the treatment of IBD in a mammal.

A preferred embodiment of present invention provides use of compound A for preparation of a low dose pharmaceutical composition for the treatment of IBD in a mammal wherein said composition is effective in the dosage range of 2.5 mg to 60 mg per day.

[Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp ation] jennyp Unmarked set by jennyp 1313 Another preferred embodiment of present invention provides use of compound A in a range of 2.5mg to 60mg for preparation of a low‘dose pharmaceutical composition for the treatment of IBD in a mammal. Said low dose pharmaceutical composition further comprises at least one ceutically acceptable carrier ed from diluent, binder, disintegrant, pH adjusting agent and lubricant.

Preferably, t ion ”provides a low dose pharmaceutical composition for non- parenteral administration, such as oral administration of compound A.

Therefore, another embodiment of the present invention provides a low dose pharmaceutical composition for oral administration comprising compound A and at least one pharmaceutically acceptable carrier selected from diluent, binder, egrant, pH adjusting agent and lubricant, wherein said composition is effective in the dosage range of 2.5 mg to 60 mg per day for the treatment of IBD in a mammal.

A preferred embodiment of the present invention provides a low dose pharmaceutical composition for oral administration comprising compound A in an amount of 2.5 mg to 60 mg and at least one pharmaceutically able carrier selected from diluent, binder, disintegrant, pI-l adjusting agent and lubricant, wherein the said composition is effective in treating inflammatory bowel disease in a .

Effective dose of compound A for the treatment of IBD according to present invention ranges from 2.5 mg to 60 mg per day or its equivalent dose when present as salt or ester. Preferably, dose of the compound A is 4.5 mg to 50 mg, most preferably dose of nd A is 4.5 mg to 40 mg; EffectiVe dose as defind herein also include the obvious modification of dosage range on either side, which does not render'additional significant eutic benefit and / or reduce the adverse effects over specified dosage range.

[Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp ation] jennyp ed set by jennyp 1414 It has been observed that above ned dosage range provide optimally efficacious dosage range wherein maximally effective concentration at desired site is achieved with minimal systemic exposure and thus provide therapeutic s in treatment of IBD with improved safety margin. Therefore, present invention provides a low dose pharmaceutical composition where least systemic exposure and more local . exposure of compound A is provided upon administration.

Thus, another embodiment of the present invention provides a low dose pharmaceutical composition comprising nd A and at least one pharmaceutically acceptable carrier ed from diluent, binder, disintegrant, pH adjusting agent and lubricant for the treatment of [BD in a mammal, wherein the said composition es at least 50% local exposure of the total amount of said compound stered. r embodiment of the present invention provides a method of treating IBD in a mammal by administering a low dose pharmaceutical composition comprising therapeutically effective amount of compound A wherein the said composition provides at least 50% local exposure of the total amount of said compound administered. Said low dose pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier selected from diluent, binder, disintegrant, pH adjusting agent‘and lubricant.

Anether embodiment of the present invention provides use of compound A for preparation of a low dose pharmaceutical composition for the treatment of IBD in a mammal, n the said composition provides at least 50% local exposure of the total amount of said compound administered. Said low dose pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier selected from diluent, binder, disintegrant, pH adjusting agent and lubricant.

The pharmaceutical composition according to present invention es at least 50% local least _ exposure of the amount of compound A stered. ably, composition provides at [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp ionNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 1515 60% local exposure of the amount of compound A administered. Most preferably, composition es at least 70% local exposure of the amount of compound administered.

Preferably, low dose pharmaceutical composition according to present invention is non- parenteral ition; most ably low dose pharmaceutical composition is oral composition.

One embodiment of the present invention provides a low dose pharmaceutical composition for immediate e comprising compound A and at least 'one pharmaceutically acceptable r selected from t, binder, disintegrant, pH adjusting agent and lubricant, for the treatment of IBD in a mammal.

Preferably, present invention provides a low dose non-parenteral composition, particularly oral composition for immediate release of nd A for the treatment of IBD in a mammal comprising compound A and at least one pharmaceutically acceptable carrier selected from diluent, binder, disintegrant, pH adjusting agent and lubricant, wherein nd A is effective in the dosage range of 2.5 mg to 60 mg.

It was noticed that the composition according to present invention shoWs synergistic effect when pharmaceutically acceptable carrier selected from diluent, binder, disintegrant, pH adjusting agent and ant are used in formulating compound A.

Another embodiment of the present invention provides a low dose pharmaceutical composition comprising compound A in an amount of 2.5 mg to 60 mg and at least one pharmaceutically acceptable carrier selected from diluent, binder, disintegrant, pH adjusting agent and ant wherein diluent, when used, is present in the amount of 10-98% w/w of the total composition; binder, when used, is present in the amount of 1-20% w/w of the total composition; disintegrant, when used, is t in the amount of 0.1—15% w/w of the total composition; pH adjusting agent, when used, is present in the amount of 0.01-20% w/w of the total ition and lubricant, when used is present in the amount of 0.02—5% w/w of the total composition. ation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp ionNone set by jennyp [Annotation] jennyp ed set by jennyp 1616 Additionally, pharmaceutical acceptable carrier according to present invention can be any excipients required for formulating composition according to present invention such as glidant, crystal growth inhibitor, surfactant, film g polymer, plasticizer, buffering agent or complexing agent. Any of such excipients may be used alone or in combination of same/other excipients. Any of the said carriers can be used in the quantity which is suitable for formulating the composition according to present invention.

A diluent is selected from powdered cellulose, rystalline cellulose, silicified rystalline ose, starch, dibasic calcium phosphate, dibasic sodium phosphate, tribasic sodium phosphate; sugars such as dextrose,,lactose or sucrose; sugar alcohols such as mannitol, sorbitol, xylitol or erythritol; or mixtures thereof. The diluent may be present in an amount ranging from 10-98% w/w of the total composition.

A binder is selected from starches such as maize starch, corn starch, pregelatinised starch; cellulose tives such as cellulose powder, microcrystalline cellulose, hydroxypropyl methylcellulose, ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, polyethylene glycol, hydroxyethyl cellulose; polyvinyl. pyrrolidone, gelatin, polymethacrylates, sodium alginate, gums, synthetic resins or mixtures thereof. The binder may be present in an amount ranging from 1-20% w/w of the total composition.

A crystal growth inhibitor‘is the agent which substantially inhibits the precipitation of compound A. Crystal growth tor may be meglumine, po'lyoxyethylene-polyoxypropylene block copolymers and the like. The crystal growth inhibitor may be present in an amount ranging from‘0.0l-20% w/w of the tOtal composition.

. A ant or t is selected from talc, metallic stearate, such as magnesium te, ‘ calcium stearate, zinc te; colloidal silicon dioxide, finely divided silicon dioxide, stearic acid, hydrogenated vegetable oil, yl palmitostearate, glyceryl monostearate, glyceryl [Annotation] jennyp None set by jennyp ation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 1717 behenate, sodium stearyl fumarate, magnesium trisilicate; or mixtures thereof. The lubricant or glidant may be present in an amount ranging from % w/w of the total composition.

A surfactant is selected from one or more non-ionic or ionic (i. e., cationic, anionic and Zwitterionic) surfactants suitable for use in pharmaceutical compositions. Suitable surfactants include mono fatty acid esters of polyoxyethylene sorbitan such as those sold under the brand name Tween®; sodium lauryl e, polyoxyethylene castor oil derivatives such as those sold under the brand name hor®, polyethoxylated fatty acids and their derivatives, prOpylene glycol fatty acid esters, sterol and sterol derivatives; an fatty acid esters and their derivatives, sugar esters, polyoxyethylene-p0lyoxypropylene block copolymers such‘as those sold under the brand name Poloxamer® , soy lecithin, or mixtures f. The surfactant may be‘present in an amount ranging from 0.01-20% w/w of the total composition.

A pH adjusting agent according to present invention is any agent which maintains the pH of the composition, ably above 3. A pH adjusting agent is either organic or inorganic agent.

The non limiting examples of such agent are meglumine, NaOH, KOH, NH3, ammonium hydroxide, carbonates such as sodium carbonate or potassium carbonate and the like..pH . adjusting agent also includes buffering agent. The pH ing agent may be present in an amount g from 0.01-20% w/w of the total composition.

A ing agent is selected from phosphates such as sodium phosphate, sodium dihydrogen . phosphate, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate, disodium hydrogen phosphate dodecahydrate, potassium phosphate, ium dihydrogen phosphate and dipotassium hydrogen phosphate; boric acid and borates such as, sodium borate and potassium borate; citric acid and citrates such as sodium citrate and disodium citrate; acetates such as sodium acetateand ium acetate; carbonates such as sodium carbonate and, sodium hydrogen carbonate and the like. The buffering agent may be present in an amount ranging from 0.01-20% w/w of the total composition.

[Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 1818 A disintegrant is sodium starch glycolate, crospovidone, croscarmellose sodium and the like.

The disintegrant may be present in an amount ranging from 01-15% w/w of the total composition.

A complexing agent is selected from cyclodextrin class of les, such as cyclodextrins containing from ‘six to twelve glucose units, especially, alpha- cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, 'or their derivatives, such as hydroxypropyl beta cyclodextrins, or mixtures thereof. The complexing agent may be present in an amount ranging from % w/w of the total composition.

Film forming polymers is ed from hydroxpropyl cellulose, methylcellulose, ethylcellulose, polyethylene glycol, ypropyl cellulose, povidOne, polydextrose, lactose, maltodextrin, acrylic polymer, or mixtures f. Film forming polymers may be present in an amount ranging from 01-10% w/w of the total composition.

The immediate release composition according to present invention can be uncoated or coated with a suitable coating agent.

An alternative embodiment of the present invention provides a low dose pharmaceutical composition comprising compound A for the treatment of IBD in a mammal, wherein composition provides controlled release of the compound A.

The controlled release composition according to present invention is d release composition, extended release composition, delayed release composition or composition for site specific delivery of compound A. A controlled release composition is prepared by using suitable amount of at least one pharmaceutical acceptable carrier which es controlled release of nd A. Preferably, a controlled release composition according to t invention is delayed release composition.

[Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp ation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 1919 Thus, another embodiment of the present invention provides a low dose pharmaceutical composition for oral administration comprising compound A and at least one pharmaceutically acceptable carrier selected from diluent, , disintegrant, pH adjusting agent and lubricant for the treatment of IBD in a mammal, wherein the said composition is substantially insoluble at pH below 5.

Another embodiment of the present invention provides a low dose pharmaceutical composition for oral administration comprising nd A and at least one pharmaceutically acceptable carrier selected from diluent, binder, disintegrant, pH adjusting agent and lubricant for the treatment of IBD in a mammal, n the said composition releases at least 50% of the said compound in vivo at pH above 5.

A pharmaceutical composition according to present invention es at least 50% of the said compound in vivo at pH above 5, preferably ceutical composition according to t invention releases at least 60% of the said Compound in vivo at pH above 5, most preferably pharmaceutical composition according to present invention releases at least 70% of the said compound in viva at pH above 5. r embodiment of the present invention provides a low dose pharmaceutical composition for oral administration comprising compound A and at least one pharmaceutically acceptable carrier for the treatment of IBD, wherein the said carrier is suitable for controlled release of the said compound. Preferably, said carrier is le for delayed release of the nd A.

A red embodiment of the present invention provides a low dose pharmaceutical composition for oral administration comprising compound A and at least one pharmaceutically acceptable carrier suitable for delayed release for the treatment of IBD in a mammal, wherein the said carrier is present in an amount of at least 1% w/w of tal ition. Preferably said carrier is present in amount of at least 2 % w/w of the total composition, more preferably ation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp ation] jennyp Unmarked set by jennyp ation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp ation] jennyp Unmarked set by jennyp 2020 said carrier is present in an amount of at least 10% w/w of the total composition, most preferably said carrier is present in an amount ofat least 20% w/w of the total composition.

Present invention provides a low dose pharmaceutical composition for delayed release comprising compound A and at least one pharrnaceutically acceptable carrier suitable for d release for the treatment of [BB in a mammal.

“Pharmaceutically acceptable carrier suitable for controlled release” includes one or more excipients which facilitates controlled release of the compound A. Such excipients include polymeric or non-polymeric compoUnds, preferably polymeric nds. Such polymeric compounds may further are water soluble polymers or water insoluble polymers.

“Water-soluble polymer” used in the t composition is polymer which is soluble or ble in water. Preferably, it dissolves and/or swells in water at room temperatures. Non ng es include the cellulose ethers, hydrocolloid (gum), polyvinyl alcohol and nyl , 'pyrrolidone.

The cellulose ethers include carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, ' hydroxypropyl cellulose, hydroxybutyl cellulose, hydroxyethylmethyl cellulose, hydroxyethylethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylethyl cellulose, hydroxybutylmethyl cellulose, hydroxybutylethyl cellulose, carboxymethyllcellulose and salts thereof. hydrocolloid (gum) includes guar gum, alginic acid and its pharmaceutically acceptable - salts e.g., sodium alginate and xanthan gum “Water insoluble polymer” used in present invention are the polymer which are substantially insoluble in water and include cellulose ethers such as ethylcellulose, butylcellulose, cellulose acetate, ose acetate butyrate, ne vinyl acetate copolymer, polyvidone acetate, polyvinyl acetate, polyvinyl butyrate, polymethacrylates, including ethylacrylate/methylmethacrylate copolymers, and ammonia methacrylate mers, thus including commercially available dispersions such as Kollicoat® MAE30DP, it® RL3OD, Eudragit® NE30D, and Eudragit® RS30D.

[Annotation] jennyp None set by jennyp ation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 2121 A pharmaceutical acceptable carrier suitable for delayed release is any carrier which facilitate the release of the compound A in lower GI tract or which substantially s the release of compound A in upper GI tract.

“Pharmaceutically acceptable carrier suitable for delayed e” includes one or more excipients which facilitates delayed release of the compound A. Such excipients include polymeric or non-polymeric nds, preferably polymeric compounds. Such excipients may be pH dependent or pH independent; preferably pH dependent nds are used. These excipients include cellulose derivatives, acrylic acid derivatives, maleic acid copolymer, polyvinyl derivatives and the like.

Cellulose derivatives include hydroxypropylmethylcellulose acetate succinate, hydroxypropylmethylcellulose ate, hydroxymethylethylcellulose phthalate, cellulose acetate ate, cellulose acetate succinate, cellulose acetate maleate, cellulose acetate trimelliate, cellulose benzoate phthalate, cellulose propionate ate, methylcellulose phthalate, carboxymethylethylcellulose, ethylhydroxyethylcellulose phthalate and the like.

Acrylic acid derivatives include styrene, acrylic acid copolymer, methyl acrylate, acrylic acid mer, methyl acrylate, methacrylic acid copolymer, butyl acrylate e acrylic acid copolymer, methacrylic acid, methyl methacrylate copolymer (e.g. Trade-names: it® L 100 and Eudragit® S), methacrylic acid polymers, ethyl acrylate copolymer (e.g. Trade-name: Eudragit L® 100-55), methyl acrylate, methacrylic acid octyl acrylate copolymer and the like.

Maleic acid copolymer based polymers include vinylacetate maleic acid anhydride copolymer, styrene maleic acid anhydride copolymer, styrene maleic acid monoester copolymer, vinylmethylether maleic . acid anhydride mer, ethylene maleic acid anhydride copolymer, vinylbutylether maleic acid anhydride copolymer, acrylonitrile, methyl acrylate maleic acid anhydride mer, butyl acrylate styrene maleic acid. anhydride copolymer and the like.

, Polyvinyl derivative based rs includes polyvinyl alcohol phthalate, polyvinylacetal phthalate, nyl butylate phthalate, nylacetoacetal phthalate and the like.

[Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp ation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 2222 The pharmaceutical compositions may additionally contain excipients such as colorants selected from known F.D. & C. and D. & C. dyes, titanium e and the like.

A, preferred route. of administration of ceutical composition according to present invention to the mammals is non parenteral route, most preferably oral route but alternatively intrarectal route can also be used for administration of the composition.

Pharmaceutical composition of the present invention which is free from any pharmaceutically acceptable carrier also forms part of this invention.

The pharmaceutical composition as described herein may be obtained in any suitable form such as tablet, capsule, powder, oral on, suspension, rectal gel,_rectal foam, rectal enema or suppository and the like.

Further embodiment of the t invention provides a process of preparation of a low dose pharmaceutical composition of present ion.

Composition according to present invention can be prepared by any method known in the art such as by 'mixing the compound A‘with ceutically acceptable rs. Alternatively wet granulation or dry granulation techniques may be employed for the preparation of ition according to present invention.

Alternatively, composition of present invention can be prepared as matrix based formulation in which compound A is dispersed within a matrix. Alternatively, compound A containing particles may be coated by suitable pharmaceutically acceptable carriers. Types of particles include granules, s, minitablets, microparticles or beads.

Rectal composition may be prepared by a skilled person as known in the art.

Another embodiment of t invention provides method of treating [BD in a mammal by administering pharmaceutical ition of present invention.

[Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 2323 Another embodiment of present invention provides use of compound A for the preparation of low dose pharmaceutical composition according to present invention.

Low dose pharmaceutical composition according to present invention can beadministered once a day or twice a day to achieve therapeutic effect. Preferably, low dose pharmaceutical composition according to t invention is administered twice a day to achieve therapeutic effect.

The low dose pharmaceutical compositiOn comprising compound A according to present invention may further comprise r agent suitable for treatment of IBD including immune modifier or anti-inflammatory drugs. atively, pharmaceutical composition can be administered along with other agents suitable for treatment of IBD including immune r or anti-inflammatory drugs.

The ion according to present ion may be illustrated by the following examples which are not to be ued as limiting the scope of the invention: Example 1a: Component Amount of excipients in mg Compositions i ii iii Compound A 2.5 10 40 Mannitol 20.63 - , 22.5 Meglumine - 0.63 2.50 10.00 Microcrystalline 55.63 42.50 10.00 cellulose Magnesium stearate 1.0 1.0 1.0 ‘ Polysorbate - - 2.0 HPMC 1.0 1.0 1.0 Sodium Starch 4.63 . 6.50 14.0 [Annotation] jennyp None set by jennyp [Annotation] jennyp ionNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 2424 Glycolate Hydroxypropyl 1.0 1.0 1.0 cellulose Talc . 0.40 0.40 0.40 Titanium Dioxide 1.00 1.00 1.00 Microcrystalline cellulose (MCC), mannitol, meglumine and sodium starch glycolate (SSG) were sifted with compound A and dry mixed in Rapid Mixer Granulator (RMG) for 10 minutes followed by granulation with water in‘RMG. After drying the granules at 60-70°C in fluid bed dryer (FBD), granules were sized using oscillating ator followed by blending in conta blender for about 5 minutes. These granules were blended with mixture of MCC, mannitol and SSG for compositions i and ii. Obtained blend was lubricated with Mg stearate using conta blender for about 5. minutes followed by ssion in rotatory compression machine to form tablet. Seal coating of HPMC, HPC, talc and titanium dioxide solution in water was applied over prepared tablet. Similar procedure was followed for composition iii.

Example 1b: Component ' Amount of excipients in mg Compositions ~ i ii iii nd A 2.5 10 40 Microcrystalline 55.63 42.50 10.00 cellulose ol 20.63 22.50 10.00 Meglumine - 0.63 2.50 10.00 Sodium Starch 6.50 14.00 4'63 Glycolate Magnesium te 1.00 1.00 1.00 HPMC 1.00 1.00 1.00 Hydroxy Propyl 1.00 1.00 ~ 1.00 [Annotation] jennyp None set by jennyp ation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 2525 ose (HPC) Talc 1.63 1.63 1.63 Titanium Dioxide 1.00 1.00 1.00 Methacrylic acid- 2.46 2.46 2.46 Ethylacrylate copolymer (1:1) " “ ___.___..__ '""‘ dispersion 30% ene Glycol 0.74 0.74 0.74 Microcrystalline cellulose (MCC), mannitol, meglumine and sodium starch ate (SSG) were sifted with compound A and dry mixed in Rapid Mixer Granulator (RMG) for 10 minutes followed by granulation with water in RMG. After drying the granules at 60-70°C in fluid bed dryer (FBD), granules were sized using ating granulator followed by blending in coma blender for about 5 minutes. These granules were blended with mixture of MCC, mannitol and SSG for compositions i and ii, while with SSG only for composition iii. Obtained blend was lubricated with Mg stearate using conta blender for about 5 minutes ed by compression in rotatory compression machine to form tablet. Seal coating of HPMC, HPC, talc and titanium dioxide solution in water was applied over prepared tablet, which was further coated with enteric coating comprising Methacrylic acid-Ethylacrylate mer (1:1) sion 30%, propylene glycol and talc. Prepared tablets were stored in HDPE bottle.

In-vitro dissolution profile of the tablets prepared according to Example 1b was tested in 900 mL of Fasted State Simulated Intestinal Fluid having pH 5, i.e SOmM Sodium acetate/FaSSIF at 37°C. To mimic in-vivo state, dissolution was checked in Simulated Intestinal Fluid having pH 5 with enzymes such as FaSSIF. FaSSIF or Fasted state simulated intestinalfluid is prepared as per USP. In-vitro dissolution at lower pH such as 0.1 N HCl was also assessed. Results are . summarized as Table 1: [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp ionNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 2626 Table 1 Composit Dissolution ion in 0.1 N Dissolution in 50mM Sodium acetate/FaSSIF. pH 5.0, 75 HCI, 75 RPM, 900 mL, 37°C at time interval in min RPM, 900 mL, 37°C Time ”II-“fl“ Ex lb (i) Sodium 16.1 78.8 78.8 101.2 acetate Ex1b(i -----n Ex1b(iiv Sodium 18.4 59.0 75.7 93.4 99.1 acetate ' ‘ ----nmm Ex 1b (iii) Sodium “III-II7.7 43.9 76.1 98.6 98.0 Ex 1b (iii) mammal Above given results in table 1 show that composition according to present invention is substantially insoluble at acidic pH, specifically at pH below 5 and composition would es Compound A in lower GI tract, i.e. above pH 5.

Example 2. Intrarectal composition Component Amount of excipients in % nd A ' 2.5-60mg mer 407 10-30% Polyethylene Glycol 15-30% Potassium hydrogen Phosphate 0-5% [Annotation] jennyp None set by jennyp [Annotation] jennyp ionNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 2727 Example 3: Preparation of TNBS induced colitis model Animal with TNBS (2, 4, 6 trinitrobenzene sulfonic acid) induced colitis is an established model for IBD. Male BALB/c mice were kept for 16 hours fasting and were administered 0.1 ml TNBS solution (containing 1.5 mg of TNBS in 50% ethanol) rectally 4 cm inside the anus with the help of flexible polyethylene catheter under isoflurane esia. Immediately after TNBS administration, mice were held vertically in a head-down position for an additional 45- 60 sec to ensure retention and distribution of TNBS solution within the colon of animal.

(Fiorucci et al, Immunity, 2002; Vol. 17; 769—780) Example 4: cokinetic evaluation of compound A in TNBS induced colitis model (Intraperitoneal (IP) administration and Peroral (PO) administration) Pharmaceutical composition comprising compound A was stered to mice with TNBS induced s (as ped according to example 3) at the dose of 1 mg/kg of compound A twice a day, at 10 ml/kg dosing volume by intraperitoneal route and peroral route. Dosing was initiated a day prior to ion (day -1) of TNBS colitis. Blood samples of treated animals were ted before administration of fifth dose (0 min) and at various time points after fifth dose administration. Blood samples were centrifuged within one hour of collection at 4000 rpm for 10 min at 4°C and plasma was separated. These plasma samples were analyzed for compound A levels using LC—MS/MS method. Pharmacokinetic parameters of the compound A were calculated by non-compartmental is method using Phoenix WinNolin version 6.2. Pharmacokinetic results are summarized in table 2.

Table 2 Parameter Unit Per Oral Intraperitoneal Cmax ng/mL 13.20 399.24 AUC0_12 hr*ng/mL 63.08 255.91 [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp ation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 2828 Example 5: Effectiveness of pharmaceutical composition of nd A in TNBS induced s model (Peroral (PO) administration) Pharmaceutical composition comprising compound A was administered orally to mice with TNBS induced colitis (as deVeloped according to example 3) at the dose of 1 mg/kg of compound A twice a day, at 10 ml/kg dosing volume. Dosing was initiated a day prior to ion (day -l) of TNBS colitis. Treatment related attenuation in body weight loss, improved disease activity index (DAI, a composite score ranging from 0 to maximum 12 considering the loss in body weight, fecal conSistency and presence or absence of occult blood in feces), healthier colon (macroscopy score) and rate of survival were established in the 6 days treated animals (from day -1 to day 4) to check efficacy of compound A. Body s‘were monitored daily, DAI was ed on day 2 and day 4 whereas colonic damage scopy score and histopathological score) was scored based on extent cf colonic damage.

Histopathological evaluation of colonic damage was performed in formalin fixed colon tissue sections, stained with hematoxylin and eosin, at 10X magnification using Leica® DM2500 microscope, after study completion. Placebo group was given r composition without compound A. Results are given in Fig la-ld._ (*p<0.05 vs placebo/vehicle {composition without compound A}), Fig 5 and Fig 8.

Observation: Data of table 2 clearly shows that at equal dose of compound A, oral administration was found to provide significantly lower systemic concentration compared to IP administration but still oral dose was found efficacious (Figla-ld). Results of Fig 1, Fig 5 and Fig 8 show that ition of compound A provided therapeutic benefit by improving the disease ty index, attenuating the extent of colon , reducing rate of weight loss and improving survival rate in diseased animals.

Example 6: Comparative efficacy - of pharmaceutical composition of nd A administered through oral route and intraperitoneal route Similar to procedures given above in Example 5, pharmaceutical composition of compound A was administered, twice a day, in mice with TNBS induced colitis through intraperitoneal route [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 2929 and oral route. Treated animals were analyzed for improved disease activity index and healthier colon (macroscopy score). Results are summarized in Fig 6a and 6b.

Observation: Data given in Fig 6a and 6b show that oral administration of compound A trated better therapeutic efficacy as compared to IP administration even at lower doses.

Example 7: Effectiveness of pharmaceutical composition of nd A in DSS (Dextran sodium e) induced colitis model % DSS 000-50000) dissolved in drinking water was provided to female BALB/c mice for 11 days (Gunther et al, The Journal ofPharmacology and Experimental Therapeutics, 1999; Vol. 292, N0. 1; 22-30) followed by DSS free period till day 14. Pharmaceutical composition sing compound A was administered to these animals from day 5 to day 14 at the dose of l and 2.5 mg/kg of nd A twice a day, at 10ml/kg dosing volume by oral route (PO). Placebo group was given similar composition without compound A. Placebo (composition without nd A) was also administered to healthy animals, which were given only drinking water and represented as placebo (water). Treatment related attenuation in body weight loss, improved disease activity index (DAI, a composite score ranging from 0 to maximum 12 considering the loss in body weight, fecal consistency and presence or absence of occult blood in feces) and colon histopathology (Score) were established in 10 days treated animals (from day 5 to 'day 14) to check efficacy of compound A. Body weights were red daily, DAI was captured every alternate day whereas histopathological evaluation of colonic damage was performed in'formalin fixed colon tissue after study completion, similar to example 5. Results are summarized in Fig 2a-2c. (*p<0.05 vs o (5% D88» and Fig 4.

Observation: These results from Fig 2 and 4 show that composition of nd A provided therapeutic benefit by reducing the rate of weight loss, improving the disease activity index, ating extent of colon damage and preserving tissue architecture in diseased animals.

[Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp ed set by jennyp 3030 Example 8: Tissue distribution of compound A in DSS induced colitis model.

% DSS (MW-3600060000) dissolved in drinking water was provided to female BALB/c mice for 7 days. Pharmaceutical composition sing cOmpound A was administered twice a day to these animals from day 5 orally at the dose of 1mg/kg of compound A and intraperitoneally at the dose of.025 mg/kg of compound A, at 10 ml/kg dosing volume. 6 hours after first dose on 7th day administration of composition, s were sacrificed; whole body perfusion through heart using cold phosphate buffered saline was med followed by collection of tissues of interest, which were further rinsed with PBS buffer for removal of any residual GI content. Plasma and tissue levels of compound A were estimated using LC-MS/MS method. The results are ized in Fig 3.

Observation: Data clearly shows that at four fold differences in dose administered through oral and intraperitoneal route, mean exposure of compound A in tissue of interest via oral stration was found to be 6 to d higher than achieved with peritoneal administration.

Example 9: Excretion of compound A in feces .

% DSS (MW50000) dissolved in- drinking water was provided to female BALB/c mice throughout the study duration of 9 days. Single administration of pharmaceutical composition comprising compound A at the dose of 1 mg/kg of compound A, at 10m1/kg .dosing volume was performed orally on day 5. Feces were collected. for 96 hours post administration of the composition. Feces collected was ed for compound A levels using LC-MS/MS method. Results are summarized in Table 3.

Table 3 Animals Total. Amount of % Recovery No amount of compound A' in feces compound administered A in feces (pg) [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp [Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 3131 (I19) 1 12.71 25.00 50.88 2 12.08 19.00 63.60 3 15.34 24.00 63.92 4 13.71 22.00 62.35 .5 12.63 24.00 52.66 Mean %Recovery in feces 58.68 so 6.37 Observation: Above results from table 3 show that more than 50% of the compound A is recovered from feces, when an oral composition according to'present invention was administered to diseased animals.

Example 10: HSP70 sion in colon Effect of oral stration of pharmaceutical composition of compound A on sion of HSP70 was checked in DSS model of colitis.

% D88 (MW50000) dissolved in drinking water was provided to female BALB/c mice for 5 days. Single dose of compound A was stered to these animals on day 5 Composition sing compound A was administered at the dose of 2.5 mg/kg, at 10 ml/kg dosing volume by oral route (PO). Placebo group was given r composition without compound A. Six hours after administration, animals were sacrificed and colon were processed for whole tissue extract preparation. Proteins were separated on SDS-PAGE followed by immunoblotting, employing HSP7O antibody. The results are presented in Fig. 2d.

Observation: It was obServed that oral administration of pharmaceutical composition of Compound A resulted in a nced induction of HSP70 in colon as compared with the respective placebo (composition without compound A).

[Annotation] jennyp None set by jennyp [Annotation] jennyp MigrationNone set by jennyp [Annotation] jennyp ed set by jennyp [Annotation] jennyp None set by jennyp ation] jennyp MigrationNone set by jennyp [Annotation] jennyp Unmarked set by jennyp 3232 Example 11: Inflammatory marker expression in colon Effect of oral administration of pharmaceutical ition of compound A on expression of interleukin (IL)-10, TNF-a, and interferon (INF)-y mRNA was assessed in DSS model of colitis.

Pharmaceutical composition sing compound A was administered 2.5 mg/kg twice a day from day 5 to day 14 to DSS induced s mice (as developed according to example 7) by oral route (PO). Animals were sacrificed on day 14 and colon were processed for whole tissue extract preparation. Expression of interleukin O, TNF-a, and interferon (lNF)-y mRNA along with expression of 18S rRNA was monitored employing gene specific primer and probes (Applied Biosystems, Foster City, CA, USA) by quantitative real~time polymerase chain reaction on ABI 7900 HT (Applied Biosystems, Foster City, CA, USA). mRNA expression was normalized relative to the expression of 188 rRNA. Placebo group was given r ition without compound A. The results, as given in Fig 7, were expressed as fold induction relative to placebo (composition without compound A).

Observation: It was observed that oral administration of pharmaceutical composition of Compound A resulted in reduction of expression of pro-inflammatory cytokines, TNFa and INFy and elevation in the expression of the anti-inflammatory cytokine IL—lO.

Claims (4)

We Claim:

1. Use of [(2-hydroxyoxo-6,7,8,9-tetrahydro-4H,5Hthia-1,4a-diazabenzo [a]azulenecarbonyl)-amino]-acetic acid or its pharmaceutically acceptable salt for preparation of a pharmaceutical composition for the ent of inflammatory bowel disease in a human being wherein said nd is for non-parenteral administration.

2. Use according to claim 1, wherein [(2-hydroxyoxo-6,7,8,9-tetrahydro-4H,5Hthia- 1,4a-diaza-benzo[a]azulenecarbonyl)-amino]-acetic acid or its pharmaceutically acceptable salt is in the range of 2.5 mg to 60 mg.

3. Use ing to claim 1, wherein [(2-hydroxyoxo-6,7,8,9-tetrahydro-4H,5Hthia- 1,4a-diaza-benzo[a]azulenecarbonyl)-amino]-acetic acid or its pharmaceutically acceptable salt is effective in the range of 2.5 mg to 60 mg per day.

4. Use according to claim 1 wherein said ceutical composition further comprises at least one pharmaceutically acceptable carrier selected from diluent, binder, disintegrant, pH adjusting agent and lubricant.