NOVEL SALTS OF lH-l-BENZAZEPINE-l-ACETIC ACID, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITION

FIELD OF THE INVENTION

The present invention relates to novel water soluble amino acid salts of 1H-1- benzazepine-1 -acetic acid, process of preparation of such novel salts and methods of using them. The present invention also provides pharmaceutical compositions comprising the same and method of using such novel compositions for the prophylaxis, amelioration and/or treatment of cardiovascular and other associated disorders which comprises the administration of an effective amount of such composition to a subject in need thereof. Preferably the 1 H- 1-benzazepine-l -acetic acid is an endothelin conversion enzyme (ECE) inhibitor and/or neutral endopeptidase (NEP) inhibitor having the general formula (Formula- 1) as stated below:

Formula-1

Wherein:

R] is a selected from the group consisting Of (Ci-C ₆ ) alkoxy, (Ci-C ₆ ) alkyl, phenyl-(C _r

C ₆ )-alkyl and phenyloxy-(Ci-C ₆ )-alkyl wherein the phenyl group may be substituted with (Ci-Ce)alkyl, (Ci-C ₆ ) alkoxy or halogen, and/or naphtyl-(Ci-C ₆ )-alkyl,

R ₂ and R ₃ are both independently hydrogen or halogen,

R ₄ is a biolabile ester forming group, acid, amide or a substituted aryl or alkyl amide,

M is a hydrogen or a metal ion, preferably a bivalent metal ion, and n is 1, 2 or 3.

More preferably, the IH- 1-benzazepine-l -acetic acid used in the present invention is (3S, 2"R)-3-{ l-[2 ^λ -(ethoxycarbonyl)-4"-phenylbutyl]cyclopentane-l-carbonylamin o}-2, 3, 4, 5-tetrahydro-2-oxo- I H- 1-benzazepine-l -acetic acid having Formula-II.

FormuIa-II

The novel salts of the present invention possess high solubility in water and are amenable into any pharmaceutically acceptable dosage form such as solid oral, drinkable or injectable compositions.

BACKGROUND OF THE INVENTION

Cardiovascular disorders such as coronary artery disease (CAD) and congestive heart failure (CHF) are the leading cause of mortality in the developed world. The patients with CAD have several concomitant conditions, including hypertension, diabetes and hyperlipidemia. Cardiovascular disorder such as Angina pectoris is a severe constricting pain in the chest, often radiating from the precordium to the left shoulder and down the left arm. Often angina pectoris is due to ischaemia of the heart and is usually caused by coronary disease. The symptomatic management of angina pectoris involves the use of a number of drugs, frequently as a combination of two or more of the following classes: beta-blockers, nitrates and calcium channel blockers. Most, if not all, of these patients require therapy with a lipid-lowering agent as well. Hypertension is a major risk factor for cardiovascular disease. Both systolic and diastolic blood pressure has been shown to be related to cardiovascular disease leading to morbidity and mortality. Hypertension frequently coexists with hyperlipidemia and both are considered to be major risk factors for developing cardiac disease. It would therefore be advantageous for patients to have a single therapy which treats both of these conditions. Atherosclerosis is a condition characterized by irregularly distributed lipid deposits in the intima of arteries, including coronary, carotid and peripheral arteries. Many individuals are at an elevated risk of suffering serious to life-threatening cardiovascular events, such as myocardial infarction (heart attack), cardiac arrest, congestive heart failure, stroke, peripheral vascular disease and/or claudication. The risk factors are numerous and widespread throughout the world population. They include cigarette smoking, diabetes, hypercholesterolemia (high serum

cholesterol), hypertension, angina, systemic lupus erythematosus, prior heart attacks or strokes, hemodialysis, hyperhomocysteine levels, obesity, sedentary lifestyle, receiving an organ transplant, atherosclerosis, and others.

Endothelin (ET) is a peptide which is composed of 21 amino acids that is synthesized and released by the vascular endothelium. Endothelin is produced by enzymatic cleavage of a Trp-Val bond in the precursor peptide big endothelin (Big ET). This cleavage is caused by an endothelin converting enzyme (ECE). Endothelin exists as three isoforms, ET-I , ET-2 and ET-3 (hereinafter, unless otherwise stated, 'endothelin' shall mean any or all of the isoforms of endothelin). Endothelin acts on two pharmacologically distinct subtypes of receptors, termed ET.sub.A and ET.sub.B that are expressed on a wide variety of vascular and non-vascular target cells, eliciting, for example, contraction and proliferation of vascular smooth muscle cells and release of nitric oxide from endothelial cells. Endothelin is associated with smooth muscle contraction which is involved in the pathogenesis of, inter alia, cardiovascular, cerebrovascular, respiratory and renal pathophysiology. An agent which suppresses endothelin production, such as an endothelin converting enzyme (ECE) inhibitor, or which inhibits the binding of endothelin to an endothelin receptor, such as an endothelin receptor antagonist, antagonizes various physiological effects of endothelin and produces beneficial effects in a variety of therapeutic areas. Endothelin receptor antagonists and ECE inhibitors are therefore useful in treating a variety of diseases affected by endothelin. A non-exhaustive list of such diseases includes chronic heart failure, myocardial infarction, cardiogenic shock, systemic and pulmonary hypertension, ischemia-repurfusion injury, atherosclerosis, coronary and systemic vasospastic disorders, cerebral vasospasm, and subarachnoid hemorrhage and the like.

Neutral Endopeptidase (NEP) is a cell surface enzyme that cleaves and inactivates neuropeptides. It is well established that the natriuretic peptide system is intimately involved in the control and regulation of blood pressure and plasma volume in the body. Natriuretic peptides can also have antimitogenic actions within the cardiovascular tree. Natriuretic peptides are degraded by the metallo peptidase neutral endopeptidase enzyme (NEP). Thus NEP inhibitors primarily increase the levels of vasodilatory peptides including atrial natriuretic peptide and also increase the half-life of other vasodilator peptides, thereby decreasing vascular tone and lowering blood

pressure. The compound (3S, 2 ^> R)-3-{ l -[T-(ethoxycarbonyl)-4 ^λ -phenylbutyl] cyclopentane-l-carbonylamino}^, 3, 4, S-tetrahydro^-oxo-lH-l-benzazepine-l-acetic acid (SLV-306) also known as Daglutril and represented by Formula-II is an orally active inhibitor of neutral endopeptidase (NEP) and endothelin conversion enzyme (ECE). It is a Benzazepine-N-acetic acid derivative which contains an oxo group in alpha position to the nitrogen atom and are substituted in position 3 by a 1- (carboxyalkyl) cyclopentyl-carbonylamino radical, and have NEP-inhibitory effects on the heart, as described in Waldeck et al., U.S. Pat. No. 5,677,297. The benzazepine-N- acetic acid compounds used in the present invention are known from EP 0733642, EP 0830863, WO 00/48601 and WO 01/03699, and can be produced by the methods described in said U.S. Pat. No. 5,677,297.

The compounds of Formula-I and Formula-II, preferably Formula-II as stated herein, are poorly bio-available drugs due to their poor solubility in the gastric fluid. Even when they are used in the form of salts and when they are dissolved in a buffer, it precipitates in the gastric fluid. The precipitate formed is very difficult to solubilize again, leading to a low overall bioavailability. EP0733642 is related to these compounds and their physiologically acceptable salts as such and to the use of the compound in heart insufficiency. EP0830863, WO00/48601 and WO01/03699 are related to the use of the above compounds in the improvement of gastrointestinal blood flow, in the treatment of hypertension and in the treatment and prophylaxis of cardiac damages induced by adriamycin and comparable anti-cancer drugs, respectively. PCT publication no. WO2004062692 discloses a formulation with enhanced bioavailability consisting of a thermodynamically stable liquid or semisolid solution of the poorly water-soluble biologically active substance having the Formula-II. Compounds of Formula-I, particularly the compound having the Formula-II are obtained as sticky paste-like substance which are very difficult to handle and also are insoluble in aqueous medium. The monovalent metal ionic salts such as sodium and potassium salt of the compound of Formula-II, although water soluble, are obtained as solid foam which are difficult to formulate and lack reproducibility relating to its release in vivo and in turn a variable and low bioavailability. Other bivalent metal ions salts such as magnesium, calcium and zinc salts of the compound of Formula-II, although can be obtained in the powder form, have a comparatively low aqueous solubility in comparison to the monovalent metal ionic salts

and thus have poor bioavailability. Hence there still exists a need to develop novel salts particularly of the compounds of Formula-II which are easy to handle, have an appreciable aqueous solubility and bioavailability, and could be formulated into any pharmaceutically acceptable dosage form such as solid oral, drinkable or injectable compositions without difficulty. The present invention provides novel salts of 1H-1- benzazepine-1 -acetic acid which overcomes limitations of prior art.

SUMMARY OF THE INVENTION

It is an objective of the present invention to provide novel amino acid salts of 1H-1- benzazepine-1 -acetic acid.

It is another objective of the present invention to provide novel amino acid salts of the compound of Formula-1 as stated herein.

It is also another objective of the present invention to provide novel amino acid salts of the compound of Formula-II as stated herein.

It is another objective of the present invention to provide novel salts of 1H-1- benzazepine-1 -acetic acid, the compound of Formula-I or the compound of Formula-II as stated herein with an organic basic amino acid or a neutral amino acid ester.

It is another objective of present invention to provide novel salts of lH-1-benzazepine- 1 -acetic acid, the compound of Formula-I or the compound of Formula-II as stated herein with an organic basic amino acid preferably selected from group comprising arginine, lysine and ornithine.

It is another objective of present invention to provide novel salts of lH-1-benzazepine- 1 -acetic acid, the compound of Formula-I or the compound of Formula-II as stated herein with a neutral amino acid ester preferably selected from group comprising glycine, leucine and serine.

It is another objective of the present invention to provide process of preparation of such novel amino acid salts of I H-I -benzazepine-1 -acetic acid, the compound of Formula-I or the compound of Formula-II as stated herein.

It is another objective of the present invention to provide pharmaceutical compositions comprising such novel amino acid salts of 1 H- 1-benzazepine-l -acetic acid, the compound of Formula-I or the compound of Formula-II as stated herein.

It is a further objective of the present invention to provide a method of using such novel salts or pharmaceutical compositions comprising such novel salts which comprises administering to a subject in need thereof an effective amount of such novel salt or composition thereof.

The novel amino acid salts of IH- 1-benzazepine-l -acetic acid, the compound of Formula-I or the compound of Formula-II are obtained in highly pure solid form; possess appreciable aqueous solubility and good bioavailability; and can be formulated into any pharmaceutically acceptable dosage form such as solid oral, drinkable or injectable compositions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes novel amino acid salts of IH- 1-benzazepine-l -acetic acid preferably novel amino acid salts of the compound of Formula-I more preferably novel amino acid salts of the compound of Formula-II as stated herein.

In an embodiment of the present invention, the novel salts of IH- 1-benzazepine-l - acetic acid or the compound of Formula-I or the compound of Formula-II as stated herein are formed with an amino acid selected from but not limited to a group comprising organic basic amino acid or a neutral amino acid ester.

In an embodiment, the organic basic amino acid is selected from but not limited to a group comprising racemic arginine, lysine and/or ornithine or their stereoselective enantiomers such as L and/or R-Lysine, L and/or R-arginine and L and/or R-ornithine and the like, or mixtures thereof. Preferably the organic basic amino acid is L-lysine, L- arginine, L-ornithine or the like, or mixtures thereof.

In an embodiment of the present invention, the novel amino acid salts of IH- 1 - benzazepine-1 -acetic acid or the compound of Formula-I or the compound of Formula- II as stated herein are highly pure and can be formulated into pharmaceutical compositions without substantial loss of chiral and/or chemical purity.

In a further embodiment of the present invention, the novel amino acid salts of 1H-1- benzazepine-1 -acetic acid or the compound of Formula-I or the compound of Formula- II as stated herein have solid state properties which permit easy handling and allow preparation of a pharmaceutical composition with pharmaceutically acceptable excipients and preferably by using conventional methods and equipments.

In an embodiment, the novel salts of the compound having Formula-I is formed with L- arginine having the formula-HI and L-lysine having the formula-IV. The novel salts with L-arginirie and L-lysine have the form u Ia-V and formula-VI respectively as stated below.

Formula - III

Formula - IV

Formula - V

Formula - VI

In another embodiment of the present invention is provided the novel salts of the compound of Formula-II as stated herein with an organic basic amino acid such as arginine, lysine, or ornithine in a racemic form represented by Formula- VII, Formula- VIII and Formula-IX respectively as stated below:

Formula - VII

Formula - IX

In another embodiment of the present invention is provided novel salts of 1H-1-

benzazepine-1 -acetic acid, the compound of Formula-I or the compound of Formula-II as stated herein with a neutral amino acid ester preferably selected from the group comprising glycine, leucine and serine represented by Formula-X, Formula-XI and Formula-Xll respectively as stated below:

Formula - X

Formula - XI - OH

Formula - XII

Where R is selected from the group of lower alkyl containing Ci-C ₆ and lower aromatic group.

The novel salts of the present invention possess improved stability in aqueous solution in comparison to the alkali metal salts, for example, sodium and potassium salts. In another embodiment, the present invention provide process of preparation of such novel salts of IH-I -benzazepine-1 -acetic acid, the compound of Formula-I or the compound of Formula-II as stated herein. Preferably the salts of the present invention can be

prepared in a manner known per se by reacting (3S, 2"R)-3-{l-[2"-(ethoxycarbonyl)-4'- phenylbutyl]cyclopentane-l-carbonylamino}-2, 3, 4, 5-tetrahydro-2-oxo-lH-l- benzazepine-1 -acetic acid with a substantially equimolar amount of a compound of amino acid in an aqueous solution or in a suitable protic or aprotic organic solvent such as an aliphatic or cycloaliphatic ether, for example dioxane or a lower alcohol, such as methanol or ethanol at a temperature of from 0 ⁰ C to the boiling temperature of the solvent. The salts are recovered by standard methods such as filtration if they are insoluble in the original medium or, if they are soluble in that medium, the salt is precipitated by evaporation of the solvent or by addition of a non-solvent for the salt.

In another embodiment of the present invention is provided a process for preparation of pharmaceutical compositions comprising such novel amino acid salts(s) particularly the salts of 1 H- 1-benzazepine-l -acetic acid, the compound of Formula-I or the compound of Formula-II as stated herein along with one or more pharmaceutically acceptable excipient(s). In an embodiment, the process comprises treating the novel amino acid salt(s) of the present invention with one or more pharmaceutically acceptable excipient(s). In an embodiment, the novel amino acid salts of the present invention may be administered as an aqueous solution for parenteral use such as for an LV. (intravenous) injection or an oily suspension for intra-muscular injection or can be formulated into a drinkable aqueous or hydro-alcoholic solution.

In an embodiment, the novel amino acid salts of the present invention can be formulated into suitable pharmaceutically acceptable dosage forms, such as solid, semisolid or liquid dosage forms, for e.g. tablets, mini-tablets such as such as layered or monolithic mini-tablets, capsules, suppositories, patches, topical gels or sprays, or the like. The solid dosage forms can be produced by known methods such as direct compression, granulation, compaction, extrusion, molding, or the like or mixtures thereof. In a preferred embodiment of the present invention, the oral composition is prepared by direct compression or compaction granulation. The composition prepared by granulation technique is either by aqueous or non-aqueous technique or melt granulation technique. The non-aqueous ^' solvent used is selected from a group comprising dehydrated alcohol, isopropyl alcohol, methylene chloride or acetone. In an embodiment, the compositions of the present invention are in the form of granules,

beads or pellets that may be further compacted, compressed, or moulded, or made into capsules. In a preferred embodiment, the compositions of the present invention are formulated as solid oral dosage form compositions possessing good bioavailability comprising one or more alkaline substance and/or surfactants. In another preferred embodiment, the solid oral dosage form composition preferably as capsule is formulated as SMEDDS. Liquid dosage forms for oral administration can include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise excipients such as wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents. Pharmaceutical compositions suitable for buccal or sublingual administration include lozenges comprising a compound of the present invention in a flavored base, usually sucrose, and acacia or tragacanth, and pastilles comprising the drugs in an inert base such as gelatin and glycerin or sucrose and acacia. The compositions may be coated with a functional coating. By the term 'functional coating' it is herein implied that the coating composition comprises a part of the active agent(s) and/or the composition comprises excipients which aid in controlling the rate of release of the active agent(s) and/or the composition comprises additionally another active agent which is different from the active agent present in the core composition. The composition may be formulated as layered tablets comprising at least two layers wherein the same active agent is present in all the layers exhibiting different release profiles or one or more additional active agent(s) is present in the layers exhibiting different release profiles. The coating composition employed in the present invention may be an aqueous, non-aqueous or a hydro-alcoholic system. The solvents used to prepare a non-aqueous coating composition is selected from but not limited to a group comprising dehydrated alcohol, isopropyl alcohol, methylene chloride, acetone or any other solvent known to the art for such use, or mixtures thereof.

The pharmaceutical excipients that can be used for preparation of such compositions are selected from but not limited to diluents, disintegrants, binders, fillers, bulking agents, anti-adherents, anti-oxidants, buffering agents, colorants, flavoring agents, coating agents, plasticizers, organic solvents, stabilizers, preservatives, lubricants, glidants, chelating agents, and the like known to the art used either alone or in

combination thereof. In an embodiment, the filler(s) used in the present invention is selected from but not limited to a group comprising lactose, mannitol, sorbitol, starch, microcrystalline cellulose, xylitol, fructose, sucrose, dextrose, dicalcium phosphate, calcium sulphate and the like or mixtures thereof. The disintegrants used in the present invention include but not limited to starch or its derivatives, partially pregelatinized maize starch, croscarmellose sodium, sodium starch glycollate, and the like used either alone or in combination thereof. The lubricants used in the present invention include but not limited to talc, magnesium stearate, calcium stearate, stearic acid, hydrogenated vegetable oil and the like used either alone or in combination thereof. The tablets may be coated with a material such as glyceryl monostearate, glyceryl distearate, cellulose acetate phthalate, hydroxypropylcellulose phthalate, polyvinylacetate phthalate, methylmethacrylate polymer, a polymer mixture such as Eudragit®, cellulosic polymers, carbomers, zein, wax or similar material, or any other dissolvable coat, to delay absorption in the gastrointestinal tract. Capsules for oral use include hard gelatin capsules in which the components are mixed with a solid diluent or soft gelatin capsules wherein the fill material exist as aqueous or hydroalcoholic or oily systems.

The pharmaceutical compositions according to the invention, which comprises the novel salts are intended for enteral, such as oral, and also rectal and parenteral administration to a warm blooded mammal, especially human and comprise the novel salts as the pharmacological active ingredient on its own or together with a pharmaceutically acceptably carrier. Additionally other pharmaceutically active agent(s) may be used in combination with the novel salts of the present invention. The amount of the novel salts of the present invention to be incorporated into the pharmaceutical composition of the present invention can vary over a wide range depending on known factors such as, for example, the disorder to be treated, the severity of the disorder, the patient's body weight, the dosage form, the chosen route of administration and the number of administration per day. However, selection of optimum amount is simple and routine for a person skilled in the art. Typically the amount of such novel salts would be equivalent to about 10 mg to about 1000 mg of the free acid form of the 1 H- 1-benzazepine-l -acetic acid or compound of formula-I or compound of formula-II. The pharmaceutical compositions of the present invention can be manufactured by techniques that are well-known to the art and comprise operations

such as mixing, granulating, compressing, dissolving, sterilizing and the like or combinations thereof. According to the present invention, the pharmaceutical composition can be prepared by mixing the novel salts of the present invention with other drug(s) or with pharmaceutically acceptable carrier(s). In accordance with the present invention it has been found that acidic hydrogen atom of the carboxylic group of 1 H- 1 -benzazepine-l -acetic acid can be interacted with amino group of particularly basic amino acids, preferably corresponding to formula-Ill and formula-IV, and the product and/or ionic complex or said salt thus obtained is stable in solidified form. The salts are water soluble even at the pH range between about 4 to about 8 and hence are well tolerated at the gastrointestinal level. Furthermore, they do not have any unpleasant taste and/or odor.

In an embodiment, the present invention provides method of using such novel amino acid salts or pharmaceutical compositions comprising such novel amino acid salts which comprises administering to a subject in need thereof an effective amount of such novel salt or composition thereof. In a further embodiment of the present invention, the novels salts may be used for treatment or prophylaxis of cardiovascular disorders, for the promotion of blood circulation in the mesenteric vascular system in humans and is suitable for the treatment and/or prophylaxis of gastrointestinal disorders associated with reduced gastrointestinal blood circulation due to a wide range of causes, in particular for the treatment and/or prophylaxis of abdominal angina.

The present invention further relates to a pharmaceutical composition for the prevention or treatment of cardiovascular diseases/disorders such as hypertension, heart failure, angina, gastrointestinal disorders, and the like, comprising the novel salts, their solvate, hydrate or a mixture thereof with pharmaceutically acceptable carrier(s)/excipient(s). The novel salts of the present invention can be formulated in combination with other drugs which preferably includes antihypertensives, cardiovascular drugs, diuretics, antihyperlipidemics, antidiabetics, and/or other drugs which are known to the art for the treatment of hypertension or other cardiovascular disorders or other associated disorders such as coronary artery disease, congestive heart failure, angina, atherosclerosis, hyperlipidemia, diabetes, myocardial infarction, cardiogenic shock, systemic and pulmonary hypertension, ischemia-reperfusion injury,

coronary and systemic vasospastic disorders, cerebral vasospasm, and subarachnoid hemorrhage, hyperglycemia, metabolic disorders, renal disorders, neurodegenerative disorders or the like.

In an embodiment, the present invention provides use of the novel salts for the preparation of a pharmaceutical composition for the treatment/prophylaxis of cardiovascular diseases/disorders and/or for the promotion of blood circulation in the mesenteric vascular system and/or for the treatment and/or prophylaxis of gastrointestinal disorders which are associated with reduced gastrointestinal blood circulation and/or for the treatment/prophylaxis of abdominal angina.

According to the present invention, the mixture of novel salts of the present invention can be in the form of an associate or a complex or inclusion compounds with the pharmaceutically acceptable excipient(s). For example, a mixture of the novel salts and povidone can be in the form of an associate of the novel salts with povidone, a mixture of novels salts and phospholipid can be in the form of a complex, and a mixture of the salts with cyclodextrin can be in the form of an inclusion of the novel salts in cyclodextrin.

Yet another object of the present invention is to provide a pharmaceutically acceptable salt of compound, corresponding to formula-I and formula-II, which is suitable as medicament for larger mammals, especially humans, for in the treatment of heart failure and for promoting diuresis/natriuresis, especially in patients suffering from heart failure, for the improvement of gastrointestinal blood flow, in the treatment of hypertension and in the treatment and prophylaxis of cardiac damage induced by adriamycin and comparable anti-cancer drugs. For this purpose the said salts of the present invention can be used in medicinal forms which can be administered parenterally, especially through the IV route, orally or as suppository.

Yet another object of the present invention is to provide a pharmaceutical composition comprising the said physiologically and pharmaceutically acceptable water soluble novel salts of compound, corresponding to formula-I or formula-II, which is suitable and useful for the prevention or treatment of heart failure and for promoting

diuresis/natriuresis, especially in the patients suffering from heart failure, for the improvement of gastrointestinal blood flow, in the treatment of hypertension and in the treatment of prophylaxis of cardiac damage induced by adriamycin and comparable anti-cancer drugs. 5

Pharmacokinetic Study

A comparative pharmacokinetic study has been carried out to study the comparative pharmacokinetic profile of different salts of (3S, 2 ^* R)-3-{l-|T-(Ethoxycarbonyl)-4"- phenylbutyl]cyclopentane-l -carbonylamino}-2, 3, 4, 5-tetrahydro-2-oxo-lH-l- 10 benzazepine-1 -acetic acid (Formula-II) in rats.

Female Wistar rats (160-180 g) were selected for the study comprising 5 animals in each group for a period of 6 hours with sampling intervals of 0, 15, 30, 60, 90, 120, 180, 240 and 360 minutes. 17.63 mg of calcium salt of (3S, 2 ^λ R)-3-{l-[T-

15 (Ethoxycarbonyl)-4'-phenylbutyl]cyclopentane-l-carbonylamino }-2, 3, 4, 5-tetrahydro- 2-oxo-lH-l-benzazepine-l-acetic acid and 22.50 mg of L-arginine salt of (3S, 2 ^λ R)-3- {l-[2 ^λ -(Ethoxycarbonyl)-4"-phenylbutyl]cyclopentane-l-carbonylamin o}-2, 3, 4, 5- tetrahydro-2-oxo- IH-I -benzazepine-1 -acetic acid were used with a dose of 100 mg/kg body weight through oral route. Heparin (0.2 mg/ml of blood) was used as an

20. anticoagulant. Blood samples were collected from rats at 0 hour. One capsule of calcium salt or two capsules of the L-arginine salt of (3S, 2"R)-3-{l-[2 ^λ - (Ethoxycarbonyl)-4'-phenylbutyl]cyclopentane-l-carbonylamino }-2, 3, 4, 5-tetrahydro- 2-oxo- IH-I -benzazepine-1 -acetic acid were administered orally to each rat. One capsule of calcium salt was equivalent to two capsules of L-arginine salt of (3S, 2"R)-3-

25 {^^'-(EthoxycarbonyO^'-phenylbutylJcyclopentane-l-carbonylam ino}^, 3, 4, 5- tetrahydro-2-oxo- IH-I -benzazepine-1 -acetic acid. The blood samples (120μl) were collected at regular intervals as mentioned above, and the whole blood were analyzed for (3S, 2'R)-3-{ l-[2 ^" -(Ethoxycarbonyl)-4'-phenylbutyl]cyclopentane-l- carbonylamino}-2, 3, 4, 5 -tetrahydro-2-oxo- IH-I -benzazepine-1 -acetic acid and its 0 metabolite by LC-MS/MS. Out of 5 animals in each of the two groups used for the study, 2 animals from each group showed erroneous/abnormal profiles and hence were not considered in arriving at the result. The concentration (ng/ml) of the calcium salt of compound of Formula-II and L-arginine salt of compound of Formula-II in blood were

calculated for remaining 3 animals in each group and the average values were noted. The result is presented in table- 1.

Table-1: Comparative pharmacokinetic profile Time Cone, of calcium salt of Cone, of L-arginine salt of

(in minutes) compound of FormuIa-II (ng/ml) compound of FormuIa-II (ng/ml)

0 0 ^' 0

15 9046.67 10920.00

30 10163.33 13010.00 60 7896.67 35450.00

90 6306.67 15023.33

120 4626.67 7216.67

180 5323.33 4520.00

240 3780.00 1292.33 360 0 0

The result shows that the arginine salt of the compound of Formula-II showed a significantly higher drug concentration in the blood thus indicating a comparatively higher bioavailability compared to the calcium salt of the compound of Formula-II. The peak blood concentration (C _max ) of the arginine salt is significantly higher (35450.00 ng/ml) in comparison to the peak blood concentration (C _maλ ) of the calcium salt (10163.33 ng/ml).

The following examples are only intended to further illustrate the invention, in more details, and therefore these examples are not deemed to restrict the scope of the invention in any way.

Example 1 : Preparation of the L-arginine salt of (3S, 2'R)-3-{ l -[2'-(Ethoxycarbonyl)- 4"-phenylbutyl]cyclopentane-l-carbonylamino}-2, 3, 4, 5-tetrahydro-2-oxo-lH-l- benzazepine-1 -acetic acid

To a solution of 3.26 g (18.7 mmol) of L-arginine in 150 ml of DM water, 10.0 g (18.7 mmol) of (3S, 2 ^λ R)-3-{ l-[2 ^{^} -(Ethoxycarbonyl)-4'-phenylbutyl]cyclopentane-l- carbonylamino}-2, 3, 4, 5-tetrahydro-2-oxo-l H-l -benzazepine-l-acetic acid was added with stirring and the resulting mixture was stirred for about 3 to 4 hours. The solution was filtered through a glassfibre filter and the aqueous filtrate was lyophilized. The

resulting product was isolated. The L-arginine salt of (3S, 2'R)-3-{l-|_2'- (Ethoxycarbonyl)-4'-phenylbutyl]cyclopentane-l-carbonylamino }-2, 3, 4, 5-tetrahydro- 2-oxo-l H- 1-benzazepine-l -acetic acid was obtained as a white solid which melts at about 135-14O ⁰ C. The morphological purity of the salt formed was confirmed by powder X-ray diffractogram of the solid (Figure 1). The powder X-ray diffractogram of the solid (Figure 1) having a broad peak centered at about 20 degrees 2 theta (20), showed that the resulting substance is in amorphous form.

Example 2: Preparation of the L-lysine salt of (3S, 2 ^v R)-3-{l-[T-(Ethoxycarbonyl)-4 ^λ - phenylbutyl] cyclopentane-l-carbonylamino}-2, 3, 4, 5-tetrahydro-2-oxo-lH-l- benzazepine-1 -acetic acid

To a solution of 2.74 g (18.7 mmol) of L-lysine in 150 ml of DM water, 10.0 g (18.7 mmol) of (3S, 2'R)-3-{l-[2'-(Ethoxycarbonyl)-4'-phenylbutyl]cyclopentane-l - carbonylamino}-2, 3, 4, 5-tetrahydro-2-oxo- IH- 1-benzazepine-l -acetic acid was added with stirring and the resulting mixture was stirred for about 2 to 3 hours. The solution was filtered through a glassfibre filter and the aqueous filtrate was lyophilized. The resulting product was isolated. The L-lysine salt of (3S, 2ε)-3-{l-[2"- (Ethoxycarbonyl)-4'-phenylbutyl] cyclopentane-l-carbonylamino}^, 3, 4, 5- tetrahydro-2-oxo- IH- 1-benzazepine-l -acetic acid was obtained as a white solid. The morphological purity of the salt formed was confirmed by powder X-ray diffractogram of the solid (Figure 2). The powder X-ray diffractogram of the solid (Figure 2) having a broad peak centered at about 20 degrees 2 theta (2θ), showed that resulting substance is in amorphous form.

In the examples stated below describing composition comprising the novel salts of the present invention, the quantity stated for unit dosage form is the equivalent dose of the compound of Formula-II. The tablet/capsule compositions disclosed herein may optionally be coated by film forming polymer or enteric polymer known to the art.

Example-3: Tablet

S. No. Ingredient Quantity/tablet (mg)

1. Compound of Formula- V 300.0

2. Sodium carbonate 270.0

3. Sodium bicarbonate 30.0

4. Microcrystalline cellulose 310.0

5. . Croscarmellose sodium 20.0

6. lsopropyl alcohol Lost in processing 7. Hydrogenated castor oil 7.5

8. Purified talc 7.5

9. Colloidal silicon dioxide 7.5 Procedure: i) Compound of Formula-V, Sodium carbonate, Sodium bicarbonate, Microcrystalline cellulose and Croscarmellose sodium were sifted through #40 sieve and were mixed together. ii) The blend of step (i) was granulated by using lsopropyl alcohol, iii) The wet mass of step (ii) was sifted through #24 sieve and granules obtained were dried. iv) Hydrogenated castor oil, Purified talc and Colloidal silicon dioxide were sifted through #40 sieve and were mixed together, v) Granules of step (iii) were mixed with the mixture of step (iv). vi) The material of step (v) was compressed into tablets by using a tablet compression machine.

Example-4: Capsule

S. No. Ingredient Quantity/capsjile (mg)

1. Compound of Formula-X 150.00

2. Magnesium carbonate 150.00 3. Dicalcium phosphate 131.25

4. Sodium starch glycolate 30.00

5. Magnesium stearate 10.00 Procedure: i) Compound of Formula-X, Magnesium carbonate, Dicalcium phosphate, Sodium starch glycolate and Magnesium stearate were sifted through #40 sieve and were mixed together, ii) The blend of step (i) was compacted and the compacts were passed through #30 sieve.

iii) The granules of step (ii) were lubricated with #60 sieve passed Magnesium stearate. iv) The material of step (iii) was filled into hard gelatin capsule.

Exaraple-5: Mucoadhesive Tablet

S. No. Ingredient Quantity/tablet (mg)

1. Compound of Formula-VII 600.00

2. Hydroxypropyl beta cyclodextrin 12.00

3. Sodium carboxymethylcellulose 15.00 4. Povidone 5.00

5. Maltodextrin 20.00

6. Saccharin sodium 1.00

7. Sodium stearyl fumarate 0.50

8. Lake Erythrosine 0.08 9. Chocolate flavor 0.70

Procedure: i) Compound of Formula-VII was complexed with Hydroxypropyl beta cyclodextrin. ii) The complex of step (i) was mixed with Sodium carboxymethylcellulose,

Povidone, Maltodextrin, Saccharin sodium, Lake Erythrosine and Chocolate flavor. iii) The mixture in step (ii) was mixed with part of Sodium stearyl fumarate and was roller compacted to obtain compacts, iv) The compacts in step (iii) were broken into granules which were passed through

#20 sieve and were retained on #30 sieve. v) The granules in step (iv) were mixed with the remaining part of Sodium stearyl fumarate. vi) The granules in step (v) were compressed to obtain tablets.

Example-6: Injection S. No. Ingredient Quantity/ 100 ml

1. Polyethylene glycol (PEG-400) 30.0 ml

2. Propylene glycol 20.0 ml

3. Glycine Buffer pH 1 1 .3 35.0 ml

4. Compound of Formula- Vl 2.5 g

5. Sodium hydroxide (NaOH) solution 4.0% w/v 10.0 ml Procedure: i) Specified quantity (30.0 ml) of PEG-400 was taken into a vessel. ii) Propylene glycol (20.0 ml) was added to step (i) with continuous stirring using mechanical stirrer, iii) About 30.0 ml of the Glycine Buffer pH 1 1.3 was added to the step (ii) with continuous stirring to form a homogeneous mixture. iv) Weighed amount of Compound of Formula- VI (2.5 g) was passed through #60 sieve and was added to the step (iii) with continuous stirring. v) Specified quantity (10.0 ml) of NaOH 4.0% solution was added to the step (iv) with continuous stirring to form a homogeneous solution. vi) The solution of step (v) was mixed for about 30 minutes by continuous stirring, vii) Remaining quantity of Glycine Buffer pH 11.3 was added to make up volume to 100 ml. viii) The solution of step (vii) was mixed for about 10 minutes by continuous stirring. ix) Final pH was adjusted to 10.0 by adding NaOH 4.0% w/v solution. x) The solution of step (ix) was mixed for about 10 minutes by continuous stirring.

Example-7: Modified release Tablet

S. I So. Ingredient Quantity (mg)

1. Compound of Formula-XI 300.00

2. Valsartan 40.00

3. Lactose 120.00

4. Sodium starch glycollate 30.00

5. Hydroxypropyl methylcellulose 67.00

6. Isopropyl alcohol Lost in processing

7. Croscarmellose sodium 3.00

8. Colloidal silicon dioxide 2.00

9. Magnesium stearate 2.00

Procedure: i) Compound of Formula-XI, Valsartan, Lactose and Sodium starch glycollate were sifted through #30 sieve and were mixed together.

ii) Hydroxypropyl methylcellulose was dissolved in Isopropyl alcohol to obtain a homogeneous dispersion. iii) The blend of step (i) was granulated with the dispersion of step (ii). iv) The granules of step (iii) were dried and were sifted through #24 sieve. v) Croscarmellose sodium. Colloidal silicon dioxide and Magnesium stearate were sifted through #40 sieve, vi) The material of step (v) was mixed with the material of step (iv) and compressed into tablets.

Example-8: Oral Liquid

S. No. Ingredient Quantity (mg/100 ml)

1. Compound of Formula-VIII 150.0

2. Citric acid monohydrate 1.5

3. Hydroxyethyl cellulose 20.0 4. Sorbitol solution (70% w/v) 50.0

5. Saccharin sodium . 0.5

6. Sodium benzoate 1.0

7. Raspberry flavor q.s.

8. Purified water q.s. to 100 ml Procedure: i) Compound of Formula-VIII and Hydroxyethyl cellulose were sifted through #40 sieve and were blended together, ii) Citric acid monohydrate, Saccharin sodium, Sodium benzoate, Raspberry flavor and Sorbitol solution were dispersed together in Purified water. iii) The material of step (i) was added with continuous stirring to the material of step

(ii) and a homogeneous suspension was obtained.

Example-9: Oral Suspension

S. No. Ingredient Quantity (mg/100 ml) 1. Compound of formula-IX 40.0

2. Citric acid monohydrate 1.5

3. Hydroxyethyl cellulose 20.0

4. Sorbitol solution (70% w/v) 50.0

5. Saccharin sodium U.5

6. Sodium benzoate 1.0

7. Raspberry flavor q.s.

8. Purified water q.s. to 100 ml Procedure: i) Compound of formula-IX and Hydroxyethyl cellulose were sifted through #40 sieve and were blended together, ii) Citric acid monohydrate, Saccharin sodium, Sodium benzoate, Raspberry flavor and Sorbitol solution were dispersed together in Purified water. iii) The material of step (i) was added with continuous stirring to φe material of step

(ii) and a homogeneous suspension was obtained.