TECHNICAL FIELD OF INVENTION:

The present invention relates to synergistic pharmaceutical co-crystals/salts comprising Mesalamine as active ingredient in combination with amino acids, nutraceuticals or flavonoids.

BACKGROUND AND PRIOR ART:

Mesalazine also known as Mesalamine or 5-aminosalicylic acid (5-ASA), is an antiinflammatory drug used to treat inflammation of the digestive tract, ulcerative colitis and mild-to-moderate Crohn's disease. Mesalazine is a bowel-specific aminosalicylate drug that acts locally in the gut and has its predominant actions there, thereby having few systemic side effects. The common side effects are: (a) Diarrhea, (b) Nausea, (c) Cramping & (d) Flatulence.

Mesalamine occurs as white to slightly grey crystals or a light tan to pink crystalline powder and has a solubility of 1 mg/mL in water at 20°C and also is slightly soluble in alcohol. The drug has pKa of 3, 6, and 13.9.

There is ample literature which discloses pharmaceutical compositions containing 5- amino salicylic acid as active ingredient to treat ulcerative colitis. However these compositions are pH dependent and require high dosage. The half-life of 5-ASA is 7 to 9 hours, and consequently 4 to 5 days are required to achieve steady-state plasma concentrations. Further, mesalamine is unstable in the presence of water and light, since oxidation and, to a lesser extent, light-catalyzed degradation of the drug occurs thus affecting its physico chemical properties.

Preparation of pharmaceutical co-crystals offers an attractive option because they offer multiple opportunities to modify the chemical and/or physical properties of an active pharmaceutical ingredient (API) without compromising the structural integrity of the active pharmaceutical ingredient (API).

Pharmaceutical co crystals are defined as hydrogen bonded complexes between an active pharmaceutical ingredient (API) and a co-former (CCF, benign partner molecule), usually having a fixed API: CCF stoichiometry. The co-crystal formers can be selected from the group consisting of phenolics, flavonoids, monoterpenes, aminoacids, alkaloids, vitamins, neutraceuticals, which works synergistically along with active pharmaceutical ingredient (API).These co-crystals have utility in imparting desirable physical properties and stability, which are otherwise not achievable for the pure active agent or in combination as a simple formulation using the excipients incorporated with the active agent.

The poor bioavailability, adherence property, low stability, low dissolution rate and to enhance the efficacy of the parent molecule in lower doses, synergistic action of API with the co-crystal former selected from the group consisting of amino acids, flavonoids or neutraceuticals is desired.

OBJECT OF THE INVENTION:

It is therefore the object of the invention to prepare novel mesalamine co-crystals having improved bioavailability, physicochemical properties to be used in pharmaceutical composition.

The other object of the invention is to prepare mesalamine co-crystal in 1 : 1 combination with coformers selected from amino acids, flavonoids or neutraceuticals.

SUMMARY OF THE INVENTION:

In accordance with the above objective, the present invention discloses novel synergistic pharmaceutical co-crystal comprising of Mesalamine as API with alpha amino acids, flavones or nutraceuticals. The inventive co-crystal of Mesalamine can be used in Mesalamine sustained release dosage forms. The word 'Mesalamine' as described herein also encompasses Mesalamine salts including Mesalamine hydrochloride.

In one aspect, the invention provides novel pharmaceutical composition comprising mesalamine and amino acid co-crystals in association with one or more pharmaceutical carriers. Alpha amino acids are selected from Leucine, isoleucine, lysine, methionine, phenyl alanine, threonine, tryptophan, valine, alanine, asparagines, aspartic acid, cystenine, glutamic acid, glutamine, L-glutamine, glycine, proline, serine, tyrosine, arginine, and histidine, especially L-glutamine.

In a further aspect, the invention provides novel pharmaceutical composition comprising mesalamine and flavone co-crystals of the current invention in association with one or more pharmaceutical carriers.

In another aspect, the invention provides novel synergistic Mesalamine co-crystals which comprise Mesalamine and flavones belonging to a class of 3-hydroxy flavones such as quercetin.

In a further aspect, the invention provides novel pharmaceutical composition comprising mesalamine and nutraceuticals co-crystals of the current invention in association with one or more pharmaceutical carriers.

The Bioavailability / blood plasma binding of Mesalamine is poor, hence these co- crystals will enhance the blood plasma binding ability of Mesalamine, thereby enhancing the 'time of residence' of Mesalamine in the blood. This would naturally translate to improving the efficacy, and hence lowering the dose of Mesalamine.

In another aspect, the invention provides the mesalamine -amino acid co-crystals, wherein said method comprises grinding of mesalamine free base and amino acid in 1 : 1 ratio in acetonitrile.

The invention also provides methods for the treatment of the disorder discussed above. Mesalamine co-crystals and pharmaceutical compositions containing them may, according to the invention, be administered using any amount, any form of pharmaceutical composition and any route of administration effective for the treatment. After formulation with an appropriate pharmaceutically acceptable carrier in a desired dosage, as known by those of skill in the art, the pharmaceutical compositions of this invention can be administered by any means that delivers the active pharmaceutical ingredient (s) to the site of the body whereby it can exert a therapeutic effect on the patient.

BRIEF DESCRIPTION OF DRAWINGS:

Fig.l shows IR spectra of Mesalamine hydrochloride

Fig.2 shows IR spectra of Glutamine

Fig.3 shows IR spectra of Mesalamine hydrochloride- Glutamine co-crystal

Fig.4 shows the PXRD spectra of Mesalamine hydrochloride

Fig.5 shows the PXRD spectra of Glutamine

Fig.6 shows the PXRD spectra of Mesalamine hydrochloride- Glutamine co-crystal Fig.7 shows NMR peaks of Mesalamine hydrochloride

Fig.8 shows NMR peaks of glutamine

Fig .9 shows NMR peaks of Mesalamine hydrochloride- Glutamine co-crystal

Fig.10 depicts DSC of Mesalamine hydrochloride

Fig.l 1 depicts DSC of Glutamine

Fig.12 depicts DSC of Mesalamine hydrochloride- Glutamine co-crystal

Fig.13 depicts TGA of Mesalamine hydrochloride

Fig.l4depicts TGA of Glutamine

Fig.15 depicts TGA of Mesalamine hydrochloride- Glutamine co-crystal

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated. The embodiments as described are not limiting or restricting the scope of the invention. DETAILED DESCRIPTION OF INVENTION:

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

The present invention relates to novel synergistic pharmaceutical mesalamine co-crystals with high purity, adequate stability, good flow ability and good dissolution properties that can be used in sustained release dosage forms.

Mesalamine or 5-aminosalicylic acid (5-ASA), is an anti-inflammatory drug used to treat inflammation of the digestive tract, ulcerative colitis and mild-to-moderate Crohn's disease. However, a longstanding obstacle in its effective ulcerative colitis treatment has been the heavy pill burden mandated by 5-ASA therapy, and the resultant difficulties with adherence to treatment regimens.

In accordance with the above, the present invention discloses novel synergistic pharmaceutical co-crystal comprising of Mesalamine as API with alpha amino acids, flavones or nutraceuticals. The inventive co-crystal of Mesalamine can be used in Mesalamine sustained release dosage forms. One preferable conformer is amino acid.

In an embodiment, the present invention provides novel pharmaceutical composition comprising Mesalamine and amino acid co-crystals in association with one or more pharmaceutical carriers.

The a-amino acids which are naturally occurring biomolecules, apart from being essential building blocks for the proteins, have a wide range of biological action. The amino acids being lipophilic helps Mesalamine in binding with blood plasma protein thereby increasing the plasma protein binding effect of the drug which in turn helps slow release of the drug from the co-crystals in unbound form yet maintaining the equilibrium of the drug levels in the body. They are further classified as essential and non essential amino acids. Types of Non Essential Amino Acids:

L-Alanine - acts as a producer of energy and it regulates blood sugar. Also, intervenes in the metabolism of glucose.

L-Asparagine - is important in the metabolic process of the nervous system.

L-Aspartic Acid - is essential for the transformation of carbohydrates into muscular energy.

L-Citruline - immune system stimulator helps in the production of body energy and helps to detoxify the liver from ammonia substance.

L-Cysteine - stimulates the growth of hair and protects against the damages that can be caused by alcohol and cigarettes.

L-Glutamine - helps the memory, concentration, and the correct functioning of mental activity.

L-Glutamic Acid - helps in the production of energy and brain function.

L-Glycine - slow the muscle degeneration by supplying addional creatine. It is vital for structuring red blood cells and providing amino acids to the body. Glucose and creatine are 2 essential substances in the production of energy and require glycine in their synthesis process.

L-Histidine - important in the production of red and white blood cells, and is vital for the formation of body tissues.

L-Proline - is important ingredient in the formation of tissues. L-Serine - aid the memory, the nervous system function, and is very important in the production of cell energy.

L-Tyrosine - helps in the treatment of insomnia, anxiety and depression, as well as allergies and is very important in the function of the thyroid and hypophysis glands. Deficiency of this amino acid is associated to hyperthyroidism (can cause fatigue).

L-Carnitine - helps control weight and body fat, as well as reduce the risk of heart problems. Lysine and vitamin B l and B6 along with iron are needed for the body to produce this amino acid.

GABA (Gamma- Aminobutyric Acid) - helps stop anxiety and hyperactivity.

L-Taurine - important for the muscle and disorders of the heart, helps the digestion of fats (it is found in bile), and also helps in hypoglycemia and hypertension.

Essential Amino Acids and their role in biological functions are given below:

L-Isoleucine - is needed in regulating sugar and the energy levels, as well as in the formation of hemoglobin. This amino acids is transformed and converted into muscle tissue. Lack of this AA produces a symptom similar to hypoglycemia or low blood sugar. L-Leucine - an important amino acid, which is found in animal and vegetable proteins. It is important for controlling the blood sugar level.

L-Lysine - an important for the construction of proteins principally in muscles and bones. Helps the assimilation of calcium, to obtain greater mental concentration and helps to lessen the effects of colds, flu and the herpes virus. It helps in the production of hormones, antibodies, enzymes and also in the formation of collagen. The deficiency of this amino acid produces: fatigue, irritability, anemia, and hair loss.

L-Methionine - helps to remove poison wastes from the liver and take part in the formation of the liver and kidney tissues. Help the digestive system, weak muscles, fragile hair, and is beneficial for osteoporosis. L-Phenylalanine - helps against depression, obesity, and loss of memory. It is an important element in the production of collagen, the principal fibrous protein in the body. Due to its action in the central nervous system, these amino acids decrease the pain associated with migraines, menstruation, and arthritis. L-Phenylalanine should not be taken by pregnant women or those who suffer from high blood pressure.

L-Tryptophan - helps control hyperactivity in children, alleviate stress, is good for the heart. It helps in weight control and allows the growth of the hormones necessary for the production of Vitamin B6 and Niacin. The brain utilizes this amino acid to produce Seratonina and Melatonina, neurotransmitters necessary for transferring nervous impulses from one cell to another. Lack of them (viz. Serotonine and Melatonine) produces depression, loss of sleep and other mental disorders.

L-Threonine - found in the heart, central nervous system and muscles. It is beneficial in the formation of collagen and elastin. Helps the liver and maintain the body's proteins in balance.

L-Valine - has a stimulating effect. It maintains the metabolism of muscles, repairs tissues and balances nitrogen. Valine should be combined with Leucine and isoleucine.

Glutamine is especially recommended for people who suffer from problems such as irritable bowel syndrome, colitis, Crohn's Disease and anyone under heavy stress (including strenuous exercise) or recovering from injury or other trauma. It is felt that lack of L-glutamine leads to a loss of epithelial cell integrity in the lining of the intestines. This, in turn, may allow toxins and infectious agents to enter the body. Most research studies concerning glutamine and the gastrointestinal system involve the addition of L-glutamine.

L-glutamine the biologically active isomer of glutamine is widely used as a dietary supplement. It accounts for 30-35 percent of the amino acid nitrogen in the plasma. It contains two ammonia groups, one from its precursor, glutamate, and the other from free ammonia in the bloodstream. One of glutamine's roles is to protect the body from high levels of ammonia by acting as a "nitrogen shuttle." Thus, glutamine can act as a buffer, accepting, and then releasing excess ammonia when needed to form other amino acids, amino sugars, nucleotides, and urea. This capacity to accept and donate nitrogen makes glutamine major vehicle for nitrogen transfer among tissues. Hagen and her coauthors had previously shown that glutamine protects against cell death from H. pylori-produced ammonia. They observed that the damaging effects of ammonia on gastric cells could be reversed completely by the administration of L-glutamine.

Thus, in a preferred embodiment, the invention provides novel synergistic mesalamine co-crystals which comprise mesalmine and L-glutamine. The said co-crystal improves the bioavailability of mesalamine and also increases the amount of mesalamine available at the colon, as L-glutamine has the tendency to accumulate in the colon. Further, the other side effects of mesalmine viz, flatulence will be mitigated as L-glutamine has the acid - base stabilization effect in the gastrointestinal fluids.

The novel synergistic pharmaceutical co-crystals comprising Mesalamine and L- glutamine is useful in treating Crohn,s disease and Ulcerative colitis either alone or together with other modalities of treatment selected from corticosteroids, immunomodulators such as methotrexate, azathiopurine; monoclonal antibodies such as infliximab, certolizimab, natalizumab and other novel small molecule and biologic drugs.

In an embodiment, novel synergistic pharmaceutical co-crystals comprising Mesalamine and L-glutamine is useful for the treatment caused due to infection by mycobacterial species especially Mycobacterium avium subspecies paratuberculosis (MAP).

In another embodiment, the synergistic pharmaceutical co-crystals comprising Mesalamine and L-glutamine is useful against inflammatory bowel disease caused due to infection by various mycobacterial species especially Mycobacterium avium subspecies paratuberculosis (MAP).

In yet another embodiment, the synergistic pharmaceutical co-crystals comprising Mesalamine and L-glutamine is useful for the treatment caused due to infections by bacteria such as E.coli, Clostridium Difficile Co\\\Xs,Helicobacter pylori etc. In yet another embodiment, the synergistic pharmaceutical co-crystals comprising Mesalamine and L-glutamine is useful for the treatment of viral infections, microbial, fungal infections.

In another embodiment, the present invention provides novel pharmaceutical composition comprising mesalamine and flavone co-crystals in association with one or more pharmaceutical carriers, wherein flavones are belonging to a class of 3-hydroxy flavone such as Quercetin.

Quercetin, a plant derived flavonoid belongs to a class of 3-hydroxy flavone. It is used as a nutritional supplement and has demonstrated significant anti-inflammatory activity by inhibiting both manufacture and release of histamine and other allergic/inflammatory mediators. In addition, it acts as an anti-tumor agent; induces apoptosis and inhibits synthesis of heat shock proteins. It inhibits many enzyme systems including tyrosine protein kinase, phospholipase A2, phosphodiesterases, mitochondrial ATPase, PI 3-kinase and protein kinase C and can also activate Ca2+ and K+ channels [Merck Index]. It is also known as a potent inhibitor of CYP3A4and CYP2C9, which are enzymes that break down most drugs in the body.

Quercetin is insoluble in water and sparingly soluble in aqueous buffers. However it is soluble in organic solvents such ethanol, DMSO, DMF and the solubility in these solvents is 2mg/ml in ethanol and 30mg/ml in DMSO and DMF. One characteristic feature of quercetin bioavailability is that the elimination of quercetin metabolites is quite slow, with reported half-lives ranging from 1 1 to 28 h. This could favor accumulation in plasma with repeated intakes.

In another preferred embodiment, the present invention relates to novel synergistic mesalamine co-crystals which comprise mesalamine and quercetin. This co-crystal improves the bioavailability of mesalamine. Also, the anti-inflammatory effect of Mesalamine will be synergistically enhanced by Quercetin, thus effectively reducing the dosage levels. In another embodiment, the present invention provides novel pharmaceutical composition comprising mesalamine and nutraceuticals co-crystals in association with one or more pharmaceutical carriers.

Nutraceutical, a term combining the words "nutrition" and "pharmaceutical," is a food or food product that provides health and medical benefits, including the prevention and treatment of disease. Such products may range from isolated nutrients, dietary supplements and specific diets to genetically engineered foods, herbal products, processed foods such as cereals, soups, beverages and include but not limited to Alanine, Arginine, Ascorbic acid, Aspartic acid, Biotin, Calcium carbonate, Calcium citrate, Calcium glycerophosphate, Calcium oxide, Calcium pantothenate, Calcium phosphate, Calcium pyrophosphate, Calcium sulfate, Carotene, Choline bitartrate, Choline chloride, Copper gluconate, Cuprous iodide, Cysteine, Cystine, Ferric phosphate, Ferric pyrophosphate, Ferric sodium pyrophosphate, Ferrous gluconate, Ferrous lactate, Ferrous sulfate, Glycine, Histidine, Inositol, Iron reduced, Isoleucine, Leucine, Linoleic acid, Lysine, Magnesium oxide, Magnesium phosphate, Magnesium sulfate, Manganese chloride, Manganese citrate, Manganese gluconate, Manganese glycerophosphate, Manganese hypophosphite, Manganese sulfate, Manganous oxide, Mannitol, Methionine, Methioninehydroxy analogue, Niacin, Niacinamide, D-pantothenyl alcohol, Phenylalanine, Potassium chloride, Potassium glycerophosphate, Potassium iodide, Proline, Pyridoxine hydrochloride, Riboflavin, Riboflavin-5-phosphate, Serine, Sodium pantothenate, Sodium phosphate, Sorbitol, Thiamine hydrochloride, Thiamine mononitrate, Threonine, Tocopherols, Tocopherol acetate, Tyrosine, Valine, Vitamin A, Vitamin A acetate, Vitamin A palmitate, Vitamin B12, Vitamin D2, Vitamin D3, Zinc sulfate, Zincgluconate, Zinc chloride, Zinc oxide and Zinc stearate.

In yet another preferred embodiment, the present invention discloses novel synergistic mesalamine and nutraceutical co-crystals which can be used as medically valuable compounds.

In another embodiment, the present invention discloses preparation of mesalamine hydrochloride glutamine co-crystal in 1 : 1 ratio. Mesalamine exists as zwitter ion which did not interact with glutamine under different conditions. Accordingly Mesalamine and conc.HCl taken in 1 :1 molar ratio are dissolved in methanol and then heated up to 55°C for half an hour. The resulting solution is filtered and kept for solvent evaporation to isolate mesalamine hydrochloride.

In an embodiment, Mesalamine hydrochloride obtained from above is mixed with Glutamine and grounded in acetonitrile for 10 minutes using a mortar and pestle and left for solvent evaporation to obtain free flowing solid. The resultant solid is subjected to analytical studies

The formation of these co-crystals was confirmed by powder X-ray diffractometry, IR spectrometry, NMR, DSC and TGA.

The physical characteristics of the co-crystals of Metformin and glutamine prepared according to the current invention are as tabulated below,

Table 1: I R Data

The Infrared spectrum of co-crystal as shown in Table 1 above clearly indicates that there is a proton transfer from NH3+ of Mesalamine hydrochloride to Glutamime which is giving a new carbonyl stretching frequency at 1733 cm-land also there is new carboxylate peak in co-crystal at 1650 cm-1. Table 2: PXRD data

The PXRD pattern shows that the co crystal is a different crystalline form as compared to the two formers (Table 2).

Table 3: 1H NMR ANALYSIS:

The NMR analysis indicates that the co-crystal was formed by 1 : 1 ratio of mesalamine hydrochloride and glutamine.

DSC ANALYSIS:

Generally, the co-crystal melts in-between the melting points of co-crystal formers (more often) or below their melting points (less often). DSC analysis of the co-crystal in 1 :1 ratio of the present invention begins to decompose at 162.57°C which is lower than the melting point of mesalamine hydrochloride free(263°C) and glutamine (187.14°C).

TGA ANALYSIS:

In Glutamine the weight loss was observed at 200°C is around 19.6 mass which roughly corresponds to the H20 moiety and another weight loss was observed at 247.5°C is around 30.6 which roughly corresponds to CO moiety. In Mesalamine hydrochloride the weight loss was observed at 194.8°C is around 42.5 mass which roughly corresponds to C02 moiety. In co-crystal the weight loss observed at 192.3°C is around 36.9 mass which roughly corresponds to the HC1 moiety.

In a further embodiment, the invention provides pharmaceutical compositions comprising a therapeutically effective amount of mesalamine-L glutamine co-crystals of the current invention in association with one or more pharmaceutical carriers. The co-crystals of the invention have the same pharmaceutical activity as its API. Further, the pharmaceutical composition of the invention may be any pharmaceutical form which maintains the crystalline form of a co-crystal of the invention.

In another embodiment, the invention provides pharmaceutical compositions comprising a therapeutically effective amount of mesalamine and quercertin co-crystals with one or more pharmaceutical carriers.

In yet another embodiment, the invention provides pharmaceutical compositions comprising a therapeutically effective amount of mesalamine and nutraceutical co- crystals with one or more pharmaceutical carriers.

The carriers/ excipients are added to the composition for variety of purposes. Dosage forms include solid dosage forms such as tablets, powders, capsules, liquid dosage forms as well as parenteral dosage forms. The dosage forms can also be prepared as sustained, controlled, modified and immediate dosage forms, preferably the dosage form is sustained release.. The active ingredient(s) and excipients can be formulated into compositions and dosage forms according to methods known in the art.

The pharmaceutical composition of the instant invention may be prepared in the form of raw powders or granules dispersed in a suitable aqueous or non-aqueous liquid(s), pellets, beads, micro or nano particles, micro or nano crystals or a solvated powders, sachets, semisolids, an Injectable preparations, a tablets, a capsules or a suitable specific two- or three-dimensional matrix compositions. The composition may be prepared by the techniques of dry granulation, wet granulation and / or direct compression using aqueous / Non aqueous solvent further fabricate into the single layer, bilayer, and multilayer and / or multicoated capsule or tablet dosage forms, preferably, the pharmaceutical composition is a multi-layer. Further the pharmaceutical composition of instant invention may be prepared in the form of suppositories such as enema, douche, pessary the parental composition comprises intravenous, Intradermal (ID) Intramuscular (IM) Intraosseous (10) Intraperitoneal (IP) Intravenous (IV) Subcutaneous (SC) Intrathecal (IT) injections and the topical formulation comprises cream, gel, liniment or balm, lotion, or ointment, drops, transdermal patch etc.

The pharmaceutical composition of the instant invention may be extended to the development of micelle, emulsion and liposome formulations, including small molecules, peptides, proteins, nucleic acids, antisense and siRNA.

In another embodiment, the novel co-crystal of Mesalamine hydrochloride and glutamine is useful for the treatment of Crohn's disease, ulcerative colitis, indeterminate colitis, inflammatory bowel disease caused due to mycobacterial species especially Mycobacterium avium subspecies paratuberculosis (MAP), viral infections, microbial, fungal infections, bacterial disease caused due to bacteria such as E.coli, Clostridium Difficile Colitis, Helicobacter pylori etc.

In yet another embodiment, the novel co-crystal of Mesalamine hydrochloride and glutamine may be administered through abdomen intraperitonealy; abdominal stents that open into colon, laproscopic guided direct delivery to colon; also may be delivered in the form of rectal enema, rectal suppository and other forms of delivery through rectum.

The invention also provides methods for the treatment of the disorder discussed above. Mesalmine co-crystals and pharmaceutical compositions containing them may, according to the invention, be administered using any amount, any form of pharmaceutical composition and any route of administration effective for the treatment. After formulation with an appropriate pharmaceutically acceptable carrier in a desired dosage, as known by those of skill in the art, the pharmaceutical compositions of this invention can be administered by any means that delivers the active pharmaceutical ingredient(s) to the site of the body whereby it can exert a therapeutic effect on the patient.

The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown by way of example, for purpose of illustrative discussion of preferred embodiments of the invention, and are not limiting the scope of the invention

Examples:

Example 1: Preparation of Mesalamine hydrochloride from Mesalamine:

Mesalamine (1 g) and cone. HC1(2.4 ml) were taken (1 : 1 molar ratio) in methanol (40 ml) then heated up to 55°C for half an hour. The resulting solution was filtered and kept it for solvent evaporation. The white solid precipitated was mesalamine hydrochloride.

Example 2: Preparation of Mesalamine hydrochloride-Glutamine co-crystal:

Mesalamine hydrochloride (18.9 mg, and O. lmmol) and Glutamine ( 14.6mg, O. l mmol) were ground in Acetonitrile (2mL) for 10 minutes using a mortar and pestle and left it for solvent evaporation to obtain free flowing co crystal solid.