TECHNICAL FIELD:
This invention relates to pharmaceutical co-crystals of Quercetin in combination with L-arginine to improve HDL elevation and decreases the levels of Oxidized LDL, thereby useful in the treatment of Atherosclerosis (CVD). The invention also relates to making a more soluble (water), thereby more bioavailable and stable form of quercetin that can be used as OTC. The invention further relates to process for preparation of the same and also relates to pharmaceutical compositions comprising these synergistic co-crystals.
BACKGROUND AND PRIOR ART:
Eventhough co-crystals were known as early as 19th century; the pharmaceutical industry has recognized the potential for their applications only recently. Pharmaceutical co-crystals are crystalline molecular complexes containing therapeutic molecules. These co-crystals represent emerging class of pharmaceutical materials offering the prospects of optimized physical properties.
Pharmaceutical co crystals are defined as hydrogen bonded complexes between an active pharmaceutical ingredient (API) and a coformer (CCF, benign partner molecule), usually having a fixed API: CCF stoichiometry. The utility of pharmaceutical co-crystals in solving stability, solubility, bioavailability, filtration, hydration, tableting, etc. issues is highlighted in recent papers.
The application of co-crystallization can further be extended to neutral molecules, amorphous compounds, to improve their physio-chemical properties. Further, 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.
Quercetin is a plant-derived flavonoid, specifically a flavonol, used as a nutritional supplement.
The American Cancer Society says that Quercetin "has been promoted as being effective against a wide variety of diseases, including cancer. As high dietary intake of fruits and

vegetables is associated with reduction in cancer, and therefore scientists suspect Quercetin may be partly responsible. Quercetin is the aglycone form of a number of other flavonoid glycosides, such as rutin and quercetrin, found in citrus fruit, buckwheat and onions. Quercetin forms the glycosides quercetrin and rutin together with rhamnose and rutinose, respectively. Quercetin is classified as IARC group 3 (no evidence of carcinogenicity in humans).
Further, Quercetin is an Anti-tumor agent; induces apoptosis and inhibits synthesis of heat shock proteins. Quercetin, one of the most widely distributed flavonoids in the plant kingdom inhibits many enzyme systems including tyrosine protein kinase, phospholipase A2, phosphodiesterases, mitochondrial ATPase, PI 3-kinase and protein kinase C; can also activate Ca2+ and K+ channels [Merck Index].
Quercetin, one of the most widely distributed flavonoids in the plant kingdom, inhibits various enzymes. This study examined its inhibitory effect on the angiotensin-converting enzyme activity through the cardiovascular response to bradykinin and angiotensin I. Quercetin pretreatment (88.7 µmol/kg p.o., 45 min; 14.7 µmol/kg i.v., 5 min) significantly potentiated the hypotensive effect of bradykinin (10 nmol/kg i.v.). This association was significantly attenuated by an antagonist of the B2 receptor. In addition, the hypertensive response to angiotensin I (0.1 nmol/kg iv) was significantly reduced by Quercetin pretreatment using the same parameters as before. These results suggest an inhibitory effect of Quercetin on the angiotensin-converting enzyme activity, similar to that of captopril. Quercetin was equally effective when given orally or intravenously. [Inhibition of Angiotensin-Converting Enzyme by Quercetin Alters the Vascular Response to Bradykinin and Angiotensin, L.P.N. Hackl, G. Cuttle, S. Sanches Dovichi, M.T. Lima-Landman, M. Nicolau, Pharmacology, 65(4), 2002].
Pre-incubation of cells with Quercetin followed by cisplatin treatment appeared to be the most effective and was correlated with strong activation of caspase-3 and inhibition of both heat shock proteins (Hsp72) and multi-drug resistance proteins (MRP) levels. The results indicate that Quercetin pretreatment sensitizes HeLa cells to cisplatin-induced apoptosis in HeLa cells [The effect of Quercetin on pro-apoptotic activity of cisplatin in HeLa cells, J. Jakubowicz-Gil et al., Biochemical Pharmacology, 69(9), 1343-1350, 2005].

The results indicate that Quercetin and rutin may be useful in the treatment of IAR and LAR in asthma via inhibition of histamine release, PLA2, and EPO, and reduced recruitment of neutrophils and eosinophils into the lung [Anti-asthmatic Action of Quercetin and Rutin in Conscious Guinea-pigs Challenged with Aerosolized Ovalbumin, Chan Hun Jung, Ji Yun Lee, Chul Hyung Cho, and Chang Jong Kim, Arch Pharm Res. 30(12), 1599-1607, 2007].
Gabriela Suchankova et al examined in HepG2 cells whether glucose-induced changes in AMP-activated protein kinase (AMPK) activity could be mediated by SIRTl, an NAD+-dependent histone/protein deacetylase that has been linked to the increase in longevity caused by caloric restriction. Incubation with 25 vs. 5 mM glucose for 6 h concurrently diminished the phosphorylation of AMPK (Thr 172) and ACC (Ser 79), increased lactate release, and decreased the abundance and activity of SIRTl. In contrast, incubation with pyruvate (0.1 and 1 mM) for 2 h increased AMPK phosphorylation and SIRTl abundance and activity. The putative SIRTl activators resveratrol and Quercetin also increased AMPK phosphorylation. None of the tested compounds (low or high glucose, pyruvate, and resveratrol) significantly altered the AMP/ATP ratio. Collectively, these findings raise the possibility that glucose-induced changes in AMPK are linked to alterations in SIRTl abundance and activity and possibly cellular redox state [Concurrent regulation of AMP-activated protein kinase and SIRTl in mammalian cells, Gabriela Suchankova et al., Biochemical and Biophysical Research Communications, 378(4), 836-841, 2009].
Recent findings: Quercetin bioavailability has been underestimated in the past and can be improved by food matrix components or particular delivery forms. Among the biological effects of particular relevance, the antihypertensive effects of Quercetin in humans and the improvement of endothelial function should be emphasized. Together with its antithrombotic and anti-inflammatory effects, the latter mainly mediated through the inhibition of cytokines and nitric oxide; Quercetin is a candidate for preventing obesity-related diseases. Most exiting are the findings that Quercetin enhances physical power by yet unclear mechanisms. The anti-infectious and immunomodulatory activities of Quercetin might be related to this effect [Quercetin: potentials in the prevention and therapy of disease, Bischoff, Stephan C, Current Opinion in Clinical Nutrition and Metabolic Care: 11(6), 733-740, 2008].

Quercetin increased mRNA expression of PGC-1 alpha and SIRT1 (P<0.05), mtDNA (P<0.05) and cytochrome C concentration (PO.05). These changes in mitochondrial capacity were associated with an increase in both maximal endurance capacity (P<0.05) and voluntary wheel running activity (P<0.05). These benefits of querectin on fitness without exercise training may have important implications for enhancement of athletic and military performance and may also extend to prevention and/or treatment of chronic diseases [Quercetin increases brain and muscle mitochondrial biogenesis and exercise tolerance. Am J Physiol Regul Intesr Comp Physiol, 2009-3-12].
Over-the counter (OTC) drugs can be purchased without a prescription and are commonly used to treat symptoms of common illnesses that may not require the direct supervision of a physician. Occupying a space somewhere between essential nutrients (those nutrients critical to normal health, such as vitamins) and drugs with defined impacts on specific diseases, nutraceuticals are bioactive chemicals derived from foods but taken as supplements at much higher concentrations than diet alone could provide. Natural health products include nutraceuticals as well as herbs and functional foods that have got a therapeutic value due to presence of some bioactive substances. Nutraceuticals are natural and bioactive products with food value to keep energy balance in the body and promise substantial therapeutic value in several diseases. Major Nutraceuticals are now part of nutrition supplements at non-prescription counters and their self- prescription is increased at large scale. The current literature suggests the nutraceuticals are used in the prevention and management of cardiovascular disease, ageing, osteoporosis and cancer as well as diabetes.
Nutraceuticals can be broadly classified as antioxidants, polyphenols, omega-3 fatty acids, amino acids, vitamins, minerals and potentially disease-fighting compounds from common spices such as cinnamon and turmeric.
There is ample patented literature available on Quercetin. WO/2008/011364 discloses a composition containing Quercetin, vitamin B3, vitamin C, and folic acid. Also disclosed is a method of using the composition for enhancing physical or mental performance or treating various diseases or disorders.

Most of the nutraceuticals are derived from plants and animal origin and act as biochemical metabolites either by direct intermediary metabolism or regulating immunity. In Ayurveda, medicinal plants are the basis for the treatment of diseases. Gum guggul, shilajeet, psyllium, fenugreek, garlic, triphala, are common medicines obtained from plant sources in Ayurveda.
Many a times these bioactive compounds when taken as a traditional ayurvedic preparation may have the necessary effect as the compounds are in an intricate matrix of interactions thereby making them more bioavailable. On the contrary when these bioactive molecules are isolated pure, their solubility (in water) is poor, as most of them are lipophillic molecules arising out of the Shikimate / Acetate-mevalonate pathways. To overcome this poor solubility these nutraceuticals are taken in large amounts often leading to unwanted side effects.
For example, Vitamin E capsules that are sold in market are of 100 to 400 i.u, while the health regulations recommend a daily intake of 40 i.u. These bioactive compounds in their pure form undergo aerial oxidation and / or photoreactions, thereby making these molecules very unstable. Hence, a formulation strategy is essential to make these nutraceuticals effective Over the Counter Drugs. This formulation strategy makes the molecules more shelf stable and also water soluble.
a-amino acids are naturally occurring biomolecules. Apart from being essential building blocks for the proteins, alpha-amino acids have a wide range of biological action.
Amino acids are critical to life, and have a variety of roles in metabolism. One particularly important function is as the building blocks of proteins which are linear chains of amino acids. Amino acids are also important in many other biological molecules, such as forming parts of coenzymes, or as precursors for the biosynthesis of molecules such as heme. Due to this central role in biochemistry, amino acids are very important in nutrition.
Arginine is an a-amino acid. The L-form is one of the 20 most common natural amino acids. Its codons are CGU, CGC, CGA, CGG, AGA, and AGG. In mammals, arginine is

classified as a semi-essential or conditionally essential amino acid, depending on the developmental stage and health status of the individual. Infants are unable to meet their requirements and thus arginine is nutritionally essential for infants. Arginine was first isolated from a Lupin seedling extract in 1886 by the Swiss chemist Ernst Schultze.
US20090197942 discloses a composition for inhibiting hypertension comprising quercetin and arginine. However, this application fails to explain the co-crystal formation of quercetin and L-arginine and the pharmaceutical compositions comprising the same.
Method for preventing or treating elevated blood lipid level-related diseases by administering rutin and Quercetin is disclosed in US 6509372.
WO/2002/076473 describes Quercetin, its preparation and the medicinal composition containing the same and their application for preventing or treating diseases related to 5HT14 receptor or neure damage, including preventing or treating Alzeheimer's disease, drug or alcohol dependence, sleep disorders or panic state, delaying senility or improving memory function and preventing or treating neure damage caused by brain injury.
Further, another WO2004/078163 publication describes a pharmaceutical composition comprising a co-crystal of an API and a co-crystal former; wherein the API has at least one functional group selected from ether, thioether, alcohol, thiol, aldehyde, ketone, thioketone, nitrate ester, phosphate ester, thiophosphate ester, ester, thioester, sulfate ester, carboxylic acid, phosphinic acid, phosphonic acid, sulfonic acid, amide, primary amine, secondary amine, ammonia, tertiary amine, imine, thiocyanate, cyanamide, oxime, nitrile diazo, organohalide, nitro, S-heterocyclic ring, thiophene, N-heterocyclic ring, pyrrole, 0-heterocyclic ring, furan, epoxide, peroxide, hydroxamic acid, imidazole, pyridine and the co-crystal former has at least one functional group selected from amine, amide, pyridine, imidazole, indole, pyrrolidine, carbonyl, carboxyl, hydroxyl, phenol, sulfone, sulfonyl, mercapto and methyl thio, such that the API and co-crystal former are capable of co-crystallizing from a solution phase under crystallization conditions
Another WO2008/153945 discloses co-crystals comprising at least one nutraceutical compound and at least one co-crystal former with or without impurities. These co-crystals

may be included in compositions (optionally also including other components such as pharmaceutically acceptable excipients, other nutritional supplements, etc.) having utility as pharmaceuticals, nutraceuticals, nutritional supplements, and foodstuffs.
US20070299033 disclose pharmaceutical compositions comprising a co-crystal of an API and a co-crystal former, and methods of making and using the same.
Article titled "In vitro solubility, stability and permeability of novel quercetin-amino acid conjugates" discloses quercetin prodrug with improved bioavailability. The author of the article synthesized nine quercetin-amino acid conjugates and estimated their pharmacokinetic properties including water solubility, stability against chemical or enzymatic hydrolysis, and cell permeability. Among the synthesized quercetin prodrugs, quercetin-glutamic acid conjugate Qu-E (4g/5g) showed remarkable increases in water solubility, stability, and cell permeability compared with quercetin, which warrants further development as a quercetin prodrug.
However, prior art failed to identify pharmaceutical co-crystals of Quercetin and L-arginine, useful in effective treatment of disease conditions. Pharmaceutical co-crystals are crystalline molecular complexes containing therapeutic molecules. These co-crystals represent emerging class of pharmaceutical materials offering the prospects of optimized physical as well as therapeutic properties.
Therefore, it is an objective of the present invention is to provide synergistic pharmaceutical co-crystals comprising Quercetin and L-arginine to provide effective treatment of diseased condition with reduced dosage of the active.
DESCRIPTION OF DRAWINGS:
Fig 1 shows IR spectra of Quercetin
Fig 2 shows the IR spectra of L-Arginine
Fig 3 shows the IR spectra of co-crystal of Quercetin + L-Arginine
Fig 4 shows PXRD of Quercetin
Fig 5 shows PXRD of L-Arginine
Fig 6 shows PXRD of cocrystal of Quercetin + L-Arginine

Fig 7 shows DSC of Quercetin
Fig 8 shows DSC of L-Arginine
Fig 9 shows DSC of co-crystal of Quercetin - L-Arginine
Fig 10 shows TGA of Quercetin
Fig 11 shows TGA of L-Arginine
Fig 12 shows TGA of co-crystal of Quercetin - L-Arginine
Fig 13 shows schematic diagram of TLC (50%EtOAc+Hexane).
SUMMARY OF THE INVENTION:
In accordance with the above objective, the present invention provides synergistic pharmaceutical co-crystals of Quercetin and L-arginine as combination drugs is provided herein which is further analyzed and characterized using PXRD, IR and Mass spectra.
The co-crystals of Quercetin and L-arginine shows good HDL elevation, decreased levels of Oxidized LDL and hence, could also be used in the treatment of Atherosclerosis (CVD). The said co-crystal also has mild anti-hypertensive effect, insulin sensitization effect and also regulates fat metabolism.
In another aspect, the invention provides the Quercetine- L-arginine co-crystals, wherein said method comprises neat grinding of Quercetine and L-arginine in 1:1 ratio.
The inventors have surprisingly noted that the melting point of the Quercetin co-crystal formed is much lower than the Quercetin. Further, the solubility and shelf stability of Quercetin is also substantially improved when it is delivered as a co-crystal with water soluble drug.
DETAILED DESCRIPTION OF THE 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.

In one preferred embodiment, a co-crystal of Quercetin - L-Arginine, as a combination drug for treatment of various ranges of disease is provided herein. The co-crystal formed is further analyzed and characterized using PXRD, IR and Mass spectra.
In one preferred embodiment, a co-crystal of Quercetin - L-Arginine, as an OTC having a more bioavailable form of Quercetin.
In another preferred embodiment, the invention provides a process for preparation the aforementioned co-crystals of Quercetin and L-arginine or their derivatives.
The process comprises grinding of Quercetin and L-arginine or their derivatives taken together (1:1 ratio) in mortar pestle and hand grinded for 10 minutes in suitable solvent for making it homogenous. The resultant mixture was crystallized in solvents selected from methanol, ethanol, isopropyl alcohol, dimethyl formamide, acetonitrile, dimethyl formamide- acetonitrile, tetrahydrofuran, or dioxane under ambient conditions. Crystallization was accomplished by heating the solution over hot plate at 50-100°C and then the saturated solution was slowly cooled. Generally co-crystals appear in 2-3 days. The formation of these co-crystal or salt was confirmed by powder X-ray powder difractometry (PXRD) and IR spectrometry. These co-crystals were further characterized by thermal analysis.
In another aspect, the invention provides synergistic pharmaceutical compositions comprising the co-crystals of Quercetin -L-arginine and its derivatives as described above.
In yet another aspect, the invention provides process for production of pharmaceutical preparation, wherein said process comprises preparing Quercetin + L-arginine suitable amino acids as mentioned above; isolating co-crystal comprising Quercetin + L-arginine incorporating into pharmaceutical composition along with one or more suitable pharmaceutical carriers. Excipients are added to the composition for variety of purposes. Dosage forms include solid dosage forms like tablets, powders, capsules, sachets, troches and lozenges as well as liquid syrups, suspensions and elixirs. The active ingredient(s)

and excipients can be formulated into compositions and dosage forms according to methods known in the art.
The invention also provides methods of treating Atherosclerosis, hypertension, metabolic syndrome, or diabetes Type II, which comprises administering 'an effective amount' of the 'Quercetin and L-arginine cocrystals' to the subject suffering affected Atherosclerosis, hypertension, metabolic syndrome, or diabetes Type II. The subject mentioned herein is human.
'An effective amount' according to present invention is 100 mg/day to 200 mg/day. The invention further discloses use of the 'composition of the present invention comprising Quercetin and L-arginine cocrystals' in preparing the medicament intended to treat symptoms associated with Atherosclerosis, hypertension, metabolic syndrome, or diabetes Type II.
EXAMPLES: Example 1:
Co-crystals of the present invention are prepared by using the following method. Materials used: L-arginine Quercetin (1:1 ratio)
Grinding of Quercetin( heated to 120°C, 30.3 mg, 0.1mmol) and L-arginine (17.4mg, 0.1mmol) were taken together (1:1 ratio) in mortar pestle and hand grinded for 10 minutes in acetonitrile for making it homogenous. The resultant solid was subjected to following analytical studies. The co-crystals so formed was analysed using thin layer precoated chromatographic plates. One liter of HPLC grade acetonitrile was stirred with 2 g of Potassium Carbonate (LR grade, RANKEM) for twelve hours. This was then refluxed for two hours under nitrogen atmosphere. Then dry acetonitrile was obtained by distillation under nitrogen atmosphere. The dried acetonitrile was used in analyzing quertcetin and L-arginine co-crystals via thin layer precoated chromatographic plates (Silicagel 60 F254 from Merck) the results of which are depicted in Figure 13.

The physical characteristics of the co-crystals of L-arginine - Quercetin so formed is tabulated below:
L-arginine + Quercetin co-crystal (1:1)

IR DATA:

PXRD DATA (2Theta):

(Table Removed)
DSC Analysis:
While the L-arginine shows an endotherm at 228°C and Quercetin was at 320°C. But in the case of co-crystal the endotherm was at 191 -195 °C.
TGA Analysis:
In Quercetin no weight loss was observed below 300°C. In L-arginine weight loss observed at 258°C is around 48 mass which roughly corresponds to the NH2-C=NH moiety, where as in co-crystal the same weight loss observed at 260.3°C which roughly corresponds to the NH2-C=NH moiety.

We claim,
1. A co-crystal comprising Quercetin and L-Arginine characterized by PXRD and IR.
2. The co-crystal according to claim 1, wherein the said co-crystal is characterized by IR spectra having values 3298, 3155, 1665, 1654, 1608, 1559,1512, 1421, 1361, 1315, 1245, 1199, 1164 and 932 cm-1.
3. The co-crystal according to claim 1, wherein the said co-crystal characterized by PXRD having 2Θ values 10.82, 12.54, 12.71, 13.62, 14.49, 16.14, 18.90, 22.73 ± 0.2.
4. The co-crystal according to claim 1, wherein the said co-crystal is having endotherm at 191-195 °C.
5. A pharmaceutical composition comprises of co-crystal of Quercetin and L-Arginine according to claim 1.
6. The pharmaceutical co-crystal composition according to claim 1, further comprises one or more pharmaceutical carrier.
7. A process for preparing pharmaceutical co-crystal composition according to claim 1, comprising (a) neat grinding of Quercetin and L-arginine in 1:1 ratio; (b) isolating the co-crystals.
8. Method of reducing symptoms associated with Atherosclerosis, hypertension, metabolic syndrome, or diabetes Type II, which method comprises administering 'an effective amount' of the 'Quercetin and L-arginine cocrystals' according to claim 1 to the subject suffering from said symptoms.
9. The method according to claim 5, wherein said subject is human.

10. Use of Quercetin and L-arginine co-crystal according to claim 1 in preparing the
medicament intend to reduce symptoms associated with Atherosclerosis,
hypertension, metabolic syndrome, or diabetes Type II.
11. Use of the said cocrystal as an OTC drug.