Technical field:
This present invention relates to pharmaceutical synergistic co-crystals of Quercetin, or its salts or its polymorph such as hydrate and Isoniazid or its salts thereof, useful for the treatment of tuberculosis. Further, the present invention relates to process for preparation of the same and pharmaceutical compositions comprising the said stable co-crystals.
Background and prior art:
Even though 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. 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 Quercetin, found in citrus fruit, buckwheat and onions. Quercetin forms the glycosides Quercetin 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 nmol/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].
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 (PO.05), mtDNA (P<0.05) and cytochrome C concentration (P<0.05). These changes in mitochondrial capacity were associated with an increase in both maximal endurance capacity (PO.05) and voluntary wheel running activity (P<0.05). These benefits of Quercetin 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 Integr Comp Physiol, 2009-3-12].
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.
US 20080031940 describes a composition includes 10-50 wt. % Quercetin along with papain; calcium salt; zinc salt; bee pollen; pumpkinseed; bromelain; and saw palmetto; wherein the composition is a sustained release composition in tablet or capsule form suitable for oral administration to a human. Methods of making and using the composition are provided.
Method for preventing or treating elevated blood lipid level-related diseases by administering rutin and Quercetin is disclosed in US 6,509,372. 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.
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, O-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
Isoniazid (Laniazid, Nydrazid) also known as isonicotinylhydrazine (1NH), is an organic compound that is the first-line antituberculosis medication in prevention and treatment. It was first discovered in 1912, and later in 1951 it was found to be effective against tuberculosis. Isoniazid is never used on its own to treat active tuberculosis because resistance quickly develops.
The compound was first synthesized in the early 20th century, but its activity against tuberculosis was first reported in the early 1950's and three pharmaceutical companies attempted unsuccessfully to simultaneously patent the drug (most prominently, Roche, who launched their version, Rimifon, in 1952). With the introduction of isoniazid, a cure for tuberculosis was first considered reasonable. Isoniazid is available in tablet, syrup, and injectable forms (given intramuscularly or intravenously). Isoniazid is available worldwide, is inexpensive and is generally well tolerated. It is manufactured from isonicotinic acid, which is produced from 4-methylpyridine.
Article by Lechner et. al. (Volume 1, Issue 2, 21 August 2008, Pages 71-75) titled Modulation of isoniazid susceptibility by flavonoids in Mycobacterium discloses that plant natural products modulates the susceptibility of different strains of fast-growing mycobacteria to the first-line antituberculotic isoniazid (INH). The flavonoids epicatechin, isorhamnetin, kaempferol, luteolin, myricetin, quercetin, rutin and taxifolin were assessed for their ability to modulate isoniazid susceptibility in fast-growing mycobacteria. Myricetin was the most potent compound and could reduce the minimum inhibitory concentration of isonizid 64-fold at a concentration of 16ug/ml, followed by Quercetin. Structure-activity relationships of flavonoids as intensifiers of INH

susceptibility in mycobacteria indicate that they overlap with SARs for their radical-scavenging properties, however the potentiation of INH activity cannot only be explained by their radical-scavenging activity alone.
The prior art failed to provide pharmaceutical co-crystals of Quercetin with Isoniazid, useful in effective treatment of diseased conditions such as Tuberculosis and will act as antimycobacterial agent or antibacterial agent. 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 to develop stable and synergistic pharmaceutical co-crystals of Quercetin and Isoniazid which act as antimycobacterial agent or antibacterial agent useful in treatment of Tuberculosis either alone or as one of the drugs in four drugs combination therapy. Cocrystallization improved the solubility and hence, the bioavailability of both Isoniazid & Quercetin thereby making co-crystal formulation an improved form of Isoniazid.
Description of drawings:
Fig 1 shows IR spectra of Quercetin
Fig 2 shows the IR spectra of Isoniazid
Fig 3 shows the IR spectra of co-crystal of Quercetin + Isoniazid
Fig 4 shows PXRD of Quercetin
Fig 5 shows PXRD of Isoniazid
Fig 6 shows PXRD of cocrystal of Quercetin + Isoniazid
Fig 7 shows DSC of Quercetin
Fig 8 shows DSC of Isoniazid
Fig 9 shows DSC of co-crystal of Quercetin - Isoniazid
Fig 10 shows TGA of Quercetin
Fig 11 shows TGA of Isoniazid
Fig 12 shows TGA of co-crystal of Quercetin - Isoniazid
Fig 13 shows NMR Peak of Quercetin
Fig 14 shows NMR Peak of Isoniazid
Fig 15 shows NMR Peak of Quercetin - Isoniazid

Summary of the invention:
In accordance with the above objective, the present invention provides stable pharmaceutical co-crystals of Quercetin and Isoniazid to act as combination drugs useful in treatment of Tuberculosis either alone or as one of the drugs in four drugs combination therapy.
Accordingly in one embodiment, a pharmaceutical co-crystal of Isoniazid/ Quercetin, as a combination drug is provided herein and process for preparation thereof. The co-crystal formed is further analyzed and characterized using PXRD, IR and Mass spectra. Further, solubility 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 this application, the inventors have achieved the stable pharmaceutical co-crystals of
Quercetin and Isoniazid or its salts thereof, which works synergistically for treating
diseased condition such as Tuberculosis and act as antimycobacterial agent or
antibacterial agent. Quercetin is present in the form of salt or polymorphic forms such as
hydrates etc. Cocrystallization improves the solubility and hence the bioavailability of
both Isoniazid & Quercetin thereby making co-crystal formulation an improved form of
Isoniazid.
Tuberculosis has been treated with combination therapy for over fifty years. Drugs are not
used singly (except in latent TB or chemoprophylaxis), and regimens that use only single
drugs result in the rapid development of resistance and treatment failure. The standard
"short" course treatment for TB is isoniazid, rifampicin, pyrazinamide, and ethambutol
for two months, then isoniazid and rifampicin alone for a further four months.
The stable pharmaceutical co-crystals of Quercetin and Isoniazid or its salts thereof can be used alone for the treatment of tuberculosis or can be used as one of the drug in four drugs combination therapy for the treatment of tuberculosis. The co-crystal so obtained is stable under accelerated condition for at least six months.

In another embodiment, a synergistic co-crystal of Isoniazid - Quercetin as a combination drug is further analyzed and characterized using PXRD and 1R.
In another preferred embodiment, the invention provides a process for preparation the aforementioned co-crystals of Quercetin and Isoniazid or its salts thereof.
The process comprises grinding of Quercetin and Isoniazid or its salts thereof 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 and IR spectrometry. These co-crystals were further characterized by thermal analysis.
In another aspect, the invention provides stable and synergistic pharmaceutical compositions comprising the co-crystals of Quercetin and Isoniazid or its salts thereof as described above.
In yet another aspect, the invention provides process for production of pharmaceutical preparation, wherein said process comprises preparing Quercetin + Isoniazid as mentioned above; isolating co-crystal comprising Quercetin dihydrate + Isoniazid 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 tuberculosis, which comprises administering 'an effective amount' of the 'Quercetin and Isoniazid cocrystals' to the subject suffering from tuberculosis. The pharmaceutical co-crystals of Quercetin and

Isoniazid or its salts thereof can be used alone for the treatment of tuberculosis or can be used as one of the drug in four drugs combination therapy for the treatment of tuberculosis. The subject mentioned herein is human. 'An effective amount' according to present invention is as per the WHO norms.
The invention further discloses use of the 'composition of the present invention comprising Quercetin and Isoniazid cocrystals' in preparing the medicament intended to treat symptoms associated with Tuberculosis.
EXAMPLES Example 1:
Co-crystals of the present invention are prepared by using the following method.
Materials used:
Isoniazid
Quercetin (1:1 ratio)
Grinding of Quercetin( heated to 150°C, 30.3 mg, O.lmmol) and Isoniazid (13.7mg,
O.lmmol) were taken together (1:1 ratio) in mortar pestle and hand grinded for 10
minutes in Acetone for making it homogenous.
The physical characteristics of the co-crystals of Isoniazid - Quercetin so formed is
tabulated below:
ISONIAZID + QUERCETIN CO-CRYSTAL (1:1)
(Table Removed)
IR DATA:
(Table Removed)

PXRD DATA (2Theta):
(Table Removed)
1H NMR Analysis:
NMR analysis shows the cocrystal was formed by 1:1 ratio.
DSC Analysis:
DSC thermogram of Isoniazid shows an endotherm at 172.07°C, where as Quercetin shows at 320°C. But the co-crystal shows a sharp endotherm at 272.98°C.

TGA Analysis:
In Isoniazid weight loss is observed at 224.49°C. In Quercetin no weight loss was observed below 300°C. But in co-crystal one water molecule weight loss was observed at 119.92°C.
From Infrared spectrum [Table 1] it is clear that the carbonyl in Quercetin has shifted
from 1670 to 1651 cm-1. Also in isoniazid there is shift corresponding to - NH2 from
3191 to 3217cm'1 indicating that this moiety is interacting with quercetin. The O-H stretching of phenolic group of Quercetin shows the absorption at 3368 cm-1 But in cocrystal this peak shifted to 3449 cm-1. This means that the one of the phenolic O-H groups involves in strong hydrogen bonding with isoniazid moiety.

We claim,
1. A co-crystal comprising Quercetin and Isoniazid characterized by PXRD having 20 values 8.0, 8.3, 10.04, 11.8, 12.1, 13.9, 15.2, 25.6, 26.2, 27.0 ±0.2.
2. The co-crystal according to claim 1, wherein the said co-crystal is characterized by IR spectra having values 3449, 3217, 1651 and 1417.85 cm-1.
3. The co-crystal according to claim 1, wherein the said co-crystal is characterized by having endotherm at 272.98°C.
4. A pharmaceutical composition comprises of co-crystal of Quercetin and Isoniazid according to claim 1.
5. The pharmaceutical co-crystal composition according to claim 4, further comprises one or more pharmaceutical carrier.
6. A process for preparing pharmaceutical co-crystal according to claim 1, comprising (a) neat grinding of Quercetin and Isoniazid in 1:1 ratio; (b) isolating the co-crystals.
7. Method of treating symptoms associated with tuberculosis, which method comprises administering 'an effective amount' of the 'Quercetin and Isoniazid cocrystals' according to claim 1 to the subject suffering from said symptoms either alone or in combination therapy with rifampicin, pyrazinamide, and ethambutol.
8. The method according to claim 7, wherein said subject is human.
9. Use of Quercetin and Isoniazid co-crystal according to claim 1 in preparing the medicament intends to treat symptoms associated with Tuberculosis.