DESC:FORM 2
THE PATENTS ACT 1970
(39 of 1970)
AND
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule13)
1. TITLE OF THE INVENTION:

“SOLID STATE FORMS OF ANDROGRAPHOLIDE”

2. APPLICANT:

(a) NAME: CRYSTALIN RESEARCH PRIVATE LIMITED

(b) NATIONALITY: Indian company incorporated under the provisions
of The Companies Act, 1956

(c) ADDRESS: Plot No. 81 A/C, Unit D, MLA Colony, Road No. 12,
Banjara Hills, Hyderabad 500 034, Telangana, India.

3. PREAMBLE TO THE DESCRIPTION:
The following specification particularly describes the invention and the manner in which it is to be formed.

Technical Field of Invention:

This invention relates to the novel cocrystals of bioactive agent Andrographolide with GRAS coformers which inhibits chemical transformation of Andrographolide in in-vitro conditions, improves solubility and dissolution rate of Andrographolide. The present invention also relates to a process for preparing the novel cocrystals of Andrographolide and pharmaceutical compositions thereof.

Background of the Invention:

The discovery of biologically active molecules and their development as active pharmaceutical ingredients (API’s) is a driver for the innovation of novel drugs and pharmaceuticals (S. R. Byrn, R. R. Pfeiffer and G. G. Stowell, in Solid State Chemistry of Drugs, SSCI, West Lafayette, IN, 1999; H. G. Brittain, in Physical Characterization of Pharmaceutical Solids, Marcel Dekker Inc. NY, 1995). Even as high-throughput screening and combinatorial chemistry (C. G. Smith and J. J. O’Donnell, in The Process of New Drug Discovery and Development, Informa, NewYork, 2006; Solid form screening-A review, J. Aaltonen, M. Allesø, S. Mirza, V. Koradia, K. C. Gordon and J. Rantanen, Eur. J. Pharm. Biopharm., 2009, 71, 23-37) has increased the number of new chemical entities (NCE’s) many-fold, often poor bioavailability of these potential drug candidates poses a major hindrance in formulating them as API’s. According to Serajuddin and coworkers, (Salt formation to improve drug solubility, A. T. M. Serajuddin, Adv. Drug Delivery Rev., 2007, 59, 603; IV-IVC considerations in the development of immediate-release oral dosage form, S. Li, H. He, L. Parthiban, H. Yin and A. T. M. Serajuddin, J. Pharm. Sci., 2005, 94, 1396) while NCE solubility of <20 µg/mL was practically unheard of until 1980s, the situation has changed completely in the current times with solubility of drug molecules lower than 1 µg/mL becoming commonplace. Therefore solid state modification of new chemical entities or existing drug molecules with an intention of developing marketable dosage forms has received greater attention in the current period than ever before. Since there is no one size fits all king of solid form modification for all drugs, various solid forms of drug molecules like Polymorphs, Amorphous forms, Salts, Solvates, Hydrates etc. are prepared to optimize the drug physicochemical properties. Among them the ideal solid form for the drug is chosen for formulation and marketing. Although these solid forms are widely used to market various drug molecules, certain impending factors such as phase transformation of metastable polymorphs to stable forms during grinding and storage (Phase transformation of Carbamazepine by the milling process, H. Kala, U. Haack, U Wenzel, G. Zessin, P.Pollandt, Pharmazie, 1986, 41, 777), conversion of metastable, high energy amorphous phases to the stable, low energy crystalline forms (Highly soluble Olanzapinium maleate crystalline salts, R. Thakuria and A. Nangia, CrystEngComm, 2011, 13, 1759-1764; The effect of temperature and moisture on the amorphous to crystalline transformation of stavudine, S. Strydom, W. Liebenberg, L.Yu, and M. de Villiers, Int. J. Pharm., 2009, 379, 72-81) and desolvation of solvates and hydrates to their guest free forms (Polymorph formation from solvate desolvation, B. Nicolai, P. Espeau, R. Ceolin, M.-A. Perrin, L. Zaske, J. Giovannini and F. Leveiller, J. Therm. Anal. Calorim. 2007, 90, 337-339) makes these solid forms less attractive for pharmaceutical development.

In the past decade, Pharmaceutical Cocrystallization has emerged as an alternate solid form strategy to modulate the physicochemical properties of drug molecules (Pharmaceutical Cocrystals and their Physicochemical Properties, N. Schultheiss and A. Newman, Cryst. Growth Des., 2009, 9, 2950). Pharmaceutical Cocrystals (Pharmaceutical Cocrystals, P. Vishweshwar, J. A. McMahon, J. A. Bis and M. J. Zaworotko, J. Pharm. Sci., 2006, 95, 499; Pharmaceutical Cocrystals: An Emerging Approach to Physical Property Enhancement, W. Jones, W. D. S. Motherwell, and A. V. Trask, MRS Bulletin, 2006, 31, 875) are stoichiometric non-ionic supramolecular complexes that can be used to address physical property issues such as stability, solubility, flowability, and bioavailability in pharmaceutical art without altering the chemical composition of the active drug molecule. Pharmaceutical cocrystals can be constructed by modifying the hydrogen bonding, pi-staking, van der Waals interactions etc. between the drug molecule and the cocrystal former. Numerous literature reports on cocrystals stand testimony to their application in pharmaceutical industry. They are primarily used to address the poor physicochemical properties such as solubility and stability which can become a serious drawback for clinical development and even cause late stage failure of the drug candidate (Can the pharmaceutical industry reduce attrition rates? I. Kola, J. Landis, Nat. Rev. – Drug Discovery, 2004, 3, 711–715). Of late, pharmaceutical cocrystals have been used to improve tabletability (Improving mechanical properties of crystalline solids by cocrystal formation: New compressible forms of Paracetamol, S. Karki, T. Frišcic, L. Fábián, P. R. Laity, G. M. Day, W. Jones, Adv. Mater. 2009, 21, 3905-3909), Color and hydrolytic degradation stability (Crystal Engineering of Stable Temozolomide Cocrystals, N. J. Babu, P. Sanphui and A. Nangia, Chem. Asian J., 2012, 7, 1) and hydration control (Pharmaceutical Cocrystallization: Engineering a remedy for Caffeine Hydration, A. V. Trask, W. D. S. Motherwell and W. Jones, 2005, 5, 1013). A Cocrystal may be defined as a multi-component chemical system of a definite stoichiometric ratio, interacting through non-covalent interactions, predominantly hydrogen bonds, and is a solid at room temperature and pressure. For pharmaceutical cocrystals one of the components must be an Active Pharmaceutical Ingredient (API) and the second preferably be a Generally Regarded as Safe (GRAS) chemical. (The FDA approved GRAS list. US Food and Drug administration. EAFUS). The utility of pharmaceutical cocrystals in solving stability, solubility, bioavailability, filtration, hydration, tableting, etc. issues is highlighted in recent reviews and papers. (The role of cocrystals in pharmaceutical science. N. Shan, and M. J. Zaworotko, Drug. Disc. Today, 2008, 13, 440-446.; Crystal engineering of the composition of pharmaceutical phases. Do pharmaceutical co-crystals represent a new path to improved medicines? Ö. Almarsson, and M. J. Zaworotko, Chem. Commun., 2004, 1889-1896). Pharmaceutical cocrystals can be prepared through solution crystallization, solid state grinding, solvent assisted grinding, melt, sublimation etc. (Crystal engineering of organic cocrystals by the solid-state grinding approach. A. V. Trask, and W. Jones, Top. Curr. Chem., 2005, 254, 41-70).

Andrographolide (AP, hereafter) is a diterpenoid lactone bio-active agent used in traditional medicine in China, India and Southeast Asian countries (Phytochemistry, pharmacology, and clinical use of Andrographis paniculata, R. P. Samy, M. M. Thwin, P. Gopalakrishnakone, Nat. Prod. Commun. 2007, 2, 607-618; Protective activity of Andrographolide and arabinogalactan proteins from Andrographis paniculata Nees against ethanol induced toxicity in mice, P. K. Singha, S. Roy, S. Dey, J. Ethnopharmacol. 2007, 111, 13-21). It is derived from the plant leaves of Andrographis Paniculata, known as “King of Bitters”, in the Acanthaceae family. The local plant name in India is Kalmegh. AP has many pharmacological actions, such as anti-viral, anti-inflammatory, anti-cancer, and anti-malarial (Andrographolide up regulates cellular reduced glutathione level and protects cardiomyocytes against hypoxia/ reoxygenation injury, A. Y. Woo, M. M. Waye, S. K. Tsui, S. T. Yeung, C. H. Cheng, J. Pharmacol. Exp. Ther. 2008, 325, 226-235; Anti-tumor activities of Andrographolide, a diterpene from Andrographolide paniculata by inducing apoptosis and inhibiting VEGF level, F. Zhao, E. Q. He, L. Wang, K. Liu, J. Asian Nat. Prod. Res. 2008, 10, 467-473; HPTLC analysis of hepatoprotective diterpenoid Andrographolide from Andrographis paniculata nees (Kalmegh) A. P. Raina, A. Kumar, S. K. Pareek, Ind. J. Pharm. Sci. 2007, 69, 473-475). Despite being safe at high doses of 17 g/kg per day in humans, the efficacy of AP is limited by poor bioavailability of 2.67% as reported in rats (Poor oral bioavailability of a promising anti-cancer agent Andrographolide is due to extensive metabolism and efflux by P-glycoprotein, L. Ye, T. Wang, L. Tang, W. Liu, Z. Yang, J. Zhou, Z. Zheng, Z. Cai, M. Hu, Z. Liu, J. Pharm. Sci. 2011, 100, 5007-5015). This is partly due to its poor aqueous solubility of 46 mg/L (Solubility of Andrographolide in various solvents from (288.2 to323.2) K, M. Chen, C. Xie, L. Liu, J. Chem. Eng. Data, 2010, 55, 5297). A significant drop in the bioavailability of AP is due to transformation to four metabolites isolated from humans and rats (Four new andrographolide metabolites in human urine, L. Cui, F. Qiu, N. Wang, X. Yao, Chem Pharm Bull. 2004, 52, 772). Three of the AP metabolites are isomers of 14-deoxy-12-sulfo-andrographolide, and the fourth product is the S-conjugate (sulfated derivative) of AP. The main metabolite of AP was identified as 14-deoxy-12-(R)-sulfo-andrographolide, which was found to be similar to the anti-inflammatory drug, Lianbizhi (Studies on the structure of the adduct of andrographolide with sodium hydrogen sulfite, Z. M. Meng, Acta Pharmaceutica Sinica, 1981, 16, 571-575). Yao et al. developed an in vitro synthetic method that mimics the in vivo biotransformation and isolated the main metabolite 14-deoxy-12-(R)-sulfo-andrographolide (Identification of a rare sulfonic acid metabolite of andrographolide in rats, X. He, J. Li, H. Gao, F. Qiu, K. Hu, X. Cui, X. Yao, Drug Metab. Dispos. 2003, 31, 983-985) as shown in Scheme 1. Several AP metabolites have been studied by mass spectroscopy (Comparative metabolism and stability of andrographolide in liver microsomes from humans, dogs and rats using ultra performance liquid chromatography coupled with triple-quadrupole and Fourier transform ion cyclotron resonance mass spectroscopy, H.-Y. Zhao, H. Hu, Y.-T. Wang, Rapid. Commun. Mass Spectrom. 2013, 27, 1385-1392).

Scheme 1: Chemical transformation of andrographolide (AP) to its main metabolite 14-deoxy-12-(R)-sulfo-andrographolide, AP-SO3H.

Creating a solution to the loss of andrographolide efficacy due to competing biotransformation to the sulfate derivative will enhance the utility of AP in medicine. Therefore, there is a need in the prior art to develop novel solid forms which would play the dual role of exhibiting improved dissolution and inhibit chemical transformation which would pave the way for improved oral bioavailability and also enhance the potential therapeutic use of the herbal medicine, andrographolide.

Object of the Invention:

Accordingly, it is the object of the present invention to develop novel cocrystals of andrographolide which would serve the dual purpose of inhibiting the chemical transformation of the parent molecule and improve its solubility and dissolution rate. The other object is to provide andrographolide in the modified cocrystal form that may be used as an anti-viral, anti-inflammatory, anti-cancer, and anti-malarial agent in pharmaceutical compositions.

Summary of the Invention:

Andrographolide (referred herein as AP) has many pharmacological actions, such as anti-viral, anti-inflammatory, anti-cancer, and anti-malarial. Despite being safe at high doses of 17 g/kg per day in humans, the efficacy of AP is limited by poor bioavailability of 2.67% (reported in rats). This is partly due to its poor aqueous solubility (46 mg/L). A significant drop in the bioavailability of andrographolide is due to transformation into four metabolites isolated from humans and rats. Hence AP is difficult to administer orally in pure form.

In an aspect, the present invention provides novel co-crystals of andrographolide with GRAS coformers which inhibits chemical transformation of Andrographolide in in-vitro conditions, improves solubility and dissolution rate of AP and enhances its bioavailability.

The GRAS coformers used in the present invention are selected from 2,6-dihydroxybenzoic acid, 3.4-dihydroxybenzoic acid, 4-aminobenzoic acid, benzoic acid, vanillin, hydroquinone, vanillic acid, catechol, acetamide, resorcinol, guaiacol, fumaric acid, nicotinamide, salicylic acid, ferulic acid, adipic acid and the like.

In a preferred aspect, the instant invention provides novel cocrystals of andrographolide with coformers selected from vanillin 1a, vanillic acid 1b, salicylic acid 1c, guaiacol 1d, and resorcinol 1e, ( as shown in scheme 2) to inhibit chemical transformation of Andrographolide in in-vitro conditions, improve solubility and dissolution rate of AP and enhance its bioavailability.

For the purpose of this instant invention, the crystalline complexes of AP with vanillin 1a, vanillic acid 1b, salicylic acid 1c, guaiacol 1d, and resorcinol 1e, are referred to as cocrystals.

Scheme 2 Chemical structure of andrographolide (AP) and coformers vanillin (a), vanillic acid (b), salicylic acid (c), guaiacol (d) and resorcinol (e). These cocrystals of andrographolide with vanillin, vanillic acid, salicylic acid, guaiacol and resorcinol are referred to as 1a, 1b, 1c, 1d and 1e respectively.

In another aspect, the present invention provides a process for preparation of novel cocrystals of andrographolide (AP) and coformers comprising grinding andrographolide and coformer, in fixed stoichiometric ratio, with a few drops of solvent in a liquid-assisted method for about 30 min followed by dissolving the grinded mixture in a solvent either alone or mixtures thereof, leaving the solution and isolating co-crystals after 4-5 days.

In another aspect, all the cocrystals of AP were characterized by FT-IR, FT-Raman, Solid state CP-MAS 13C NMR, Differential scanning calorimetry (DSC), High resolution mass spectroscopy (HRMS) and X-ray diffraction.

The biochemical transformation of AP to AP-SO3H (Scheme 1), which limits the bioavailability of the active metabolite, was carried out in vitro on AP and its cocrystals (Identification of a rare sulfonic acid metabolite of andrographolide in rats, X. He, J. Li, H. Gao, F. Qiu, K. Hu, X. Cui, X. Yao, Drug Metab. Dispos. 2003, 31, 983-985). Accordingly AP was treated with HSO3– (in the presence of HSO4– and excess Na2SO3) to generate the sulfated product by the nucleophilic attack of HSO3– ion at the ß position (C12-carbon) of the a,ß unsaturated lactone moiety in AP to isolate the primary sulfonate metabolite, 14-deoxy-12(R)-sulfo andrographolide (Scheme 3). The reaction was complete in 30 min and the product was characterized as AP-SO3H by its reported 1H and 13C NMR spectra and the M+1 peak at m/z 415.1786 (C20H30O7S, calcd. M+1 415.1790) in the HRMS of the sulfate product. Further, each of the AP cocrystals was subjected to the same reaction conditions (same molar equivalent of AP, concentration, room temperature) to study the chemical stability of AP as a hydrogen-bonded cocrystal. Andrographolide-vanillin (1a), andrographolide-Guaiacol (1d), and andrographolide-resorcinol (1e) cocrystals reacted somewhat slower than pure AP in that a mixture of AP, AP-SO3H inactive product and dissociated coformer were detected by NMR at 30 min. The conversion to the inactive sulfonate metabolite was partial in case of Andrographolide–vanillic acid (1b) cocrystal (43% by weight). Andrographolide-salicylic acid (1c) cocrystal gave the best result in that no transformation to AP-SO3H could be detected by NMR and LC-MS.

The acidity of the COOH group of salicylic acid (c) and vanillic acid (b) was reasoned to have an inhibitory effect on the chemical transformation of AP to AP-SO3H (pKa’s of the relevant species are listed in Table 1). The reactive species in the aqueous ethanol medium is bisulfite anion (HSO3–), which reacts with the conjugated lactone of AP in a 1,4-nucleophilic addition. The carboxylic acid of the coformer in andrographolide-salicylic acid (1c) (and andrographolide-vanillic acid, 1b) is able to titrate the HSO3– to H2SO3 and sodium salicylate, which will stop/inhibit the subsequent addition reaction. The o-hydroxy acid (salicylic acid, c) is stronger than the p-hydroxy acid (vanillic acid, b) by almost 2.5 pKa units and consequently much more reactive in protonating the HSO3– ion. Sodium salicylate then reacts with H2SO3 to give salicylic acid and HSO3–Na+. There will be equilibrium between the acid–conjugate base pairs of salicylic acid (c) and H2SO3 because their acid strengths are within 2 pKa units. Vanillic acid (b) is less effective in proton transfer to HSO3– being a weaker acid, and this explains its intermediate inhibition. Vanillin (a), guaiacol (d) and resorcinol (e) are phenolic and unable to participate in the acid-base protonation-deprotonation chemistry as shown in scheme 3, and these cocrystals are ineffective to make any change to the HSO3– reactivity. The complete inhibition of the undesired pathway for andrographolide-salicylic acid cocrystal (1c) is due to the higher acidity of salicylic acid (c), the so called ortho effect (Application of the Hammett equation to the Ortho substituted benzene reaction series, M. Charton, Can. J. Chem. 1960, 38, 2493; Determination of a new scale of ortho steric parameters S° from N-methylation of pyridines, U. Berg, R. Gallo, G. Klatte, J. Metzger, J. Chem. Soc., Perkin Trans. 2, 1980, 1350-1355; Analysis of the ortho effect: Acidity of 2-substituted benzoic acids, S. Böhm, P. Fiedler, O. Exner, New J. Chem. 2004, 28, 67-74). The peak at d 7.5, which is indicative of the sulfated carbon, is absent in the NMR spectrum of post-reaction product of andrographolide-salicylic acid (1c). These results and the mechanistic explanations mean that it is the acidity of salicylic acid in the andrographolide-salicylic acid (1c) cocrystal that stops the chemical transformation of AP in the flask.

Scheme 3 (a) Transformation of AP to AP-SO3H and (b) a plausible mechanism for the inhibition of the reaction by Andrographoliode–Salicylic acid (AP–SLA) cocrystal.

Table 1 pKa of active species in water
Acid pKa (water) Literature source
Salicylic acid 2.97 www.drugbank.ca/drugs/DB00936 accessed on 20-05-13
Vanillic acid 4.45 www.chemicaldictionary.org/dic/V/Vanillic-acid_1030.html accessed on 20-05-13

H2SO3 1.90 evans.harvard.edu/pdf/evans_pka_table.pdf accessed on 20-05-13
HSO3– 7.21 evans.harvard.edu/pdf/evans_pka_table.pdf accessed on 20-05-13

Having improved the stability of Andrographolide through andrographolide-salicylic acid (1c) cocrystal, the invention further provides comparative solubility and dissolution. Good solubility is essential for better pharmacokinetics and higher therapeutic efficacy. Solubility improvement is a major challenge for improving drug bioavailability (A provisional biopharmaceutical classification of the top 200 oral drug products in the united states , Great Britain, Spain and Japan, T. Takagi, C. Ramachandran, M. Bermejo, S. Yamashita, X. L. Yu, G. L. Amidon, Mol. Pharmaceutics, 2006, 3, 631-643; C. A. Lipinski, in Pharmaceutical profiling in Drug Discovery for Lead Selection, Eds: R. T. Borchardt, E. H. Kerns, C. A. Lipinski, D. R. Thakker, B. Wang; AAPS Press, Arlington, VA, 2004, 93-125) Solubility studies were performed on andrographolide-salicylic acid (1c) only in 25% EtOH-water medium, the solvent being chosen such that the less soluble AP is sufficiently soluble to make the Conc. vs. Intensity calibration curves by UV-Vis spectroscopy (using the ?max of pure AP at 225 nm and AP–SLA at ?max 227 nm). Solubility was measured at the end of 24 h. The solubility of AP–SLA (4.55 g/L) is 12 times higher than that of AP (0.38 g/L). Both AP and andrographolide-salicylic acid (1c) cocrystal forms are stable under the solubility conditions (by PXRD match of the residue at the end of the experiment).

The dissolution rate is a kinetic phenomenon which gives the peak concentration of the solute and the amount of drug dissolved in a short time period (30 min to a few hours). The dissolution method is particularly useful to estimate drug release, and it is the suitable parameter for those drugs which undergo phase transformation/dissociation (Dissolution testing as a prognostic tool for oral drug absorption: immediate release dosage forms, J. B. Dressman, G. L. Amidon, C. Reppas and V. P. Shah, Pharm Res., 1998, 15, 11) in solution.

Accordingly, in another aspect, the invention provides intrinsic dissolution studies of AP and AP-cocrystals. IDR experiments on AP and AP–SLA cocrystal was performed in 25% EtOH-water for 6 h by the rotating disk intrinsic dissolution rate (DIDR) method (Feasibility studies of utilizing disk intrinsic dissolution rate to classify drugs, X. L. Yu, A. S. Carlin, G. L. Amidon, A. S. Hussain, Int. J. Pharm. 2004, 270, 221) at 37°C. Andrographolide-salicylic acid (1c) cocrystal exhibited 3 fold higher IDR than AP. Thus in vitro experiments show that the inactivation of AP by biochemical sulfation is completely inhibited through andrographolide-salicylic acid (1c) cocrystal, which also showed 12 fold improvement in solubility.

In yet another aspect, the present invention provides cocrystal of Andrographolide with anti-oxidant coformer molecules such as curcumin, quercetin, juglone, lawsone, menadione, plumbagin, piperine and the like. These complexes of andrographolide with said coformers may be used as an anti-viral, anti-inflammatory, anti-cancer, and anti-malarial agent in pharmaceutical compositions.

The co crystal of Andrographolide with said anti-oxidant molecules are prepared using the liquid-assisted grinding method known in the art (Crystal engineering of organic crystals by the solid-state grinding approach. A.V. Trask, W. D. S. Motherwell, W. Jones, Top. Curr. Chem. 2005, 254, 41; Solvent-drop grinding: Green polymorph control of cocrystallization. A.V. Trask, W. D. S. Motherwell, W. Jones, Chem. Commun. 2004, 890).

Brief description of the drawings:

Figure 1(a) depicts X-ray powder diffraction of commercial andrographolide. Figure 1(b) depicts the calculated diffraction lines from the crystal structure.
Figure 2 depicts X-ray powder diffraction of andrographolide-vanillin (1a) cocrystal
Figure 3 depicts X-ray powder diffraction of andrographolide-vanillic acid (1b) cocrystal
Figure 4 depicts X-ray powder diffraction of andrographolide-salicylic acid (1c) cocrystal
Figure 5 depicts X-ray powder diffraction of andrographolide-guaiacol (1d) cocrystal
Figure 6 depicts X-ray powder diffraction of andrographolide-resorcinol (1e) cocrystal
Figure 7 depicts DSC thermogram of commercial andrographolide.
Figure 8 depicts DSC thermogram of andrographolide-vanillin (1a) cocrystal
Figure 9 depicts DSC thermogram of andrographolide-vanillic acid (1b) cocrystal
Figure 10 depicts DSC thermogram of andrographolide-salicylic acid (1c) cocrystal
Figure 11 depicts DSC thermogram of andrographolide-guaiacol (1d) cocrystal
Figure 12 depicts DSC thermogram of andrographolide-resorcinol (1e) cocrystal
Figure 13 (a) and (b) depicts the ORTEP diagram of novel andrographolide-vanillin (1a) cocrystal and hydrogen bonding and molecular packing in the crystal structure.
Figure 14 (a) and (b) depicts the ORTEP diagram of novel andrographolide-vanillic acid (1b) cocrystal and hydrogen bonding and molecular packing in the crystal structure.
Figure 15 (a) and (b) depicts the ORTEP diagram of novel andrographolide-salicylic acid (1c) cocrystal and hydrogen bonding and molecular packing in the crystal structure.
Figure 16 (a) and (b) depicts the ORTEP diagram of novel andrographolide-guaiacol (1d) cocrystal and hydrogen bonding and molecular packing in the crystal structure.
Figure 17 (a) and (b) depicts the ORTEP diagram of novel andrographolide-resorcinol (1e) cocrystal and hydrogen bonding and molecular packing in the crystal structure.
Figure 18 depicts FT-IR spectrum of andrographolide-vanillin (1a) cocrystal overlayed with its starting components
Figure 19 depicts FT-IR spectrum of andrographolide-vanillic acid (1b) cocrystal overlayed with its starting components
Figure 20 depicts FT-IR spectrum of andrographolide-salicylic acid (1c) cocrystal overlayed with its starting components
Figure 21 depicts FT-IR spectrum of andrographolide-guaiacol (1d) cocrystal overlayed with its starting components
Figure 22 depicts FT-IR spectrum of andrographolide-resorcinol (1e) cocrystal overlayed with its starting components
Figure 23 depicts FT-Raman spectrum of andrographolide-vanillin (1a) cocrystal overlayed with its starting components
Figure 24 depicts FT-Raman spectrum of andrographolide-vanillic acid (1b) cocrystal overlayed with its starting components
Figure 25 depicts FT-Raman spectrum of andrographolide-salicylic acid (1c) cocrystal overlayed with its starting components
Figure 26 depicts 13C ss-NMR spectrum of andrographolide-vanillin (1a) cocrystal overlayed with its starting components
Figure 27 depicts 13C ss-NMR spectrum of andrographolide-vanillic acid (1b) cocrystal overlayed with its starting components
Figure 28 depicts 13C ss-NMR spectrum of andrographolide-salicylic acid (1c) cocrystal overlayed with its starting components
Figure 29 depicts 13C ss-NMR spectrum of andrographolide-guaiacol (1d) cocrystal overlayed with its starting components
Figure 30 depicts 13C ss-NMR spectrum of andrographolide-resorcinol (1e) cocrystal overlayed with its starting components
Figure 31 depicts 1H solution NMR spectrum of andrographolide
Figure 32 depicts 13C solution NMR spectrum of andrographolide
Figure 33 depicts 1H solution NMR spectrum of andrographolide sulfate product (AP-SO3H).
Figure 34 13C solution NMR spectrum of andrographolide sulfate product (AP-SO3H)
Figure 35 HRMS spectrum of AP-SO3H shows the M+1 peak at 415.1785
Figure 36 depicts 13C solution NMR spectrum of andrographolide-vanillin (1a) cocrystal reaction mixture overlayed with andrographolide.
Figure 37 depicts 1H solution NMR spectrum of andrographolide-vanillic acid (1b) cocrystal reaction mixture overlayed with andrographolide.
Figure 38 depicts 13C solution NMR spectrum of andrographolide-salicylic acid (1c) cocrystal reaction mixture overlayed with andrographolide.
Figure 39 depicts 13C solution NMR spectrum of andrographolide-guaiacol (1d) cocrystal reaction mixture overlayed with andrographolide.
Figure 40 depicts 13C solution NMR spectrum of andrographolide-resorcinol (1e) cocrystal reaction mixture overlayed with andrographolide.
Figure 41 depicts intrinsic dissolution rate curves of andrographolide-salicylic acid (1c) cocrystal in comparision to andrographolide
Figure 42 depicts the PXRD pattern of andrographolide after 24 hrs slurry in 25% EtOH-water medium showed good match when compared with its calculated pattern indicating phase stability.
Figure 43 depicts the PXRD pattern of andrographolide-salicylic acid (1c) cocrystal after 24 hrs slurry in 25% EtOH-water medium showed good match when compared with its calculated pattern indicating phase stability.

Disclosure 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.

Since the low bioavailability of andrographolide is due to its chemical transformation and low solubility, it is necessary to inhibit its chemical transformation and improve solubility in order to enhance its therapeutic efficacy.

Therefore, in an embodiment, the present invention discloses novel co-crystals of andrographolide with GRAS coformers which inhibits chemical transformation of Andrographolide in in-vitro conditions, improves solubility and dissolution rate of AP and enhances its bioavailability.

The coformers used in the instant invention are selected from the group containing C=O, COOH, CONH2 and OH functional groups such as 2,6-dihydroxybenzoic acid, 3.4-dihydroxybenzoic acid, 4-aminobenzoic acid, benzoic acid, vanillin, hydroquinone, vanillic acid, catechol, acetamide, resorcinol, guaiacol, fumaric acid, nicotinamide, salicylic acid, ferulic acid, adipic acid and the like.

The cocrystals of andrographolide and coformer is in stoichiometric ratio varying from 1:1, 1:2 or 2:1.

In a preferred embodiment, the present invention discloses novel co-crystals of andrographolide with coformers selected from vanillin(1a), vanillic acid(1b), salicylic acid (1c), guaiacol(1d) and resorcinol(1e) which inhibits chemical transformation of Andrographolide in in-vitro conditions, improves solubility and dissolution rate of AP and enhances its bioavailability.

In another embodiment, the present invention provides a process for preparation of novel cocrystals of andrographolide (AP) and coformers comprising grinding andrographolide and coformer, in fixed stoichiometric ratio, with a few drops of solvent in a liquid-assisted method for about 30 min followed by dissolving the grinded mixture in a solvent either alone or mixtures thereof, leaving the solution and isolating co-crystals after 4-5 days.

The conformer in the instant process is selected from the group containing C=O, COOH, CONH2 and OH functional groups such as 2,6-dihydroxybenzoic acid, 3.4-dihydroxybenzoic acid, 4-aminobenzoic acid, benzoic acid, vanillin, hydroquinone, vanillic acid, catechol, acetamide, resorcinol, guaiacol, fumaric acid, nicotinamide, salicylic acid, ferulic acid, adipic acid and the like.

The solvent for the process is selected from polar organic solvents such as water, acetonitrile, lower alcohols, ethyl formate, ethylacetate, acetone and the like either alone or as mixture thereof.

According to the present invention, a novel cocrystal of andrographolide with vanillin was obtained by grinding andrographolide and vanillin in 1:1 stoichiometry with a few drops of CH3CN added in a liquid-assisted method for 30 min. This was followed by dissolving the grinded product in MeOH-H2O (5:1 v/v, 6 mL) and leaving for slow evaporation and isolating the co crystals after 4-5 days.

In another aspect, the invention provides a process for the preparation of andrographolide-vanillic acid cocrystal. Both andrographolide and vanillic acid were grinded in 1:1 stoichiometry with a few drops of CH3CN added in a liquid-assisted method for 30 min. This was followed by dissolving the grinded product in MeOH-H2O (1:2 v/v, 6 mL) and leaving for slow evaporation and isolating the co crystals after 4-5 days. Formation of cocrystal was confirmed by IR, Raman DSC, PXRD etc.

In another preferred embodiment, the invention describes a novel cocrystal of andrographolide with salicylic acid prepared by grinding andrographolide and salicylic acid in 1:1 stoichiometry with a few drops of water added in a liquid-assisted method for 30 min. This was followed by dissolving the grinded product in ethyl formate-MeOH mixture (4:1 v/v, 6 mL), leaving for slow evaporation and isolating the cocrystal after 4-5 days. Purity of the bulk material was consistent as confirmed by the XRPD of andrographolide-salicylic acid cocrystal.

In a further embodiment, the cocrystal of andrographolide with guaiacol was prepared by grinding andrographolide with guaiacol (used in excess and methanol) for 30 min. This was followed by dissolving the grinded mixture in EtOH-H2O (1:2 v/v, 6 mL), leaving for slow evaporation and isolating the cocrystals after 4-5 days. The cocrystal was further characterized by IR, Raman, PXRD and DSC.

In another aspect, the invention provides a process for the preparation of andrographolide-resorcinol cocrystal. Both andrographolide and resorcinol were grinded in 1:1 stoichiometry with a few drops of water added in a liquid-assisted method for 30 min. This was followed by dissolving the ground product in 6 mL of ethyl acetate and left for slow evaporation and isolated the cocrystals after 4-5 days. PXRD of andrographolide-resorcinol cocrystal confirmed the complete conversion of andrographolide and resorcinol to the cocrystal.

In yet another embodiment, novel cocrystals of andrographolide with vanillin (1a), vanillic acid (1b), salicylic acid (1c), guaiacol (1d) and resorcinol (1e) were characterized by X-ray Powder Diffraction (XRPD), Differential Scanning Calorimetry (DSC), FT-IR, FT-Raman spectroscopy and solid-state 13C NMR spectroscopy. Significant differences in XRPD, DSC, FT-IR, FT-Raman and ss-NMR confirmed the uniqueness of each new cocrystal of andrographolide.

In yet another embodiment, biochemical transformation of AP to AP-SO3H (Scheme 1), which limits the bioavailability of the active metabolite, was carried out in vitro on AP and its cocrystals. These experiments were monitored using 1H and 13C solution NMR and HRMS. The analysis of product at the end of the experiment indicated that while AP showed complete transformation to the sulfonate product, all the cocrystals either delayed, partially or completely inhibited the chemical transformation of andrographolide. More specifically, andrographolide-vanillin (1a), andrographolide-guaiacol (1d) and andrographolide-resorcinol (1e) cocrystals delayed the chemical transformation of andrographolide in that a mixture of AP, AP-SO3H inactive product and dissociated coformer were detected by NMR at 30 min. The conversion to the inactive sulfonate metabolite was partial in case of andrographolide-vanillic acid cocrystal, 1b (43% by weight). Andrographolide-salicylic acid cocrystal (1c) showed no transformation to AP-SO3H as detected by NMR and HRMS.

In yet another embodiment, the present invention discloses intrinsic dissolution rate and solubility measurements of the novel cocrystals of andrographolide. The intrinsic dissolution rate and solubility measurements were performed in 25% EtOH-water medium to compare the dissolution rate and solubility of the stable andrographolide-salicylic acid cocrystal with andrographolide. The intrinsic dissolution rate (IDR) of andrographolide and andrographolide-salicylic acid cocrystal was found to be 0.090 x 10-3 mg/cm2/min and 0.277 x 10-3 mg/cm2/min respectively. Thus andrographolide-salicylic acid cocrystal dissolved 3 times faster than the commercial andrographolide. The equilibrium solubility measured at 24 h of andrographolide and andrographolide-salicylic acid cocrystal in 25% EtOH-water mixture is observed to be 0.378 g/L and 4.555 g/L respectively. Thus solubility of andrographolide-salicylic acid cocrystal is about 12 times higher than that of andrographolide.

In yet another embodiment, the present invention discloses the stability of andrographolide-salicylic acid cocrystal. The andrographolide-salicylic acid cocrystal was found to be stable at the end of solubility and dissolution experiments as confirmed by the PXRD experiments. Hence, in addition to completely inhibiting the chemical transformation of andrographolide, andrographolide-salicylic acid cocrystal also exhibited higher solubility and dissolution without converting to its individual components i.e andrographolide and salicylic acid. Since chemical transformation and poor solubility are the major drawbacks of promoting andrographolide as a frontline drug, the cocrystals described in the present invention especially the andrographolide-salicylic acid cocrystal has overcome these hindrances for andrographolide to be used as a therapeutically active agent.

In yet another embodiment, the present invention relates to a pharmaceutical composition, which comprises an effective amount of novel andrographolide-coformer cocrystals of the instant invention in association with at least one pharmaceutically acceptable carrier such as stabilizers, additives, adjuvants, diluents, binder, emulsifiers known in art. Excipients are added to the composition for a variety of purposes including achieving desired dosage form with optimum therapeutic effect.

The quantity of the compound used in pharmaceutical compositions of the present invention will vary depending upon the body weight of the patient and the mode of administration and can be of any effective amount to achieve the desired therapeutic effect.

These novel solid forms of andrographolide can be formulated into a variety of compositions for administration to humans and mammals such as tablets, gels, capsules, emulsion, syrups, transdermal patches, aerosol spray, topical preparations, nanoparticles, for treating various diseases.

In another embodiment, the novel cocrystal of andrographolide are administered for the treatment or prevention of cancer, malaria, inflammations and viral infections.

In another embodiment, the present invention relates to cocrystal of Andrographolide with anti-oxidant compounds such as curcumin, quercetin, juglone, lawsone, menadione, plumbagin, piperine and the like. The complexes of andrographolide with said coformers may be used as an anti-viral, anti-inflammatory, anti-cancer, and anti-malarial agent in pharmaceutical compositions.

The co crystal of Andrographolide with said anti-oxidant molecules are prepared using the liquid-assisted grinding method known in the art.(Crystal engineering of organic crystals by the solid-state grinding approach. A.V. Trask, W. D. S. Motherwell, W. Jones, Top. Curr. Chem. 2005, 254, 41; Solvent-drop grinding: Green polymorph control of cocrystallization. A.V. Trask, W. D. S. Motherwell, W. Jones, Chem. Commun. 2004, 890).

The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

Example 1
Preparation of cocrystals of andrographolide
Andrographolide was procured from commercial suppliers and used as such without further purification. This starting material corresponds to essentially pure andrographolide guest free form.

1a: Method of preparing andrographolide-vanillin cocrystal (1a)
This cocrystal was obtained by grinding andrographolide and vanillin in 1:1 stoichiometry with a few drops of CH3CN added in a liquid-assisted method for 30 min. Bulk amount of cocrystal for other characterizations was prepared using this method. Single crystals were obtained when 40 mg of the ground product was dissolved in MeOH-H2O (5:1 v/v, 6 mL) and left for slow evaporation for 4-5 days.

1b: Method of preparing andrographolide-vanillic acid cocrystal (1b)
This cocrystal was obtained by grinding AP and vanillic acid in 1:1 stoichiometry with a few drops of CH3CN added in a liquid-assisted method for 30 min. Bulk amount of cocrystal for other characterizations was prepared using this method. Single crystals were obtained when 40 mg of the ground product was dissolved in MeOH-H2O (1:2 v/v, 6 mL) and left for slow evaporation for 4-5 days.

1c: Method of preparing andrographolide-salicylic acid cocrystal (1c)
The cocrystal was obtained by grinding AP and salicylic acid in 1:1 stoichiometry with a few drops of water added in a liquid-assisted method for 30 min. Bulk amount of cocrystal for other characterizations was prepared using this method. Single crystals were obtained when 40 mg of the ground product was dissolved in ethyl formate-MeOH mixture (4:1 v/v, 6 mL) and left for slow evaporation for 4-5 days.

1d: Method of preparing andrographolide-guaiacol cocrystal (1d)
The cocrystal with GUL (a liquid) was obtained by grinding AP with guaiacol (the latter compound used in excess and methanol). Bulk amount of cocrystal for other characterizations was prepared using this method. A single crystal of 1:1 stoichiometry was obtained when the ground mixture was dissolved in EtOH-H2O (1:2 v/v, 6 mL) and left for slow evaporation for 4-5 days.

1e: Method of preparing andrographolide-resorcinol cocrystal (1e)
The cocrystal was obtained by grinding AP and resorcinol in 1:1 stoichiometry with a few drops of water added in a liquid-assisted method for 30 min. Bulk amount of cocrystal for other characterizations was prepared using this method. Single crystals were obtained when 40 mg of the ground product was dissolved in 6 mL of ethyl acetate and left for slow evaporation for 4-5 days.

The details of the crystallization of andrographolide cocrystals are given in Table 2. All the solid phases described herein were characterized by XRPD, FT-IR, FT-Raman and DSC thermogram and solid state 13C NMR, solution NMR and HRMS.

Table 2: Experimental techniques used to obtain cocrystals of andrographolide
Solid form Condition Time
Andrographolide-vanillin cocrystal (1a) (a) Solvent assisted grinding method for bulk material
Bulk material of this cocrystal was obtained by grinding andrographolide and vanillin in 1:1 stoichiometry with a few drops of CH3CN added in a liquid-assisted method. 30 mins
(b) solution crystallization for single crystals
Single crystals were obtained when 40 mg of the ground product was dissolved in MeOH-H2O (5:1 v/v, 6 mL) and left for slow evaporation. 4-5 days
Andrographolide-vanillic acid cocrystal (1b) (a) Solvent assisted grinding method for bulk material
Bulk material of this cocrystal was obtained by grinding andrographolide and vanillic acid in 1:1 stoichiometry with a few drops of CH3CN added in a liquid-assisted method. 30 mins
b) solution crystallization for single crystals
Single crystals were obtained when 40 mg of the ground product was dissolved in MeOH-H2O (1:2 v/v, 6 mL) and left for slow evaporation. 4-5 days
Andrographolide-salicylic acid cocrystal (1c) (a) Solvent assisted grinding method for bulk material
Bulk material of this cocrystal was obtained by grinding andrographolide and salicylic acid in 1:1 stoichiometry with a few drops of water added in a liquid-assisted method. 30 mins
b) solution crystallization for single crystals
Single crystals were obtained when 40 mg of the ground product was dissolved in ethyl formate-MeOH mixture (4:1 v/v, 6 mL) and left for slow evaporation. 4-5 days
Andrographolide-guaiacol cocrystal (1d)

(a) Solvent assisted grinding method for bulk material
Bulk material of this cocrystal was obtained by grinding andrographolide with excess of guaiacol and methanol. 30 mins
b) solution crystallization for single crystals
A single crystal of 1:1 stoichiometry was obtained when the ground mixture was dissolved in EtOH-H2O (1:2 v/v, 6 mL) and left for slow evaporation. 4-5 days
Andrographolide-resorcinol cocrystal (1e) (a) Solvent assisted grinding method for bulk material
Bulk material of this cocrystal was obtained by grinding andrographolide and resorcinol in 1:1 stoichiometry with a few drops of water added in a liquid-assisted method. 30 mins
b) solution crystallization for single crystals
Single crystals were obtained when 40 mg of the ground product was dissolved in 6 mL of ethyl acetate and left for slow evaporation. 4-5 days

Example 2
Spectral analysis
(a) X-ray Powder Diffraction (XRPD)
X-ray powder diffraction is a standard method for the characterization of solid-state forms. It is a characterization tool of the solid product after grinding two solid materials. When the resulting XRPD of the final product is different from the reactants, it can be concluded that a new solid phase has formed. The unique crystalline pattern of the cocrystals was monitored by the appearance of new diffraction peaks. XRPDs were recorded on SMART Bruker D8 Focus Powder X-ray diffractometer using Cu-Ka radiation (? = 1.5406 Å) at 40 kV and 30 mA. Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and figure 6 are the XRPDs of andrographolide, andrographolide-vanillin (1a) cocrystal, andrographolide-vanillic acid (1b) cocrystal, andrographolide-salicylic acid (1c) cocrystal, andrographolide-guaiacol (1d) cocrystal and andrographolide-resorcinol (1e) cocrystal respectively.

The powder X-ray diffraction of andrographolide (Figure 1) exhibits characteristic reflections at about 2? 9.75, 11.89, 14.80, 15.68, 18.38 and 22.48 ±0.2° (Table 3). The powder X-ray diffraction of andrographolide-vanillin (1a) cocrystal (Figure 2) exhibits characteristic reflections at about 2? 11.06, 11.96, 16.50, 16.73, 21.25 and 22.30±0.2° (Table 3). The powder X-ray diffraction of andrographolide-vanillic acid (1a) cocrystal (Figure 3) exhibits characteristic reflections at about 2? 9.67, 13.84, 15.70, 16.24, 16.63 and 20.79 ±0.2° (Table 3). The powder X-ray diffraction of andrographolide-salicylic acid (1a) cocrystal (Figure 4) exhibits characteristic reflections at about 2? 12.0, 13.61, 16.49, 19.70, 21.67 and 23.06 ±0.2° (Table 3). The powder X-ray diffraction of andrographolide-guaiacol (1a) cocrystal (Figure 5) exhibits characteristic reflections at about 2? 11.55, 11.77, 13.68, 16.55, 21.46 and 23.17 ±0.2° (Table 3). The powder X-ray diffraction of andrographolide-resorcinol (1a) cocrystal (Figure 6) exhibits characteristic reflections at about 2? 11.53, 16.22, 17.49, 18.45, 18.89 and 20.89 ±0.2° (Table 3).

Table 3: X-ray powder diffraction lines of andrographolide cocrystals characterized by 2? angle
Andrographolide Andrographolide-Vanillin 1a Andrographolide-Vanillic acid 1b
Angle 2? (?) d spacing (Å) Relative
Intensity (%) Angle 2? (?) d spacing (Å) Relative
Intensity (%) Angle 2? (?) d spacing (Å) Relative
Intensity (%)
9.748 9.06567 18.7% 8.551 10.33253 4.8% 8.305 10.63814 3.7%
11.890 7.43752 14.1% 9.337 9.46379 13.6% 9.673 9.13619 53.6%
14.801 5.98033 16.6% 9.692 9.11794 34.4% 10.833 8.16046 32.5%
15.684 5.64566 100.0% 11.062 7.99184 61.4% 11.546 7.65818 20.1%
17.443 5.07997 9.1% 11.963 7.39190 91.4% 11.898 7.43235 34.9%
18.385 4.82176 13.3% 13.973 6.33303 53.7% 13.839 6.39382 46.0%
19.069 4.65041 5.8% 15.110 5.85895 18.1% 14.737 6.00607 20.0%
22.160 4.00822 6.2% 15.772 5.61428 32.3% 15.699 5.64013 45.3%
22.488 3.95043 18.7% 16.501 5.36801 100.0% 16.238 5.45430 100.0%
26.602 3.34818 9.8% 16.733 5.29401 81.6% 16.625 5.32807 50.7%
27.062 3.29224 4.8% 17.154 5.16490 48.5% 18.214 4.86681 32.4%
29.832 2.99258 4.5% 18.628 4.75938 55.5% 19.537 4.53998 22.7%
40.876 2.20596 5.5% 19.523 4.54321 34.4% 20.792 4.26878 65.2%
20.829 4.26118 29.9% 21.888 4.05734 40.0%
21.259 4.17606 88.3% 22.547 3.94032 32.9%
22.297 3.98398 72.0% 22.933 3.87489 15.9%
22.835 3.89132 28.8% 25.739 3.45842 22.3%
26.080 3.41400 34.9% 26.240 3.39354 21.6%
27.358 3.25732 13.7% 28.032 3.18058 15.4%
28.353 3.14519 14.6% 31.538 2.83449 11.9%
31.977 2.79661 26.7% 32.795 2.72864 11.3%
38.819 2.31799 12.8% 44.099 2.05189 5.7%

Andrographolide-Salicylic acid 1c Andrographolide-Guaiacol 1d Andrographolide-Resorcinol 1e
Angle 2? (?) d spacing (Å) Relative
Intensity (%) Angle 2? (?) d spacing (Å) Relative
Intensity (%) Angle 2? (?) d spacing (Å) Relative
Intensity (%)
9.204 9.60100 6.3% 9.564 9.24008 16.5% 8.843 9.99214 6.6%
9.864 8.95982 10.2% 11.550 7.65520 36.6% 10.391 8.50631 4.2%
11.375 7.77250 25.1% 11.774 7.51018 58.2% 11.530 7.66886 33.1%
12.007 7.36505 39.1% 13.675 6.47035 40.2% 12.786 6.91818 31.7%
13.605 6.50346 28.7% 16.554 5.35072 100.0% 15.040 5.88597 22.2%
16.487 5.37234 100.0% 17.458 5.07586 19.9% 15.635 5.66319 14.4%
18.150 4.88366 22.4% 18.550 4.77924 21.3% 16.224 5.45878 35.6%
18.389 4.82087 25.2% 19.009 4.66492 17.4% 17.469 5.07264 100.0%
19.695 4.50395 38.8% 19.399 4.57195 24.3% 18.447 4.80588 38.3%
20.633 4.30120 8.4% 19.800 4.48040 24.9% 18.894 4.69319 33.2%
21.669 4.09788 34.1% 20.575 4.31319 29.0% 20.894 4.24810 32.6%
22.581 3.93446 21.2% 21.457 4.13789 54.1% 22.263 3.98992 27.8%
23.056 3.85451 26.1% 22.731 3.90886 18.2% 22.971 3.86856 24.4%
25.978 3.42716 15.0% 23.170 3.83570 45.9% 23.281 3.81766 29.5%
26.746 3.33051 12.0% 25.484 3.49248 16.0% 24.429 3.64083 11.8%
27.734 3.21406 10.1% 25.881 3.43974 11.7% 28.143 3.16826 9.2%
31.757 2.81540 7.0% 26.362 3.37809 26.5% 28.956 3.08114 9.9%
39.715 2.26774 4.7% 37.046 2.42471 5.7% 34.707 2.58258 7.6%

(b) Thermal analysis of andrographolide cocrystals
Differential scanning calorimetry (DSC) was carried out to investigate the thermal behavior of andrographolide cocrystals. DSC shows the exotherm/endotherm at which the solid sample undergoes phase transition and/or melting. DSC was performed on Mettler Toledo DSC 822e module by placing the samples, typically 4-6 mg, in aluminum pans and heated in the temperature range of 30-250°C at 5°C /min. From the area of the endotherm peak, enthalpy of fusion (?Hf) was calculated. DSC of andrographolide showed one endotherm at onset temperature of 233.65 ºC (Figure 7). The endotherm (Tonset 233.65 ºC, Tpeak 234.61 ºC) for andrographolide corresponds to the melting of this material. Similarly the endotherm (Tonset 162.05 ºC, Tpeak 164.43 ºC) for andrographolide-vanillin cocrystal 1a corresponds to the melting of this material (Figure 8). Andrographolide-vanillic acid cocrystal 1b exhibited endotherm (Tonset 196.63 ºC, Tpeak 197.35 ºC) that corresponds to the melting of this material (Figure 9). Similarly, andrographolide-salicylic acid cocrystal 1c exhibited endotherm (Tonset 171.01 ºC, Tpeak 172.47 ºC) that corresponds to the melting of this material (Figure 10). Andrographolide-guaiacol cocrystal 1d exhibited two endotherms, the first one at (Tonset 103 ºC, Tpeak 104.54 ºC) that corresponds to the vaporization of guaiacol from the crystal lattice and the second one at (Tonset 228.75 ºC, Tpeak 231.53 ºC) corresponds to the melting of this material (Figure 11). Andrographolide-resorcinol cocrystal 1e exhibited a single endotherm (Tonset 124.6 ºC, Tpeak 127.67 ºC) that corresponds to the melting of this material (Figure 12). The melting point of the cocrystal correlated, in general, with the m.p. of the coformer (Physicochemical properties of Pharmaceutical cocrystals: A case study of Ten AMG 517 Co-crystals, M. K. Stanton, A. Bak, Cryst. Growth Des., 2008, 8, 3856-3862; Drug substance and former structure property relationships in 15 diverse pharmaceutical cocrystals, M. K. Stanton, S. Tufekcic, C. Morgan, A. Bak, Cryst. Growth Des., 2009, 9, 1344-1352).

Table 4: Melting Point (onset, peak), Enthalpy of fusion (?Hfus) of andrographolide cocrystals
Form Andrographolide Andrographolide-vanillin, 1a Andrographolide-vanillic acid, 1b Andrographolide-salicylic acid, 1c Andrographolide-guaiacol, 1d Andrographolide-resorcinol, 1e
Tm (onset, peak, °C) 233.65, 234.61 162.05,
164.43 196.63,
197.35 171.01,
172.47 103.00, 104.54 and
228.75,
231.53 124.60,
127.67
Hf (KJ/mol) 47.19 69.30 72.75 39.70 28.82 27.24

(c) Single crystal X-ray diffraction
Good quality single crystal obtained from the crystallization solvent(s) given in Table 2 were mounted on the goniometer of Bruker SMART CCD diffractometer equipped with Mo-Ka radiation (? = 0.71073 Å) source. Data reduction in case of andrographolide-vanillin (1a) cocrystal was performed using Bruker SAINT software. (SADABS, Program for Empirical Absorption Correction of Area Detector Data, G. M. Sheldrick, University of Göttingen, Germany, 1997). This structure were solved and refined using SHELXL-97 (SMART (Version 5.625) and SHELX-TL (version 6.12), Bruker AXS Inc., Madison, WI, 2000) with anisotropic displacement parameters for non-H atoms. X-ray reflections of andrographolide-vanillic acid (1b) cocrystal, andrographolide-salicylic acid (1c) cocrystal, andrographolide-guaiacol (1d) cocrystal and andrographolide-resorcinol (1e) cocrystal were collected on Oxford CCD X-ray diffractometer (Yarnton, Oxford, UK) equipped with Mo-Ka radiation (? = 0.71073 Å) source. Data reduction was performed using CrysAlisPro 171.33.55 software (CrysAlis CCD and CrysAlis RED, Versions 1.171.33.55, Oxford Diffraction, Oxford, 2008). Crystal structures were solved and refined using Olex2-1.0 with anisotropic displacement parameters for non-H atoms (OLEX2: A complete structure solution, refinement and analysis program, O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J.A. K. Howard, H. Puschmann, J. Appl. Crystallogr. 2009, 42, 339-341). Hydrogen atoms on O atoms were experimentally located through Fourier map in all crystal structures. All C–H atoms were fixed using H-fix command geometrically. A check of the final CIF file with PLATON (PLATON, A Multipurpose Crystallographic Tool, A. L. Spek, Utrecht University, Utrecht, Netherland, 2002; Single-crystal structure validation with the program PLATON A. L. Spek, J. Appl. Cryst. 2003, 36, 7-13) did not show any missed symmetry.

Interestingly, all the cocrystals were found to be isostructural exhibiting 1D to 3D supramolecular isostructurality. Andrographolide-vanillin (1:1) cocrystal 1a was solved in the monoclinic chiral space group P21 and the asymmetric unit contains one molecule each of andrographolide and vanillin. The ORTEP diagram of Andrographolide-vanillin (1:1) cocrystal 1a and hydrogen bonding and molecular packing in the crystal structure are displayed in Figure 13(a) and (b). Andrographolide-vanillic acid (1:1) cocrystal 1b was solved in the monoclinic chiral space group P21 and the asymmetric unit contains one molecule each of andrographolide and vanillic acid. The ORTEP diagram of Andrographolide-vanillic acid (1:1) cocrystal 1b and hydrogen bonding and molecular packing in the crystal structure are displayed in Figure 14(a) and (b). Andrographolide-salicylic acid (1:1) cocrystal 1c was solved in the monoclinic chiral space group P21 and the asymmetric unit contains one molecule each of andrographolide and salicylic acid. The ORTEP diagram of Andrographolide-salicylic acid (1:1) cocrystal 1c and hydrogen bonding and molecular packing in the crystal structure are displayed in Figure 15(a) and (b). Andrographolide-guaiacol (1:1) cocrystal 1d was solved in the monoclinic chiral space group P21 and the asymmetric unit contains one molecule each of andrographolide and guaiacol. The ORTEP diagram of Andrographolide-guaiacol (1:1) cocrystal 1d and hydrogen bonding and molecular packing in the crystal structure are displayed in Figure 16(a) and (b). Andrographolide-resorcinol (1:1) cocrystal 1e was solved in the monoclinic chiral space group P21 and the asymmetric unit contains one molecule each of andrographolide and resorcinol. The ORTEP diagram of Andrographolide- resorcinol (1:1) cocrystal 1e and hydrogen bonding and molecular packing in the crystal structure are displayed in Figure 17(a) and (b).Crystallographic data and hydrogen bond geometrical parameters for andrographolide cocrystals are summarized in Table 5 and Table 6 respectively.

Table 5: Crystallographic parameters of Andrographolide cocrystals
Andrographolide-vanillin, 1a Andrographolide-vanillic acid, 1b Andrographolide-salicylic acid, 1c Andrographolide-guaiacol, 1d Andrographolide-resorcinol, 1e
Emp form. C28H38O8 C28H38O9 C27H36O7 C27H38O7 C26H36O7
Form wt 502.58 518.58 488.56 474.57 460.55
Cryst syst Monoclinic Monoclinic Monoclinic Monoclinic Monoclinic
Sp gr P21 P21 P21 P21 P21
T (K) 100(2) 298(2) 298(2) 298(2) 298(2)
a (Å) 10.2515(18) 10.3343(4) 10.576(6) 10.4954(6) 10.3340(11)
b (Å ) 12.468(2) 12.6800(4) 12.923(6) 12.8025(7) 11.6243(11)
c (Å ) 11.0827(19) 11.3675(5) 10.544(7) 10.7318(7) 10.5375(10)
a (º) 90 90 90 90 90
ß (º) 114.246(2) 113.201(5) 114.99(7) 117.962(8) 108.050(11)
? (º) 90 90 90 90 90
Z 2 2 2 2 2
V (Å3) 1291.6(4) 1369.11(9) 1306.1(13) 1273.65(13) 1203.5(2)
Rflns collect 13278 5577 4567 5303 4300
Unique rflns 5061 3946 3390 3937 3110
Obsd rflns 4882 2779 1352 2938 1386
Parameters 356 342 330 325 303
R1 0.0397 0.0401 0.0651 0.0361 0.0823
wR2 0.0985 0.0795 0.1026 0.0800 0.0860
GOF 1.035 0.892 0.863 0.923 0.872
Diffractometer Bruker
Smart Apex Oxford
Xcalibur Gemini Oxford
Xcalibur Gemini Oxford
Xcalibur Gemini Oxford
Xcalibur Gemini

Table 6: Hydrogen bond distances and angles in AP cocrystals (neutron-normalized O?H and C?H distance)
D?H···A D···A (Å) H···A (Å) D?H···A (°) Symmetry code
Andrographolide-vanillin, 1a
O1?H1···O3 2.719(2) 1.85 171 –1+x,y,z
O2?H2C···O1 2.6067(19) 1.87 147 Intramolecular
O3?H3C ···O5 2.755(2) 1.99 147 3-x,1/2+y,2–z
O8 ?H8C ···O7 2.670(2) 2.27 111 Intramolecular
O8?H8C ···O2 2.635(2) 1.89 154 –x,–1/2+y,1–z
C15 ?H15A···O5 3.199(6) 2.51 127 3?x,1/2+y, 2?z
C18?H18B···O6 3.431(2) 2.58 144 x,y,–1+z
C19?H19C···O1 2.940(7) 2.52 106 Intramolecular
C25?H25···O5 3.524(1) 2.60 165 ?2+x,y,z
C27?H27···O2 3.280(7) 2.43 148 x,y,1+z
Andrographolide-vanillic acid, 1b
O1?H1C···O3 2.738(3) 1.94 163 1+x,y,z
O2?H2C···O1 2.591(2 1.91 140 Intramolecular
O3?H3C···O5 2.778(3) 1.97 170 –1–x,–1/2+y,–z
O7?H7C···O2 2.676(3) 1.89 159 1–x,–1/2+y,2–z
O9 ?H9C ···O2 2.722(3) 2.01 146 –1+x,y,z
O9?H9C···O8 2.666(3) 2.22 114' Intramolecular
C19?H19A···O1 2.933(3) 2.53 105 Intramolecular
C26?26 ···O7 2.710(4) 2.40 100 Intramolecular
Andrographolide-salicylic acid, 1c
O1–H1C···O2 2.595(7) 1.96 133 Intramolecular
O2–H2C···O3 2.807(8) 2.03 157 1+x,y,z
O3–H3C···O5 2.803(8) 2.24 127 –1–x,1/2+y,–z
O7 –H7C···O1 2.579(10) 1.75 171(5) 1–x,-1/2+y,1–z
O8–H8C···O6 2.582(10) 1.86 147 Intramolecular
C14–H14···O7 3.334(11) 2.52 140 –x,1/2+y,–z
C15–H15A···O8 3.364(12) 2.45 158 –1–x,1/2+y,1–z
C19–H19C···O 2.895(10) 2.53 103 Intramolecular
C24–H24•••O6 3.236(3) 2.66 120 x–1, +y, +z
C24–H24•••O1 3.460(3) 2.70 138 –x, +y+1/2, –z+2
Andrographolide-guaiacol, 1d
O1–H1C•••O3 2.796(2) 1.98 173 1+x,y,z
O2 –H2C•••O1 2.662(2) 1.91 145 Intramolecular
O3–H3C•••O5 2.789(2) 2.01 158 –1-x,–1/2+y,–z
O6–H6C•••O7 2.646(3) 2.20 115 Intramolecular
O6–H6C•••O2 2.728(3) 1.98 152 2?x,1/2+y,1?z
C14 –H14 •••O6 3.290(3) 2.56 131 1?x, ?1/2+y, ?z
C15–H15A•••O6 3.330(3) 2.60 133 1?x, ?1/2+y, ?z
C19 –H19A•••O1 2.904(3) 2.49 106 Intramolecular
Andrographolide-resorcinol, 1e
O3?H3C···O4 3.138(5) 2.47 139 ?1?x,1/2+y, ?z
O3?H3C···O5 3.020(4) 2.28 150 ?1?x,1/2+y, ?z
O24?H24···O23 2.735(4) 2.07 165 1+x,y,z
O2?H2C···O1 2.617(7) 2.00 132 Intramolecular
O6?H6···O2 2.644(5) 1.95 142 1?x, ?1/2+y,1?z
O7?H7C···O6 2.859(4) 2.09 156 ?x,1/2+y, ?z
C15?H15B···O7 3.214(9) 2.51 130 Intramolecular
C19?H19C···O2 2.819(2) 2.48 101 Intramolecular
O32?H32···O25 2.644(5) 1.95 142 1?x, ?1/2+y,1?z
O33?H33···O32 2.859(4) 2.09 156 ?x,1/2+y, ?z
C5?H5B···O33 3.214(9) 2.51 130 Intramolecular
C20?H20C···O25 2.819(2) 2.48 101 Intramolecular

(d) FT-IR and Raman spectroscopy analysis
Infrared and Raman spectroscopy provide quantitative information about the vibrational modes of a compound, and change due to the physical state of the sample, and because of hydrogen bonding and molecular conformations. Nicolet 6700 FT-IR spectrometer with a NXR FT-Raman Module was used to record IR and Raman spectra. IR spectra were recorded on samples dispersed in KBr pellet. Raman spectra were recorded on samples contained in standard NMR tubes or on compressed solids placed on a gold coated sample holder. IR and Raman bands are likely to be active for virtually all the bonds, but their relative intensities will differ, the more symmetric ones give higher Raman intensities, while the asymmetric modes exhibit higher IR intensities. Generally hydroxyl group of alcohol and phenol absorbs strongly in the stretching frequency region 3500-3100 cm–1 region. There are two type of C-O stretching frequencies in andrographolide cocrystals, one is phenolic in coformers and other is alcoholic in andrographolide. Again C-O stretching vibration in alcohol and phenol produce strong hydrogen band in the 1260-1000 cm–1 and 1430-1410 cm–1 region in the IR spectrum respectively. Infrared stretching spetra for the andrographolide cocrystals are displayed in Figure 18-22 and the values are listed in Table 7. IR spectroscopy of all the cocrystals showed differences in O–H, C=O and C-O bond vibration regions. Similarly Raman spectroscopy showed differences between the spectra of cocrystals in comparision to their starting components in carbonyl stretch, phenolic as well as alcoholic C-O and O–H stretch (Figure 23-25). FT-Raman vibrational stretching frequencies of andrographolide-vanillin (1a), andrographolide-vanillic acid (1b), andrographolide-salicylic acid (1c) cocrystals are listed in Table 8. FT-Raman spectra of andrographolide-guaiacol (1d) and andrographolide-resorcinol (1e) cocrystals could not be obtained due to the burning of the sample.

Table 7: FT-IR Vibration stretching frequency (?s, cm–1) of andrographolide cocrystals in comparison with andrographolide and coformers
Andrographolide-vanillin, 1a
AP VAN AP-VAN
O-H stretch 3397.7 3184.2 3362.7
C=O stretch 1727.5 1665.3 1729.5,
C=C stretch 1674.9 1588.7 1673.6,1596.1
C-O stretch 1074.7 1028.9 1052.2,1032.0
O-H bend 1454 1465.0 1436.5,1466.5
Andrographolide-vanillic acid, 1b
AP VLA AP-VLA
O-H stretch 3397.7 3483.5 3326.4
C=O stretch 1727.5 1684.0 1723.4,1680.9
C=C stretch 1674.9 1597.4 1673.6,1595.0
C-O stretch 1074.7 1028.9 1051.2,1028.5
O-H bend 1454 1465.0 1430.5,1464.1
Andrographolide-salicylic acid, 1c
AP SLA AP-SLA
O-H stretch 3397.7 3238.0 3376.3,3473.5
C=O stretch 1727.5 1658.1 1730.6,1668.0
C=C stretch 1674.9 1483.7 1642.7,1484.3
C-O stretch 1074.7 1031.0 1055.2,1036.2
O-H bend 1454.0 1465.5 1450.9,1468.5
Andrographolide-guaiacol, 1d
AP GUL AP-GUL
O-H stretch 3397.7 3496.8 3400.6
C=O stretch 1727.5 -- 1728.3
C=C stretch 1674.9 1596.7 1673.8,1596.5
C-O stretch 1074.7 1039.5 1078.3,1030.0
O-H bend 1454 1469.5 1445.0,1468.7
Andrographolide-resorcinol,1e
AP RES AP-RES
O-H stretch 3397.7 3261.2 3400.0
C=O stretch 1727.5 -- 1727.7,
C=C stretch 1674.9 1608.0 1674.6,1607.3
C-O stretch 1074.7 -- 1055.0
O-H bend 1454 1489.4 1465.8,1488.5

Table 8: FT-Raman vibrational frequency (?s, cm–1) of AP cocrystals
Andrographolide-vanillin, 1a
AP VAN AP-VAN
C=O stretch 1724.3 1669.1 1730.5
C=C stretch 1675.7,1649.5 1594.6 1678.9,1642.5,1595.8
C-O stretch 1035.6 1032.6 1029.8
Andrographolide-vanillic acid, 1b
AP VLA AP-VLA
C=O stretch 1724.3 -- 1707.6
C=C stretch 1675.7, 1649.5 1661.9 1680.3,1645.5,1609.5
C-O stretch 1035.6 -- 1014.4
Andrographolide-salicylic acid, 1c
AP SLA AP-SLA
C=O stretch 1724.3 3207.9 1733.8
C=C stretch 1675.7, 1649.5 -- 1669.8, 1645.3
C-O stretch 1035.6 -- 1033.9

(e) Solid state NMR spectroscopy
Solid-state 13C NMR (SS-NMR) spectroscopy (Polymorphism and Crystallization of Active Pharmaceutical Ingredients (APIs). J. Lu, S. Rohani. Curr. Med. Chem., 2009, 16, 884-905) provides structural information about differences in hydrogen bonding, molecular conformations, and molecular mobility. The solid-state 13C NMR spectra were obtained on a Bruker ultrashieldTM 400 spectrometer utilizing a 13C resonant frequency of 400 MHz (magnetic field strength of 5.87 T). Approximately 100 mg of sample was lightly packed into a zirconium rotor with a Kel-F cap. The cross polarization, magic angle spinning (CP-MAS) pulse sequence was used for spectral acquisition. Each sample was spun at a frequency of 5.0±0.01 kHz and the magic angle setting calibrated by the KBr method. Each spectrum represents between 5 and 9k transients acquired under the following conditions: 4k data set zero filled to 16k, spectral width of 20000 Hz, 5 s recycle time, 2 ms contact time, and 7.4 ms pulse width. The Hartmann–Hahn match was optimized by monitoring the 13C intensity versus 13C radio frequency field with a spinning adamantane sample. Each data set was subjected to a 5.0 Hz line broadening factor and subsequently Fourier transformed and phase corrected to produce a frequency domain spectrum. All spectra were recorded at ambient temperature and the chemical shifts externally referenced to tetramethylsilane utilizing a spinning adamantane sample [d(CH3)4Si = d(adamantane CH2) -38.3]. The peak intensity for andrographolide cocrystals were in the ss-NMR spectrum suggests their crystalline nature. Differences in the cocrystals of andrographolide were analyzed by 13C cross-polarization and magic-angle spinning (CP-MAS) ss-NMR (Figure 26-30) and their ssNMR chemical shift values are shown in Table 9.

Table 9: ss-NMR 13C chemical shifts (?, ppm) of AP cocrystals
Carbon No. Andrographolide vanillin Andrographolide
Vanillin, 1a Vanillic acid Andrographolide
Vanillic acid, 1b
1 172.6 - 174.1 - 174.1
5 148.9 - 153.5 - 148.7
8 147.0 - 148.1 - 147.6
2 128.7 - 128.7 - 129.2
17 110.2 - 129.1 - 110.9
12 84.5 - 79.3 - 82.5
4 75.0 - 76.3 - 75.5
19 64.9 - 65.8 - 66.9
3 64.2 - 64.6 - 64.8
7 57.4 - 55.2 - 57.9
16 57.3 - 53.4 - 57.9
11,15 43.0 - 41.8 - 43.5
9 38.7 - 37.7 - 39.2
14 37.4 - 34.2 - 38.0
13 28.5 - 29.1 - 29.0
10 24.9 - 23.8 - 25.5
6 23.7 - 23.4 - 24.3
20 21.2 - 22.1 - 21.7
18 17.0 - 14.1 - 17.9
7’ - 192.7 192.5 173.4 173.2
4’ - 152.4 153.5 150.8 148.7
3’ - 147.3 148.1 146.4 145.2
1’ - 128.4 129.7 123.3 121.7
6’ - 116.0 116.1 120.9 115.8
2’ - 106.3 108.2 113.2 112.1
5’ - 104.3 106.9 111.4 110.9
8’ - 56.1 55.2 56.7 55.3

Carbon No. Salicylic acid Andrographolide
Salicylic acid, 1c Andrographolide
Guaiacol, 1d resorcinol Andrographolide
Resorcinol, 1e
1 - 173.4 173.1 - 173.6
5 - 147.4 149.7 - 150.7
8 - 147.4 147.5 - 146.5
2 - 125.5 125.7 - 128.1
17 - 110.2 109.0 -- 109.8
12 - 80.7 80.7 - 79.3
4 - 76.5 77.1 - 77.3
19 - 67.4 66.9 - 67.1
3 - 65.0 65.4 - 65.5
7 - 54.7 56.0 - 55.8
16 - 53.7 53.5 - 54.3
11,15 - 41.9 42.7 - 42.1
9 - 37.2 38.5 - 39.3
14 - 36.7 37.4 - 34.0
13 - 28.0 29.7 - 28.6
10 - 24.5 24.3 - 25.7
6 - 23.2 23.7 - 24.2
20 - 22.3 21.8 - 24.2
18 - 14.9 14.6 - 17.3
7’ 177.3 173.4 57.3 - -
4’ 139.1 140.6 121.1 107.7 109.8
3’ 118.7 117.5 111.0 133.3 131.8
1’ 112.1 112.7 129.4 155.8 158.5
6’ 133.6 130.9 114.6 103.4 104.7
2’ 162.4 162.4 154.4 108.4 109.8
5’ 120.9 121.4 123.4 155.8 158.5

Example 3
In vitro Chemical Transformation studies of andrographolide and its cocrystals
The primary reason for the poor bioavailability of andrographolide is due to its chemical transformation to four metabolites isolated from humans and rats (Four new andrographolide metabolites in human urine, L. Cui, F. Qiu, N. Wang, X. Yao, Chem Pharm Bull. 2004, 52, 772). Since cocrystals are known to inhibit degradation of drug molecules and retain their color (Crystal Engineering of Stable Temozolomide Cocrystals, N. J. Babu, P. Sanphui and A. Nangia, Chem. Asian J., 2012, 7, 1), novel cocrystals of andrographolide described in the present invention were prepared to overcome this drawback of andrographolide. The biochemical transformation of AP to AP-SO3H (Scheme 1), which limits the bioavailability of the active metabolite, was carried out in vitro on AP and its cocrystals (Identification of a rare sulfonic acid metabolite of andrographolide in rats, X. He, J. Li, H. Gao, F. Qiu, K. Hu, X. Cui, X. Yao, Drug Metab. Dispos. 2003, 31, 983-985). Accordingly, to a solution of AP (250 mg) in 6 mL ethanol, a mixture of 1 mL of 1M Na2SO3, 1.2 mL H2SO4 and 2 mL water were added. The reaction mixture was stirred at room temperature for one hour. After completion of the reaction, the pH value of the reaction mixture was adjusted to neutral by adding 2% H2SO4 and evaporated to dryness. The residue was dissolved in 8 mL water and extracted with 20 mL chloroform. Both the organic and aqueous layers were evaporated to dryness in a rotary evaporator. The water layer residue was dissolved in 10 mL methanol and filtered to remove inorganic salt Na2SO4. The filtrate was evaporated to dryness to obtain 227 mg of androsulfonate (AP-SO3H) in 77% yield.

The above procedure was repeated for each cocrystal by keeping the amount of AP constant (250 mg, 0.71 mmol). Except andrographolide-vanillic acid (1b) and andrographolide-salicylic acid (1c) the other cocrystals andrographolide-vanillin (1a) andrographolide-guaiacol (1d) and andrographolide-resorcinol (1e) reacted in the same way to give AP in 70-80% yield. Andrographolide-vanillic acid (1b) gave 43% AP-SO3H by-product formed and 57% unreacted AP by solution NMR analysis. Andrographolide-salicylic acid (1c) cocrystal did not result in any side product formation (AP-SO3H), pure AP was isolated in 75% yield. The NMR spectra of Andrographolide (AP) and Andrographolide sulfate (AP-SO3H) are shown in Figure 31-34, and HRMS of AP-SO3H in Figure 35. The chemical transformation analysis of andrographolide and cocrystals Andrographolide-Vanillin (1a), Andrographolide-Vanillic acid (1b), Andrographolide-Salicylic acid (1c), Andrographolide-Guaiacol (1d), Andrographolide-Resorcinol (1e) are shown in Figures 36-40.

Solution NMR spectra were recorded on Bruker Avance 400 MHz spectrometer (Bruker-Biospin, Karlsruhe, Germany)
AP: 1H NMR (DMSO-d6, ?, ppm): 0.62 (s,3H), 1.06 (3H, d, J10.67), 1.28 (3H,d,J13.62), 1.68 (4H,dd,J 10.03-17.03), 1.89 (2H,d,J11.61), 2.27 (1H,s), 2.46 (1H,d,J14.62), 3.21 (2H), 3.84 (2H), 4.03 (1H,d,J9.32), 4.28 (1H,s), 4.38 (1H), 4.58 (1H,s), 4.78 (1H,s), 4.89 (1H), 5.82 (1H,s), 6.61 (1H,t,J7.32).
AP 13C NMR (DMSO-d6, ?, ppm): 15.1, 23.3, 24.4, 28.2, 36.8, 37.0, 42.6, 54.7, 55.8, 63.1, 64.9, 74.8, 78.9, 108.7, 129.2, 147.1, 147.9, 170.6.
AP-SO3H: 1H NMR (DMSO-d6, ?, ppm):0.052(3H,s) 0.08 (3H,s), 0.09 (1H,s), 1.00 (4H,s), 1.24 (2H,d,J11.70), 1.75-XX (6H, m,J11.32-14.62), 2.09 (1H, t, J 13.62,), 2.25(1H, d, J 12.32), 3.16 (2H, t, J 13.56), 3.47(1H, d, J 12.66), 4.90 (4H,dd,J13.37-14.62), 7.50(1H,t,J6.56)
AP-SO3H: 13C NMR (DMSO-d6, ?, ppm): 15.2, 23.2,24.3,27.0, 28.1, 36.7, 38.2,42.6, 53.0, 53.9, 54.8, 63.1, 71.0, 78.8, 107.8, 131.2, 147.6, 148.7, 174.6

Example 4
Solubility of andrographolide and andrographolide-salicylic acid cocrystal (1c)
An important goal of drug development is to maintain optimum physicochemical parameters which have a major impact on its efficacy. Solubility and permeability of a drug molecule determine its mode of administration into the body. Solubility remains a major concern for poorly soluble drugs or bioactive compounds since it largely limits their bioavailability (A provisional biopharmaceutical classification of the top 200 oral drug products in the united states , Great Britain, Spain and Japan, T. Takagi, C. Ramachandran, M. Bermejo, S. Yamashita, L. X. Yu, G. L. Amidon, Mol. Pharmaceutics, 2006, 3, 631). Hence, physical or chemical modifications are necessary for such drugs in order to enhance solubility and improve therapeutic efficacy. Solubility and dissolution experiments were performed on andrographolide and its stable andrographolide-salicylic acid (1c) cocrystal with the intention of ascertaining the solubility advantage conferred by the cocrystal. The solubility curve of andrographolide and andrographolide-salicylic acid (1c) was measured using the Higuchi and Connor method (Phase solubility techniques, T. Higuchi, K. A. Connors, Adv. Anal. Chem. Instrum. 1965, 4, 117) in 25% ethanol–water medium at 30 °C. First, the absorbance of a known concentration of the pure compound was measured at the given ?max (AP 225 nm, AP-SA 227 nm) in 25% ethanol–water on a Thermo Scientific Evolution 300 UV–vis spectrometer (Thermo Scientific, Waltham, MA). These absorbance values were plotted against several known concentrations to prepare the concentration vs. intensity calibration curve. From the slope of the calibration curves, molar extinction coefficients for AP and AP-SA were calculated. An excess amount of the sample was added to 6 mL of 25% ethanol–water. The supersaturated solution was stirred at 300 rpm using a magnetic stirrer at 30 °C. After 24 h, the suspension was filtered through Whatman’s No. 1 filter paper. The solubility of AP–SLA (4.55 g/L) was found to be 12 times higher than that of AP (0.38 g/L). The filtered aliquots were diluted sufficiently, and the absorbance was measured at the given ?max. Both AP and andrographolide-salicylic acid (1c) cocrystal forms are stable under the solubility conditions as analyzed by PXRD match of the residue at the end of the experiment. IDR experiments were carried out on USP-certified Electrolab TDT-08L dissolution tester type II (paddle) (Mumbai, India). Dissolution experiments were performed for 6 h in 25% ethanol–water at 37 °C. Prior to IDR estimation, standard curves for all the compounds were obtained spectrophotometrically at their respective ?max. The calculated molar extinction coefficients were used to determine the IDR values. For IDR measurements, 200 mg of the compound was taken in the intrinsic attachment and compressed to 0.5-cm2 disk using a hydraulic press 4.0 ton/ in2 pressure for 5 min. The intrinsic attachment was placed in a jar of 500 mL medium preheated to 37 °C and rotated at 150 rpm. 5 mL of the aliquot was collected at specific time intervals, and the concentration of the aliquots was determined with appropriate dilutions from the predetermined standard curves of the respective compounds. The IDR of the compound was calculated in the linear region of the dissolution curve (which is the slope of the curve or amount of drug dissolved/surface area of the disk) per unit time. The amount of andrographolide dissolved (mg/L) vs. time (min) are plotted in Figure 41 and solubility values and intrinsic dissolution rates for the andrographolide-salicylic acid cocrystal and the parent compound are listed in Table 10. The intrinsic dissolution rate (IDR) of andrographolide and andrographolide-salicylic acid cocrystal was found to be 0.090 x 10-3 mg/cm2/min and 0.277 x 10-3 mg/cm2/min respectively. Thus andrographolide-salicylic acid cocrystal dissolves 3 times faster than the commercial andrographolide. The identity of the undissolved material at the end of dissolution/solubility experiment was confirmed by PXRD (Figure 42, 43). The nature of the solid samples after disk compression and solubility/dissolution measurements was verified by PXRD (to ascertain any phase changes, hydration, etc).

Table 10: IDR and solubility of andrographolide-salicylic acid (1c) cocrystal. The enhancement compared to AP is given in parentheses.
Compound Molar extinction coefficient, ? /mM cm Equilibrium Solubility (g/L)
Intrinsic dissolution rate, IDR (mg/cm2)/min (x10-3) Area under the curve, AUC0-6h
(mg h)/L
AP 13.02 0.378
0.090 3025.95
Andrographolide-Salicylic acid, 1c 21.33 4.555 (x 12.04) 0.277 (x 3.01) 6137.74 (x 2.03)

Industrial Advantages:
The novel co crystals of Andrographolide with the GRAS coformers of the instant invention improved the solubility and dissolution rate of AP while inhibiting the undesired chemical transformation of AP in in vitro conditions. The cocrystal of Andrographolide-Salicylic acid, showed complete inhibition of chemical transformation of Andrographolide in invitro conditions and it also exhibited 3 times more dissolution rate and 2 times higher drug release compared to pure Andrographolide. ,CLAIMS:We claim,

1. Novel co-crystals of andrographolide comprising andrographolide and GRAS co formers in fixed stoichiometry which inhibits chemical transformation of Andrographolide in invitro conditions, improves solubility and dissolution rate of Andrographolide and enhances its bioavailability.

2. The cocrystal of andrographolide according to claim 1, wherein the GRAS coformers are selected from 2,6-dihydroxybenzoic acid, 3.4-dihydroxybenzoic acid, 4-aminobenzoic acid, benzoic acid, vanillin, hydroquinone, vanillic acid, catechol, acetamide, resorcinol, guaiacol, fumaric acid, nicotinamide, salicylic acid, ferulic acid, adipic acid and the like.

3. The cocrystal of andrographolide according to claim 2 is andrographolide-vanillin (1a) cocrystal characterized by at least;
i. powder X-ray diffraction having characteristic peaks at 2 theta angle 11.06, 11.96, 16.50, 16.73, 21.25 and 22.30±0.2°;
ii. exhibiting a melting onset endotherm at Tonset of 162.05 °C; and
iii. having a monoclinic crystal lattice system in space group P21 with the unit cell parameters a = 10.2515(18) Å, b = 12.468 (2) Å, c = 11.0827 (19) Å, a = ? = 90?, ß = 114.246 (2), V = 1291.6 (4) Å3
4. The cocrystal of andrographolide according to claim 2 is andrographolide-vanillic acid (1b) cocrystal characterized by at least;
i. powder X-ray diffraction having characteristic peaks at 2 theta angle 9.67, 13.84, 15.70, 16.24, 16.63 and 20.79 ±0.2°;
ii. exhibiting melting endotherm onset at Tonset of 196.63 °C; and
iii. having a monoclinic crystal system in space group P21 with the unit cell parameters a = 10.3343(4) Å, b = 12.6800 (4) Å, c = 11.3675 (5) Å, a = ? = 90?, ß = 113.201 (2), V = 1369.11 (9) Å3.

5. The cocrystal of andrographolide according to claim 2 is a andrographolide-salicylic acid (1c) cocrystal characterized by at least;
i. powder X-ray diffraction having characteristic peaks at 2 theta angle 12.0, 13.61, 16.49, 19.70, 21.67 and 23.06 ±0.2°;
ii. exhibiting melting endotherm onset at Tonset of 171.01 °C; and
iii. having a monoclinic crystal system in space group P21 with the unit cell parameters a = 10.576(6) Å, b = 12.923 (6) Å, c = 10.544 (7) Å, a = ? = 90?, ß = 114.99 (7), V = 1306.1 (13) Å3.

6. The cocrystal of andrographolide according to claim 2 is a andrographolide-guaiacol (1d) cocrystal characterized by at least;
i. powder X-ray diffraction having characteristic peaks at 2 theta angle 11.55, 11.77, 13.68, 16.55, 21.46 and 23.17 ±0.2°;
ii. exhibiting two melting endotherms with onsets at Tonset of 103°C and 228.75°C; and
iii. having a monoclinic crystal system in space group P21 with the unit cell parameters a = 10.4954 (6) Å, b = 12.8025 (7) Å, c = 10.7318 (7) Å, a = ? = 90?, ß = 117.962 (8), V = 1273.65 (13) Å3.

7. The cocrystal of andrographolide according to claim 2 is a andrographolide-resorcinol (1e) cocrystal characterized by at least;
i. powder X-ray diffraction having characteristic peaks at 2 theta angle 11.53, 16.22, 17.49, 18.45, 18.89 and 20.89 ±0.2°;
ii. exhibiting melting endotherm onset at Tonset of 124.6 °C; and
iii. having a monoclinic crystal system in space group P21 with the unit cell parameters a = 10.3340 (11) Å, b = 11.6243 (11) Å, c = 10.5375 (10) Å, a = ? = 90?, ß = 108.050 (11), V = 1203.5 (2) Å3.

8. A process for preparation of novel cocrystals of andrographolide comprising andrographolide and GRAS coformers comprising grinding andrographolide and coformer, in fixed stoichiometric ratio, with a few drops of solvent in a liquid-assisted method for about 30 min followed by dissolving the grinded mixture in a solvent either alone or mixtures thereof, leaving the solution and isolating co-crystals after 4-5 days.

9. The process according to claim 8, wherein the coformers are selected from 2,6-dihydroxybenzoic acid, 3.4-dihydroxybenzoic acid, 4-aminobenzoic acid, benzoic acid, vanillin, hydroquinone, vanillic acid, catechol, acetamide, resorcinol, guaiacol, fumaric acid, nicotinamide, salicylic acid, ferulic acid, adipic acid and the like.

10. The process according to claim 8, wherein the solvent is selected from polar organic solvents such as water, acetonitrile, lower alcohols, ethyl formate, ethylacetate, acetone and the like either alone or as mixtures thereof.
11. The process for preparation of andrographolide-vanillin cocrystal (1a) according to claim 8 comprises grinding andrographolide and vanillin in 1:1 stoichiometry with a few drops of CH3CN added in a liquid-assisted method for 30 min and dissolving the grinded mixture in MeOH-H2O (5:1 v/v, 6 mL) leaving the solution and isolating andrographolide-vanillin crystals after 4-5 days.

12. The process for preparation of andrographolide-vanillic acid cocrystal (1b) according to claim 8 comprises grinding andrographolide and vanillic acid in 1:1 stoichiometry with a few drops of CH3CN added in a liquid-assisted method for 30 min and dissolving the grinded mixture in MeOH-H2O (1:2 v/v, 6 mL) leaving the solution and isolating andrographolide-vanillic acid crystals after 4-5 days.

13. The process for preparation of andrographolide-salicylic acid cocrystal (1c) according to claim 8 comprises grinding andrographolide and salicylic acid in 1:1 stoichiometry with a few drops of water added in a liquid-assisted method for 30 min and dissolving the grinded mixture in ethyl formate-MeOH mixture (4:1 v/v, 6 mL) leaving the solution and isolating andrographolide-salicylic acid crystals after 4-5 days.

14. The process for preparation of andrographolide-guaiacol cocrystal (1d) according to claim 8 comprises grinding andrographolide with guaiacol (in excess) and methanol as a solvent for 30 min and dissolving the grinded mixture in EtOH-H2O (1:2 v/v, 6 mL) leaving the solution and isolating andrographolide-guaiacol crystals after 4-5 days.

15. The process for preparation of andrographolide-resorcinol cocrystal (1e) according to claim 8 comprises grinding andrographolide with resorcinol in 1:1 stoichiometric ratio with a few drops of water added in a liquid-assisted method for 30 min and dissolving the grinded mixture in 6 mL of ethyl acetate and leaving the solution and isolating andrographolide-resorcinol crystals after 4-5 days.

16. Novel co-crystals of andrographolide comprising andrographolide and an anti-oxidant selected from curcumin, quercetin, juglone, lawsone, menadione, plumbagin, piperine as anti-viral, anti-inflammatory, anti-cancer, and anti-malarial agents.

17. A pharmaceutical composition containing an effective amount of one or more of the solid forms selected from andrographolide-vanillin (1a), andrographolide-vanillic acid (1b), andrographolide-salicylic acid (1c), andrographolide-guaiacol (1d), andrographolide-resorcinol (1e) in combination with pharmaceutically acceptable excipients.

Dated this 17th day of July, 2014

Dr. P. Aruna Sree
(Regn.No.: IN/PA 998)
Agent for the Applicant
Gopakumar Nair Associates