Hepatoprotective Cocrystals of Isoniazid: Synthesis, Solid State

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Hepatoprotective Cocrystals of Isoniazid: Synthesis, Solid State Characterization and Hepatotoxicity Studies Balwant Yadav, Anilkumar Gunnam, Rajesh Thipparaboina, Ashwini K. Nangia, and Nalini R. Shastri Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.9b00541 • Publication Date (Web): 25 Jul 2019 Downloaded from pubs.acs.org on July 26, 2019

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Crystal Growth & Design

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Hepatoprotective Cocrystals of Isoniazid: Synthesis, Solid State

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Characterization and Hepatotoxicity Studies

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Balvant Yadava, Anilkumar Gunnamb, Rajesh Thipparaboinaa, Ashwini K. Nangiabc,

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Nalini R Shastria*

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aSolid

State Pharmaceutical Research Group (SSPRG), Department of Pharmaceutics,

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National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, 500037,

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India.

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bSchool

of Chemistry, University of Hyderabad, Central University PO, Prof. C. R. Rao Road, Hyderabad, India

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cCSIR-

National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411 008, India

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* Corresponding author. Nalini R Shastri

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Tel. +91-040-23423749

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Fax. +91-040-23073751

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E-mail: [email protected], [email protected]

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Address: Department of Pharmaceutics, National Institute of Pharmaceutical Education &

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Research (NIPER), Balanagar, Hyderabad, India, Pin code - 500037

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Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Isoniazid (INH) is one of the first line drugs used in combination with pyrazinamide and

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rifampicin for the management of tuberculosis. Idiosyncratic hepatotoxicity is one of the

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most common side effects of anti-tubercular therapy worldwide. The current study explores

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solid phase modification of INH by cocrystallization with various hepatoprotective

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coformers, namely chrysin, hesperetin, silibinin, syringic acid (SYRA) and quercetin (QUE)

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to address hepatotoxicity concerns. Cocrystals were obtained with SYRA and QUE.

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Supramolecular synthons based on pyridine-carboxyl and pyridine-hydroxyl synthon enabled

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the formation of cocrystals. INHSYRA and INHQUE cocrystals were characterized by FT-

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IR, DSC and PXRD. Single crystal X-ray analysis of INHSYRA revealed that it crystallised

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in triclinic system with P -1 space group. Intrinsic dissolution rate studies (IDR) showed slow

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drug release from both the cocrystals. Hepatoprotective effects of INHSYRA and INHQUE

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cocrystals were evaluated by single toxic dose study and sub chronic study for 28 days.

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Results from sub chronic study indicated significant increase in ALT, AST and ALP enzyme

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levels in INH treated group whereas the enzyme levels in INHSYRA and INHQUE cocrystal

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treated group were comparable to that of the untreated group. This study demonstrates in vivo

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hepatoprotective effects of coformers SYRA and QUE provides promising evidence for

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utility of nutraceutical based coformers, for tackling hepatotoxicity associated with various

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drugs.

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Keywords: Syringic acid, Quercetin, Hepatoprotective, solubility, IDR, anti-tuberculosis

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1

Introduction

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Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis1. In 2017,

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TB caused an estimated 1.3 million deaths among HIV negative people with an additional 3

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lakh deaths in HIV positive people. It is one of the top 10 causes of death globally. An

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estimated 54 million lives were saved through TB diagnosis and treatment between 2000 and

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2017. Intensified research and innovation is one of the strategic pillars laid down by WHO

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for culminating the TB epidemic by 20302. Treatment of TB involves a dosage regimen of a

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combination of drugs like isoniazid (INH), pyrazinamide, rifampicin, ethambutol and

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streptomycin. These are first line anti-TB drugs.

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Mycobacterium tuberculosis, the antimicrobial agents (INH and rifampicin) are insufficient

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to kill the bacteria due to which rifampicin-resistance TB and multidrug resistance TB occurs.

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Hence to avoid resistance, TB treatment uses multiple drugs in combination for chronic

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therapy. First line anti-TB drugs are given with second line drugs to avoid resistance, which

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includes at least one flouroquinolones, injectables agent (amikacin, capreomycin or

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kanamycin) and less effective ethionamide, cycloserine and paraminosalicylic acid2, 3. INH a

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first line antibiotic, is used with other drugs in combination to combat the TB. In combination

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with other drugs, INH stability is a major concern due to its degradation4, 5. INH produces

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severe idiosyncratic hepatotoxicity but it is preferred due to its efficacy6. Hepatotoxicity

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associated with INH hence needs careful assessment during the chronic therapy to avoid any

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adverse effects.

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High-throughput screening in combination with combinatorial chemistry, target protein

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identification and computational chemistry has increased the understanding of the target

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binding of small molecules7. These discovery tools often result in an identification of a higher

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proportion of lipophilic, high molecular weight and poorly soluble molecules, which do not

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adhere to Lipinski’s rules8, 9. Therapeutic advantages of drugs are often limited due to

Due to emergence of new strain of



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solubility and permeability issues. A variety of approaches are adopted by a formulation

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scientist to improve solubility, which include particle size reduction10, drug dispersion using

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matrix11, salt formation12, complexation13, use of cosolvent14, solubilizers15, hydrotrophy16

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and crystal engineering17. Crystal engineering can be described as ‘exploitation of

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noncovalent interactions between molecular or ionic components for the rational design of

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solid-state structures that might exhibit interesting electrical, magnetic, and optical

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properties’17.

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polymorphism, solvate and cocrystal. Crystal engineering has emerged as a prospective tool

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to modulate physico-chemical properties of new drug molecules in recent years with proper

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scientific approach18, 19. Cocrystals can improve various properties of drug molecules like

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solubility20, permeability21, bioavailability22, hygroscopocity23, hardness24 and alleviate side

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effects25. With modulation in property of drug molecule, cocrystal synthesis has been given

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much importance26, 27. As per USFDA guideline, cocrystal is considered as new polymorph of

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parent drug molecule which will open new avenue in pharmaceutical industry28. Salts are also

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produced by crystal engineering approach which tend to increase the dissolution and

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bioavailability29, 30.To modify the property of drug molecules suitable coformers are selected

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to address the above problems. These drug-drug and drug-nutraceutical cocrystals are

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extensively investigated to explore therapeutic advantages of individual components. Though

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various studies are reported in this perspective, in vivo evidence proving the efficacy is

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limited31, 32. Nutraceutical molecules have various therapeutic properties that could increase

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its use in cocrystallization process33,

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therapeutic advantage25, 35.

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INH (Supplementary Figure S1) is a therapeutic agent enlisted in WHO list of essential

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medicines and is generally selected as a model drug for studies in crystal engineering, due to

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its structural ability to form a variety of solid forms. Various cocrystals of INH with ferulic

Crystal

engineering

approaches

34

include

crystal

habit

modification,

concurrently tackling the side effect of drugs for


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acid, vanillic acid, caffeic acid, resorcinol, 4-hydroxy benzoic acid, fumaric acid, succinic

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acid, nicotinamide, para-aminosalicylic acid, gallic acid, 2,3-dihydroxy benzoic acid, 3,5-

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dihydroxy benzoic acid, 3-hydroxy benzoic acid, p-nitrobenzoic acid, p-cyanobenzoic acid

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and p-aminobenzoic acid have been reported36,37,38,39,40,41. de Melo et al reported sulphate salt

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and sulphate salt hemihydrate of INH42. Liu et al reported a ternary cocrystal of INH,

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pyrazinamide and fumaric acid43.

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In the current study, cocrystallization of INH was explored with novel nutraceutical

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coformers (Supplementary Figure S2); syringic acid (SYRA) and quercetin (QUE) to counter

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hepatotoxic effects of INH. SYRA is an O-methylated trihydroxybenzoic acid known for its

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hepatoprotective44 and antimicrobial effects45. Carboxylic acid functionality present in SYRA

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favours formation of carboxylic acid-pyridine heterosynthon, and hydroxy and methoxy

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groups facilitates strong adhesive interactions favouring formation of multicomponent

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systems46. QUE is a polyphenolic compound known for its antioxidant properties and

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hepatoprotective effects47. Structurally it enables formation of multicomponent systems

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through O-H···O and O-H···N interactions35, 48, 49 (Supplementary Figure S2). To the best of

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our knowledge, no hepatototoxicity study has been performed for cocrystal of INH. Hence,

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the two cocrystals of INH with hepatoprotective coformer SYRA and QUE was synthesized,

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characterized and evaluated for in vivo hepatoprotective effects. Single dose hepatotoxicity

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study was performed for INHSYRA and INHQUE cocrystal in male BALB/c mice. In

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addition, for the first time, a subchronic study for 28 days was conducted in male BALB/c

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mice for INH cocrystal.

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2

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2.1

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INH was kindly gifted by Macleod Pharma, Mumbai, India. SYRA and QUE were purchased

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from Alfa Aesar, India and Sigma-Aldrich, India respectively. HPLC grade methanol was

Experimental section Materials



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purchased from Merck, India. Double distilled water was generated in house. All other

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solvents and chemicals were of analytical grade.

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2.2

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2.2.1

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Preliminary screening was carried out by grinding unit components, drug and the coformers

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in 1:1 ratio for 30 min in an agate mortar in the presence of 120 μL of methanol

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(Supplementary Table S1).

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2.2.2

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2.2.2.1 INHSYRA

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Single crystals were obtained by anti-solvent crystallization using isopropanol and n-hexane.

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INH (68 mg) and SYRA (99 mg) were added to 7 volumes of isopropanol at 70°C. As the

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drug and coformers dissolved, the mixture was filtered and 2 volumes hexane was added to

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the solution. This mixture was kept for slow evaporation at room temperature. Single crystals

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were obtained after 2-3 days from the solution.

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2.2.2.2 INHQUE

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Single crystals suitable for structural elucidation were not obtained for the INHQUE system

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in various solvent systems. INH and QUE were taken in 1:1, 2:2 and 1:2 molar ratios to

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generate cocrystal in methanol, ethanol, isopropanol, acetone, ethyl acetate and

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dichloromethane separately but single crystals of INHQUE cocrystal was not formed. Anti-

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solvent crystallisations (methanol /hexane) as well as ternary solvent systems (water: ethyl

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acetate: methanol) were attempted, which also failed to give acceptable single crystals of

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INHQUE. All crystallization experiments are presented in Supplementary Table S2.

Solid form screening Liquid assisted grinding

Anti-solvent crystallization


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2.3

Characterization

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2.3.1

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Melting points of all solid forms were identified using melting point apparatus Stuart®

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SMP30 by capillary melting method.

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2.3.2

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Mettler Toledo DSC system operating with Stare software was used for determining the

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changes upon heating of INH and its cocrystals. Indium was used for calibration of the DSC

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system. Accurately weighed samples (5-15 mg) were taken in 40 μL aluminium crimped pans

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with pinhole and were scanned at a heating rate of 10 °C/min over a temperature range of 30-

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300 °C in nitrogen gas environment with a purging rate of 60 mL/min.

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2.3.3

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Powder XRD patterns of samples were recorded at room temperature using PANalytical

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X’Pert PRO X-ray Powder Diffractometer (Eindhoven, Netherlands), using Ni-filtered Cu Kα

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radiation (λ = 1.5406 Å). The data were recorded over a scanning 2θ range of 2° to 50° at

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step time of 0.045 steps/0.5 s.

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2.3.4

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IR spectra were recorded using a Bruker Vector 22 FT-IR spectrometer (Bruker, Germany)

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equipped with a Golden Gate single reflection diamond attenuated total reflectance (ATR)

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accessory (Specac). The spectral range was set between 4000-500 cm−1 and the spectral

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resolution at 4 cm−1. Samples of 1 to 2 mg were taken to record the IR spectrum of solid

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forms.

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2.3.5

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X-ray reflections were collected on Bruker D8 QUEST, CCD diffractometer equipped with a

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graphite monochromator and Mo-Kα fine-focus sealed tube (λ = 0.71073 Å) and reduction

Melting point

Differential Scanning Calorimetry (DSC)

Powder X ray Diffraction (Powder XRD)

Fourier-transform infrared spectroscopy (FTIR)

Single crystal X-ray diffraction



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was performed using APEXIII Software. Intensities were corrected for absorption using

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SADABS and the structure was solved and refined using SHELX201850. All non-hydrogen

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atoms were refined anisotropically. Hydrogen atoms on hetero atoms were located from

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difference electron-density maps and all C–H hydrogen atoms were fixed geometrically.

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Hydrogen-bond geometries were determined in PLATON51, 52. X-Seed was used to prepare

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packing diagrams53, 54. Crystallographic cif file is available at www.ccdc.cam.ac.uk/data or as

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part of the Supporting Information (CCDC Nos. 1865066).

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2.3.6 Solubility study

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Solubility study for INH, cocrystals and their physical mixture were carried out by adding

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excess quantity in 5 ml of water, 0.1 N HCL (pH 1.2), acetate buffer (pH 4.5) and phosphate

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buffer (pH 6.8). Julabo SW22 shaker was used for continuous stirring of suspension at 37 °C

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and 100 RPM for 12 hours. Suspensions were later centrifuged at 12000 RPM for 10 minute.

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Supernatants were then filtered using MF-Millipore™

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cellulose ester of 0.45μ size, suitably diluted with mobile phase and analyzed using HPLC

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method as given below.

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membrane filter made of mixed

2.3.7 In vitro dissolution study

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USP apparatus II (paddle type) was selected for dissolution study of the plain drug, cocrystal

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and their physical mixtures in 450 ml of media. The dissolution studies were conducted at

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37± 0.5 °C, at 50 rpm. Dissolution media was selected as 0.1N HCL (pH 1.2) and phosphate

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buffer (pH 6.8). INH, INHSYRA, INHQUE, physical mixture of (INH and SYRA) and (INH

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and QUE) equivalent to 150 mg of INH were added to the dissolution media. All samples

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were passed through BSS #sieve no. 44 and retained on BSS #sieve no. 60. These sieved

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materials were added to the dissolution media. Aliquots of 5 ml were withdrawn at

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predetermined time points (5, 10, 15, 30, and 60 min) substituting the same with equal

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quantity of fresh pre-warmed dissolution media. Samples were filtered using MF-Millipore™ 8 ACS Paragon Plus Environment

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membrane filter made of mixed cellulose ester of 0.45μ size and analyzed by a validated

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HPLC method after suitable dilution with the mobile phase. Dissolution profiles of plain drug

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and novel solid forms were evaluated for the amount of drug released at 10 min (Q10), %

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dissolution efficiency (DE) at 10 min and similarity factor using DDsolver55. Each

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experiment was performed in duplicate.

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2.3.8

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IDR study for INH, SYRA, QUE, INHSYRA and INHQUE were performed on USP certified

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instrument by rotating disc method. IDR was conducted with 100 mg of INH, cocrystals with

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equivalent amount of 100 mg of INH and coformers (SYRA and QUE) equivalent amount

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present in cocrystal. Pellet was formed at by using pellet press with die and punch at pressure

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of 5 tonnes for 5 minute to form non disintegrating compacts. The intrinsic dissolution rate

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attachment was placed in jar of 500 mL of phosphate buffer (pH 6.8)56 at 37 ± 0.5 °C and

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stirred at 75 rpm. Aliquots of 5 mL were withdrawn at predetermined time points (5, 10, 15,

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30, 45 and 60 min) substituting the same with equal quantity of fresh dissolution media.

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Samples were filtered using MF-Millipore™ membrane filter made of mixed cellulose ester

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of 0.45 μ size and analyzed by a validated HPLC method after suitable dilution with the

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mobile phase.

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2.3.9

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Drug and coformers were quantified using a HPLC system (e2695 Waters) consisting of an

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autosampler, a HPLC pump and an automated injector equipped with a UV detector (2998

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PDA). Acetic acid (1% v/v) in water and methanol (50: 50) for INHSYRA and (30:70) for

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INHQUE were used as mobile phase at a flow rate of 1 mL/min in isocratic mode on

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Inertsustain C18 (5μ, 4.6 x 100 mm) column. Sample was injected after suitable dilution and

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the absorbance of the elute was recorded at specified wavelength. Details of the HPLC

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methods used are given in Supplementary Table S4.

Intrinsic dissolution rate (IDR) study

HPLC Method



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2.3.10 Stability study

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Stability studies was performed on the generated cocrystal at accelerated condition of 40°C

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and 75% relative humidity for 3 months and the integrity of cocrystal samples were analysed

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by PXRD.

232 233

2.3.11 In vivo hepatotoxicity study

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Single dose and subchronic in vivo hepatotoxiciy studies were performed on male BALB/c

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mice 6-7 weeks old, weighing 20-25 gm. Animals were obtained from the central animal

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facility, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad,

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India. All mice were housed to a 12 hr natural light/dark cycle at a temperature of 22 ± 2 °C

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and relative humidity of 50-60%. Standard pellet diet and water was given ad libitum.

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Institutional Animal Ethics Committee (IAEC), NIPER, Hyderabad, India approved the study

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protocol (NIP/12/2016/PE/220). The animals were divided into eight groups (Vehicle control

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group, INH treated group, SYRA treated group, QUE treated group, INHSYRA cocrystal

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treated group, INHQUE cocrystal treated group, INH and SYRA physical mixture treated

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group and INH and QUE physical mixture treated group). Each group comprised of 5

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animals. All treatment doses were prepared in 0.5 % sodium carboxy methyl cellulose to

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form a suspension.

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2.3.11.1 Single dose toxicity study

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Pilot studies were performed for INH to select the single hepatotoxic dose in mice. In brief,

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INH was administered orally in three doses 90, 120 and 150 mg/kg of mice in different

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animals (n=3) and blood (80-100uL) was withdrawn at times 6, 12, 24 and 48 hours via retro

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orbital route in vials prefilled with heparin solution. Based on the results, suitable dose and

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time period were selected and a single dose study was performed. Biochemical parameters

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were monitored at different time points of 6, 12, 24 and 48 hours. INH 120 mg/kg of body 10 ACS Paragon Plus Environment

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weight of mice was selected based on biochemical parameter at 24 hours. INH, INH

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cocrystals and physical mixture equivalent to hepatotoxic dose of INH 120 mg/kg of body

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weight of mice were administered. Coformers SYRA and QUE were administered in

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equivalent amount present in the cocrystals as described in Supplementary Table S5. 2.3.11.2 Subchronic toxicity study

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Sub chronic study was performed by administering INH in a human equivalent dose of 60

259

mg/kg body weight to male BALB/c mice for 28 days in above mentioned groups. Blood

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samples of 250 ul were collected via retro orbital route in vials prefilled with heparin solution

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from animals after 28 days. INH cocrystals equivalent to human equivalent dose of INH were

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administered. Physical mixtures equivalent to amount of drug and coformers were taken for

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the study. Coformers were administered in equivalent amount present in the cocrystals for

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subchronic study (Supplementary Table S5).

265

The blood samples were centrifuged at 10000 rpm for 10 min, plasma was collected and

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analysed for enzyme levels. The enzyme levels of aspartate transaminase (AST), alanine

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transaminase (ALT) and alkaline phosphatase (ALP) in plasma were estimated using

268

commercial kits (Accurex, India) as per manufacturer’s protocol.

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2.3.12 Statistical Analysis

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The data obtained in animal study were expressed as the mean of replicate determinations

271

(n=5) ± standard error of mean (S.E.M). Statistical comparisons were made using one-way

272

analysis of variance (ANOVA). The intergroup variations were analysed by “Tukey’s

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multiple comparison test” using the Graph Pad Prism software, Version 5.0.

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3

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INH is a fascinating compound from a crystal engineering view point for the design of

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various multicomponent systems38. INH possesses pyridine-N, which facilitates formation of

Results and discussion



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pyridine-carboxylic acid heterosynthon with carboxylic acids, and a carbohydrazide group

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which can form a range of homo and heterosynthons with other functionalities. The acid-

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pyridine heterosynthons, acid hydrazide heterosynthons and hydrazide homosythons are

280

prevalent in INH cocrystal structures36 (Supplementary Figure S2). Considering the amicable

281

functionalities and their structural advantages, grinding experiments of INH were performed

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with hepatoprotective coformers SYRA and QUE for generation of cocrystals. Preliminary

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results with other hepatoprotective coformers such as hesperetin, silibinin and chrysin are

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tabulated in Supplementary Table S1. ΔpKa rule is difference between pKa of base and acid

285

and is used for the prediction of salt or cocrystal. ΔpKa < 0 generally gives a cocrystal, ΔpKa

286

> 3 may form a salt and ΔpKa in the range of 0 to 3, could result in salt-cocrystal hybrids.

287

According to ΔpKa rule, negative values correspond to the cocrystal formation57. As per

288

USFDA guideline, if ΔpKa ≥ 1 then salt formation is favoured due to substantial proton

289

transfer while if ΔpKa ≤ 1, then cocrystal formation is favoured due to less substantial proton

290

transfer28. INH has pKa value of 1.858 while SYRA has pKa 3.9346. QUE have pKa values

291

5.87 and 8.4859. Based on ΔpKa difference rule, INHSYRA showed ΔpKa value -2.13 and

292

INHQUE showed -4.07 and -6.68. Negative value of ΔpKa of the generated compounds

293

predicted their cocrystal form formation tendency (Supplementary Table S3).

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3.1

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INH gave a single endotherm at 172.84 °C corresponding to its melting point. No polymorph

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is reported for INH hence the single melting endotherm confirmed its purity similar to the

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commercial form36. The melting endotherm for SYRA was observed at 210.37 °C which

298

confirm its form. DSC curve for QUE showed two endothermic peaks at 111.04 and 319.40

299

°C. The peak at 111.04 and 319.40 °C correspond to dehydration temperature and melting

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endotherm60. The generated novel solid forms such as INHSYRA and INHQUE gave single

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melting endotherms at 169.93 and 270.05 °C respectively (Figure 1). The onset of melting

DSC analysis


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302

and ΔH values for all forms along with coformers are given in (Supplementary Table S6).

303

INHSYRA system showed depression in melting point as compared to the drug and coformer

304

while INHQUE system shows melting point in between the drug and coformer melting point

305

indicating a possibility of new solid forms.

INH SYRA QUE INHSYRA

INHQUE Compound INH SYRA QUE INHSYRA INH QUE

Onset (⁰C) 170.84 208.67 100.79 315.10 167.49 265.80

306 307 308

Figure 1 Overlay of DSC thermogram of INH- Isoniazid, SYRA-Syringic acid, QUE-Quercetin, INHSYRA-Isoniazid syringic acid cocrystal, INHQUE- Isoniazid quercetin cocrystal

309

3.2

310

The experimental powder diffraction pattern of INH corresponded with the calculated

311

diffraction pattern from the reported single crystal structure of INH. INH has shown

312

characteristic peaks at 2θ values 11.91, 16.65 and 19.59 which confirm its commercial form.

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INHSYRA has shown new characteristic peaks at 2θ values 6.19, 18.75 and 20.38. New

314

characteristic peaks at 2θ values were observed at 8.68 and 10.2 for INH-QUE. INHQUE

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cocrystal formation was also confirmed by analysing its pattern form physical mixture of

316

INH and QUE. It was very clear from cocrystal INHQUE gave absence of peaks at 2θ values

Powder XRD Analysis



317

16.65 and 19.59 of INH and 12.56 in QUE. However, these peaks were present in physical

318

mixture (Supplementary Figure S4). Characteristic peaks for both cocrystals were represented

319

in Figure 2. Powder XRD patterns of INHSYRA and INHQUE are different from that of

320

individual drugs and coformers indicating towards formation of a new solid phase (Figure 2).

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Bulk purity of INHSYRA cocrystal was confirmed by comparing simulated powder pattern

322

from single crystals and experimental powder XRD pattern (Supplementary Figure S3).

INH

SYRA

Intensity

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INHSYRA

QUE

INHQUE 5

10

20

30

40

50

2 Theta

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Figure 2 Overlay of PXRD pattern of INH-Isoniazid, SYRA-Syringic acid, INHSYRA-Isoniazid syringic acid cocrystal, QUE-Quercetin and INHQUE- Isoniazid quercetin cocrystal. Red star shows emergence of new peak in cocrystal

327

3.3

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Hydrogen bonding interactions between drug and coformers were evaluated by IR

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spectroscopy. INH showed characteristic IR peaks at 3302.5 cm-1 and 3211.1 cm-1

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representing N-H stretching of primary amine (Supplementary Table S7 and Supplementary

331

Figure S5). Secondary amine N-H stretching was observed at 3103.72 cm-1 while amide C=O

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stretch and C=N pyridine ring were represented by 1661.94 and 1322.07 cm-1 respectively.

Fourier-transform infrared spectroscopy (FTIR)


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The carbonyl stretch of C=O of carboxylic acid (COOH) of SYRA was observed at 1695.07

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cm-1 while O-H stretch was observed at 3379.00 cm-1. The O-H stretch of free OH group of

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QUE was observed at 3358.60 cm-1 and the peak at 1655.39 cm-1 corresponded to stretching

336

vibration the C=O group. INH SYRA showed peak shifts at 3327.48 and 1713.24 cm-1 when

337

compared to INH and SYRA. Similarly, INHQUE also showed peak shift at 3447.77 cm-1.

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The shifts in peaks indicate hydrogen bond formation in cocrystals which are further

339

elaborated by crystal structure analysis.

340

3.4

341

The crystal structure of INH-SYRA was solved in triclinic system with P-1 space group. The

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INH-SYRA is a 1:1 stoichiometric cocrystal stabilized through N–H···O, O–H···O, N–

343

H···N, O–H···N and C–H···O hydrogen bond interactions. In the crystal structure, SYRA

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and INH are connected by O–H···O (O3–H3A···O1, 1.90 Å, 135º) as major interaction.

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Further, the 1D chain extends via O–H···N (O5–H5A···N1, 1.90 Å, 135º) between SYRA

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and INH (Figure 3). The crystal structure is also stabilized by N–H···O (N3–H3B···O5, 2.40

347

Å, 156º, N3–H3B···O6, 2.34 Å, 134º, and N3–H3C···O4, 2.48 Å, 166º) and weak C–H···O

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interactions. The inversion centre related INH molecules are connected to SYRA units on

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either side by O–H···N (O5–H5A···N1, 1.90 Å, 135º) and N–H···O (N3–H3B···O6, 2.34 Å,

350

135º) interactions forming R44(26) ring motif (Figure 4). A homosynthon formed between the

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two INH moleculesthe by N–H···N (N2–H2A···N3, 2.24 Å, 142º) interaction forming R22 (6)

352

ring motif, which further connects to the two inversion centre related SYRA molecules by N–

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H···O (N3–H3B···O6, 2.34 Å, 135º)

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interactions forming R44(26) ring motif (Figure 5). The linear chains of INH-SYRA cocrystal

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moieties are connected by dimeric homosynthons of INH forms a R66(40) ring motif (Figure

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6). Crystallographic parameter and hydrogen bond parameters for INH-SYRA are provided in

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the Table 1 and Table 2 respectively.

Single crystal X-ray diffraction

and O–H···O (O3–H3A···O1, 1.90 Å, 135º)



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Page 16 of 32

Table 1 Crystallographic Parameter INHSYRA Identification code CCDC deposition no. Empirical formula Formula weight Temperature K Wavelength Å Crystal system Space group a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) Volume Å3 Z Density (calculated) Mg/m3 Absorption coefficient mm-1 Radiation F(000) θ range Range h Range k Range l Reflections collected Observed reflections Unique reflections R1 [I > 2 σ (I) ] wR2 (all) Goodness-of-fit Parameters Data Completeness X-Ray Diffractometer

INHSYRA 1865066 'C9 H10 O5, C6 H7 N3 O' 335.32 298(2) 0.71073 Triclinic P -1 7.2104(8) 7.5232(8) 14.3534(16) 79.421(4) 86.198(4) 84.313(5) 760.68(14) 2 1.464 0.115 MoKα (λ = 0.71073) 352 2.77-25.03 -8 to 8 -8 to 8 -17 to 17 2672 2597 2406 0.0758 0.1996 1.089 224 96.4% Bruker APEX-II CCD

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Table 2 Hydrogen bond geometry in crystal structures of INH-SYRA Compound INHSYRA

Interaction N2H2A∙∙∙N3 O3H3A∙∙∙O1 N3H3B∙∙∙O5 N3H3B∙∙∙O6 N3H3C∙∙∙O4 O5H5A∙∙∙O4 O5H5A∙∙∙N1 C5H5∙∙∙O2 C15H15A∙∙∙O1 C15H15A∙∙∙O3

d(D-H) 0.87 0.97 0.90 0.90 0.90 0.85 0.85 0.93 0.96 0.96

d(H∙∙∙A) 2.24 1.90 2.41 2.34 2.49 2.36 1.97 2.46 2.43 2.53

d(D∙∙∙A) 2.9785(3) 2.6872(3) 3.2523(4) 3.0461(3) 3.3734(4) 2.6918(3) 2.7927(3) 3.3795(4) 3.3808(4) 3.1470(4)

361 16 ACS Paragon Plus Environment

>>>

20

CT RL

L

Isoniazid –syringic acid cocrystal

HO

40

0 R

CT

Isoniazid

60

ALP LEVELS (IU/L)

40

IN H SY RA QU HS YR E A IN HQ CC U IN HS E CC YR A IN HQ PM UE PM

Syringic acid

*** AST LEVEL (IU/L)

O

Isoniazid

80 ***

HO

N

150

60

OH

RL

O

ALT LEVEL (IU/L)

NH2 N H

CT

O

CT

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Page 32 of 32

CT

R

L

IN

H

SY

A R

E CC CC M M A UE A P E P R Q YR Q U SY H INH HS NH I IN IN U Q

Sub-chronic dose hepatotoxicity study C B A

751 752 753 754

Synopsis

755

Isoniazid cocrystals of syringic acid and quercetin were prepared to address hepatotoxicity

756

concerns of isoniazid. The cocrystals were stable for 3 months under accelerated stability (40

757

⁰C/ 75 % RH). Results of in vivo subchronic toxicity and single dose toxicity studies

758

indicated significant hepatoprotective effects of isoniazid cocrystals of syringic acid and

759

quercetin.

760


Hepatoprotective Cocrystals of Isoniazid: Synthesis, Solid State

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