*Corresponding author,

e-mail : kri.alatas@lecture.unjani.ac.id (F. Alatas)

https://doi.org/10.24198/idjp.v2i1.23957

Vol 2, Issue 1, 2020 (1-6)

http://journal.unpad.ac.id/idjp

Solubility Enhancement of Clozapine through Co-crystal Formation with

Isonicotinamide

Fikri Alatas

, Hestiary Ratih

, Hesti Kurnia

, Sundani N. Soewandhi

1. Pharmaceutics group, Faculty of Pharmacy, Universitas Jenderal Achmad Yani, Jl. Terusan Jenderal

Sudirman, Cimahi, West Java, Indonesia, 40521

2. Pharmaceutics group, School of Pharmacy, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung,

West Java, Indonesia.

Received: 17 Oct 2019/ Revised: 17 Nov 2019/ Accepted: 26 Nov 2019/ Published: 6 Jan 2020

ABSTRACT

Clozapine (CLO) is an eective atypical antipsychotic to control the symptoms of psychosis and

schizophrenia. Clozapine has low solubility and high permeability, so it is classied as a class II in

the biopharmaceutical classication system. The aim of this study was to improve the solubility and

dissolution rate of clozapine by clozapine-isonicotinamide (CLO-INA) co-crystal formation. CLO-

INA co-crystal was prepared by solvent-drop grinding (SDG) method using water as a solvent.

Characterization of SDG result was conducted by powder X-ray diraction (PXRD) and Fourier

transform infrared (FTIR). Solubility test was performed in water at room temperature. The dissolution

test was performed in 900 mL of pH 6.8 phosphate buer solution, 50 rotation per minute of paddle

rotation, and at 37±0.5 °C. The PXRD pattern of SDG result of CLO-INA has many dierent peaks

from its parent components, and this may indicate the co-crystal formation. The solubility of the co-

crystal clozapine was fteen folds higher than pure clozapine. The dissolution rate of CLO-INA co-

crystal increased in the rst 10 minutes compared to pure clozapine. Percentage of clozapine dissolved

after 10 minutes from CLO-INA co-crystal and pure CLO were 10.2 and 2.4%, respectively. CLO

and INA can form co-crystal by SDG method that can improve the solubility and dissolution rate of

clozapine.

Keywords: Clozapine, Isonicotinamide, Co-crystal, Solubility, Dissolution

1. Introduction

Clozapine (CLO) is a potent antipsychotic

widely used to suppress both positive and negative

symptoms of schizophrenia and some neuroleptic

responses [1]. CLO is classied as a class II drug

according to biopharmaceutics classication

systems (BCS), due to its high permeability and low

solubility [2]. The bioavailability of CLO is 27 to

50% [3].

Solubility is one of the critical physicochemical

properties in predicting the degree of active

pharmaceutical ingredients (APIs) absorption in

the gastrointestinal tract. The very low solubility

of API in water often indicates low bioavailability,

consequently, the dissolution rate becomes the rate-

limiting step in the drug absorption process [4].

The co-crystal formation is one of the alternative

methods that can be used to increase solubility and

dissolution rate in many APIs [5]. Co-crystal can

be explained as complex of two or more neutral

molecules bonded together in a crystal lattice via a

non-covalent interaction, in particular, a hydrogen

bond [6]. The formation of co-crystals involves

combining drugs with pharmaceutically acceptable

additives in the crystal lattice. The resulting

multicomponent crystal form will have dierent

physicochemical properties and can potentially

improve solubility [7, 8], dissolution rate [9, 10],

compressibility [11], chemical and physical stability

[12].

The co-crystal formation depends on the

functional group between the API and the co-crystal

former (CCF) to allow the occurrence of hydrogen

bonding. The presence of the -NH group on the

diazepine ring in the CLO chemical structure (Fig.

1a) allows the co-crystals formation with carboxylic

acid and amide groups. Isonicotinamide (INA) with

the chemical structure shown in Fig. 1b is one of

F. Alatas et al / Indo J Pharm 1 (2020) 1-6

the useful amide group compounds used in the

formation of co-crystals and is known as the GRAS

(Generally Recognized as Safe) compound. INA

has a donor and some acceptor hydrogen bond

that makes this CCF easy to form co-crystals with

some APIs [13–17]. This purpose of this study

was to know the inuence of CLO-INA co-crystal

formation on the solubility and dissolution rate of

clozapine.

a b

Figure 1. Chemical structures of (a) clozapine dan (b)

isonicotinamide

2. Method

2.1 Materials

The materials used in the present study were

clozapine (Taizhou Xingming Pharmaceutical,

China), isonicotinamide (Sigma-Aldrich), methanol

(Merck), water, potassium dihydrogen phosphate

(Merck), sodium hydroxide (Merck). While the

tools used were powder X-ray diractometer

(Philips PW1710), Shimadzu FTIR Anity-1

spectrophotometer (DRS-8000), orbital shaker (IKA

KS-260), ultraviolet spectrophotometer (Shimadzu

1800PC), and TDTF ZRS-6G Dissolution tester.

2.2 Solubility phase curve construction of CLO in

various concentration of INA solutions

A total of 50 mg CLO was inserted into ve

vials containing ve mL of an INA solution with

a concentration range of 0.4 to1.4 M in an aqueous

solvent and shaken with an orbital shaker at room

temperature. After 24 hours, the samples were

ltered. The ltrate was diluted and analyzed by

Shimadzu 1800PC ultraviolet spectrophotometer at

293 nm. The test was carried out in triplicate. The

phase solubility curve was constructed by plotting

the level of dissolved CLO to the concentration of

INA solution.

2.3 Preparation of CLO-INA co-crystal by solvent-

drop grinding (SDG) method

An equimolar mixture of CLO (326 mg) and

INA (122 mg) was milled simultaneously for ve

minutes in a mortar with the addition of two drops

of water. The grinding result allowed to dry at room

temperature.

2.4 Characterization of CLO-INA SDG result by

powder X-ray Diraction method

The SDG or wet grinding result of CLO-INA

was characterized by Philips PW1710 powder

X-ray diraction system and compared with pure

CLO and INA powder X-ray diraction patterns.

The powder X-ray diraction data collection was

performed by scanning 100-200 mg of sample

powder placed on a glass container and attened at

an angle range of 2θ = 5-45°, scan rate 2°/min, with

a voltage of 40 kV, and a current of 30 mA.

2.5 Characterization of CLO-INA SDG result by

Fourier transform infra-red (FTIR)

The FTIR spectral data collection of CLO-

INA wet grinding result was performed using the

Shimadzu FTIR Anity-1 spectrophotometer

(DRS-8000) and compared with CLO and INA pure

spectra. The samples were homogeneously mixed

with potassium bromide at a weight ratio of 1:5 and

scanned in the 4000-400 cm-1 wavenumbers range.

KBr powder was used as a blank for background

correction.

2.6 Solubility test

As much as 50 mg of CLO-INA wet grinding

result and pure CLO were placed into vials that

containing each of ve mL of water and shaken

on an orbital shaker (IKA KS-260) at room

temperature. After 24 hours, the samples were

ltered, and the ltrate was analyzed by ultraviolet

F. Alatas et al / Indo J Pharm 1 (2020) 1-6

spectrophotometer at a wavelength of 293 nm.

2.7 Dissolution test

The dissolution test was carried out on SDG

product of CLO-INA and pure CLO in a 900 mL

pH 6.8 of phosphate buer solution at 37 ± 0.5°C

using a type I apparatus with a rotation speed of

50 rotations per minute (rpm) for 60 minutes. The

dissolved clozapine in each sample was diluted and

analyzed by ultraviolet spectrophotometer at a 293

nm. The test was performed in six replicate.

3. Result and Discussion

3.1 Solubility phase curve of CLO-INA

The drug solubility study in a CCF solution

can be used to predict the formation of co-crystals

by making a phase solubility curve. The phase

solubility curve is the solubility curve that describes

the concentration of a solute in the concentration

variation of another substance solution. According

to Higuchi and Connors, there are several types

of phase solubility curves, namely AL, AP, AN,

BS, dan BI types [18]. The phase solubility test is

useful to know the type of solubility curve of CLO

in the variation of INA concentration, and this

can be an indication of the occurrence of the co-

crystal formation. Determination of dissolved CLO

was carried out by ultraviolet spectrophotometry

method. The CLO has three maximum absorption

wavelengths, namely 230, 260, and 293 nm. INA

still has absorption at wavelengths of 230 and 260

nm, but has no absorption at 293 nm wavelength,

so a wavelength of 293 nm is used to analyze CLO

concentration without interference by INA. Fig.

2 showed the CLO phase solubility curve in the

concentration variation of the INA solution in water

follows the BS type. The CLO solubility increase

sharply at INA concentrations of 0.4 to 1 M caused

by the co-crystal began to form with high solubility

in water. At INA concentration of 1-1.4 M, CLO

solubility decreased due to the achievement of

CLO-INA co-crystal solubility product constant

(Ksp) in water, accordingly, CLO-INA co-crystal

solid was formed. The higher concentration of

INA, the more co-crystal solid was formed. This is

in accordance with the explanation of Mantri et al.,

2009 [19].

Figure 2. Phase solubility curve of clozapine (CLO) in

the various concentration of isonicotinamide

(INA) solution in water. (n=3)

3.2 Preparation of CLO-INA co-crystal by solvent-

drop grinding (SDG) method

Physical interaction can occur when two

substances are milled simultaneously. The

interaction may be a mixture of eutectic or the

formation of new molecular compound or co-

crystal. In this experiment, an equimolar mixture

of CLO and INA was milled with the addition of

two drops of water, and this method known as wet

grinding or solvent-drop grinding (SDG) [20].

The purpose of solvent addition is to accelerate

the achievement of the amorphous state of the two

components causing each component to be more

reactive and faster co-crystallization [21]. At least,

the solvent can dissolve well one of the components.

Water is used as a solvent because it can dissolve

well INA and slightly CLO.

3.3 Characterization of CLO-INA SDG result by

powder X-ray Diraction method

The initial characterization of interaction in

the wet milling process is by using powder X-ray

diraction (PXRD). The dierent of PXRD

pattern of SDG result from PXRD patterns of their

respective constituent components may indicate the

co-crystal formation. The PXRD pattern of CLO-

F. Alatas et al / Indo J Pharm 1 (2020) 1-6

INA compared to its parent components (pure CLO

and INA) is shown in Fig. 3. The PXRD pattern

of CLO shows the main peaks at the angle of 2θ =

10.6; 17.5; 19.4; 21.2; and 29.9º. These results are

consistent with those reported by the previous study

[22]. The INA raw material has the main peaks at an

angle of 2θ = 17.9; 20.9; 23.4; 26.0; and 31.1º, and

this corresponds to INA form I with reference code

EHOWIH01 [23]. The powder X-ray diraction

pattern of CLO-INA wet grinding result shows

the formation of new peaks that are dierent from

the main peaks of CLO and INA. The dierences

of PXRD pattern between CLO-INA SDG result

and its pure components indicate the formation of

CLO-INA co-crystal. The main peaks of CLO-INA

co-crystal are located at an angle of 2θ = 11.1; 13.6;

17.9; 24.3; and 29.3º.

Figure 3. Powder X-ray diraction patterns of CLO,

INA, and CLO-INA

3.4 Characterization of CLO-INA SDG result by

Fourier transform infra-red (FTIR)

One spectroscopic technique that can be used

to show the formation of co-crystal is FTIR. The

presence of shifts in the number of spectral waves

in certain groups, such as carboxylates (-COOH),

carbonyl (C=O), amines (-NH

), imines (-C=NH)

can indicate the presence of hydrogen bonds

between two interacting components [24]. Fig.

4 showed the FTIR spectrum of the CLO-INA

SDG result compared with their pure constituent

components. The CLO has two sharp peaks at 3294

and 1594 cm

-1

caused by stretching vibration of

-NH and amidine (C=N) group, respectively. The

INA has two peaks at 3368 and 3186 cm

-1

(-NH

group stretching vibration) and a peak at 1668 cm-1

(carbonyl group vibration). The FTIR spectrum of

the CLO-INA wet grinding result indicated the

peaks of -NH group and amidine group of CLO

shifted to 3298 and 1597 cm

-1

, respectively, as well

as the carbonyl group vibration of INA shifted to

1678 cm

-1

. This shifting was thought caused by the

hydrogen bond between the -NH group of CLO and

the C=O group of INA that indicated the CLO-INA

co-crystal formation.

Figure 4. FTIR spectrum of CLO-INA co-crystal

compared to its pure components.

3.5 Solubility of CLO-INA Co-crystal

Solubility is an important physicochemical factor

which may indicate a dierence in the crystalline

structure of a compound, such as a polymorph,

salt, hydrate, solvate, and co-crystalline [25]. The

solubility test was performed to determine the

change of clozapine solubility in consequence of

CLO-INA co-crystal formation. The result of the

water solubility test at room temperature showed

CLO-INA co-crystal has a solubility of 378±12

μg/mL, which is 15-folds higher than pure CLO

solubility (24±1 μg/mL). This solubility increasing

was due to the creation of hydrogen bonds between

CLO and INA that changes in the crystal structure.

F. Alatas et al / Indo J Pharm 1 (2020) 1-6

In the dissolution process of a solid in a solvent,

intermolecular bond termination between the solute

and the solvent requires energy [26]. The energy

required to break the bonds between dissolved solid

depends on the structure of the solid compound and

the intermolecular attraction between the dissolved

solid compounds, therefore the existence of

hydrogen bonds in CLO-INA co-crystal can reduce

the energy required to break the bonds between

CLO-INA co-crystal and water so that the CLO-

INA co-crystal has a higher solubility than pure

CLO.

3.6 Dissolution prole of CLO-INA Co-crystal

The dissolution rate is also a physicochemical

property which can show the dierence in the

crystalline solids structure. An increasing in

dissolution rate follows the increasing of solubility.

The BCS class II drug will be absorbed well in the

gastrointestinal tract when the dissolution rate of

the drug is high. CLO has a low solubility at neutral

pH, and its solubility increases with decreasing

pH due to the ionization. In this study dissolution

test carried out at pH 6.8 which represents the

pH conditions of the intestinal tract. The result

of the particulate dissolution test in phosphate

buer media pH 6.8 (Fig. 5) shows the dissolved

percentage of clozapine after 10 min (DP10) from

the CLO-INA and CLO pure are 10.24 and 2.4%,

respectively, and this shows that the CLO-INA co-

crystal has a faster dissolution rate than pure CLO.

Figure 5. Prole dissolution of CLO-INA co-crystal in

pH 6.8 of phosphate buer solution compared

to pure CLO. (n=6)

4. Conclusion

Clozapine and isonicotinamide may form co-

crystal by a solvent-drop grinding method with the

addition of water solvent based on characterization

results by powder X-ray diraction, Fourier

transforms infrared, and solubility phase curve

methods. The formation of CLO-INA co-crystal

can enhance the solubility and dissolution rate of

clozapine.

5. Acknowledgements

We would like to thank the Research and

Community Services (LPPM) of Universitas

Jenderal Achmad Yani for the support of research

grant.

References

[1] Manu P, Sarpal D, Muir O, et al. When can

patients with potentially life-threatening

adverse eects be rechallenged with

clozapine? A systematic review of the

published literature. Schizophr Res 2012;

134: 180–186.

[2] Dias SBT, Nascimento TG, Santos AFO,

et al. Polymorphic characterization and

compatibility study of clozapine: implications

on its stability and some biopharmaceutics

properties. J Therm Anal Calorim 2014;

V.12x: X1.

[3] Stahl SM. Antipsychotics and mood stabilizer.

3rd ed. New York: Cambridge University

Press, 2008.

[4] Chadha R, Saini A, Jain DS, et al. Preparation

and solid-state characterization of three novel

multicomponent solid forms of oxcarbazepine:

Improvement in solubility through saccharin

cocrystal. Cryst Growth Des 2012; 12: 4211–

4224.

[5] Babu NJ, Nangia A. Solubility advantage

of amorphous drugs and pharmaceutical

cocrystals. Cryst Growth Des 2011; 11: 2662–

2679.

[6] Rodríguez-Hornedo N, Nehm SJ, Jayasankar

A. Cocrystals : design , properties and

formation mechanisms. Encycl Pharm

F. Alatas et al / Indo J Pharm 1 (2020) 1-6

Technol 2007; 615–635.

[7] Alatas F, Ratih H, Soewandhi SN.

Enhancement of solubility and dissolution

rate of telmisartan by telmisartan-oxalic acid

co-crystal formation. Int J Pharm Pharm Sci

2015; 7: 423–425.

[8] McNamara DP, Childs SL, Giordano J, et al.

Use of a glutaric acid cocrystal to improve

oral bioavailability of a low solubility API.

Pharm Res 2006; 23: 1888–1897.

[9] Tomaszewska I, Karki S, Shur J, et al.

Pharmaceutical characterisation and

evaluation of cocrystals: Importance of in

vitro dissolution conditions and type of

coformer. Int J Pharm 2013; 453: 380–388.

[10] Zegarac M, Leksic E, Sket P, et al. A sildenal

cocrystal based on acetylsalicylic acid

exhibits an enhanced intrinsic dissolution

rate. CrystEngComm 2014; 16: 32–35.

[11] Aher S, Dhumal R, Mahadik K, et al. Eect of

cocrystallization techniques on compressional

properties of caeine/oxalic acid 2:1 cocrystal.

Pharm Dev Technol 2011; 1–6.

[12] Babu NJ, Sanphui P, Nangia A. Crystal

engineering of stable temozolomide

cocrystals. Chem - An Asian J 2012; 7: 2274–

2285.

[13] Wang L, Tan B, Zhang H, et al. Pharmaceutical

cocrystals of diunisal with nicotinamide or

isonicotinamide. Org Process Res Dev 2013;

17: 1413–1418.

[14] Dubey R, Desiraju GR. Structural landscape

of the 1:1 benzoic acid:isonicotinamide

cocrystal. Chem Commun 2014; 50: 1181–

1184.

[15] Madeley LG, Levendis DC, Lemmerer A.

Isonicotinamide-2-naphthoic acid (1/1). Acta

Crystallogr Sect E Struct Reports Online; 67.

[16] Báthori NB, Lemmerer A, Venter GA,

et al. Pharmaceutical co-crystals with

isonicotinamide—vitamin B3, clobric acid,

and diclofenac—and two isonicotinamide

hydrates. Cryst Growth Des 2011; 11: 75–87.

[17] Sanphui P, Kumar SS, Nangia A.

Pharmaceutical cocrystals of niclosamide.

Cryst Growth Des 2012; 12: 4588–4599.

[18] Takeru Higuchi, Kenneth A. Connors.

Phase solubility techniques. In: Advances in

Analytical Chemistry and Instrumentation.

New York: Jonh Wiley & Sons, INC, 1965,

pp. 117–212.

[19] Mantri RV, Sanghvi R, Zhu H (Jim). Solubility

of Pharmaceutical Solids. In: Yihong Qiu,

Yisheng Chen, Geo G.Z. Zhang, Lirong

Liu WRP (ed) Developing Solid Oral Dosage

Forms: Pharmaceutical Theory And Practice.

New York: Academic Press, 2009, pp. 1–24.

[20] Trask A V, Motherwell WDS, Jones W.

Solvent-drop grinding: green polymorph

control of cocrystallisation. Chem Commun

(Camb) 2004; 890–891.

[21] Rehder S, Klukkert M, Löbmann K a M, et

al. Investigation of the formation process of

two piracetam cocrystals during grinding.

Pharmaceutics 2011; 3: 706–722.

[22] Mahmoud A, Ali A, Ali AA, et al. Clozapine-

carboxylic acid plasticized co-amorphous

dispersions : Preparation, characterization

and solution stability evaluation. 2015; 65:

133–146.

[23] Aakeröy CB, Beatty AM, Helfrich BA, et

al. Do polymorphic compounds make good

cocrystallizing agents? A structural case study

that demonstrates the importance of synthon

exibility. Cryst Growth Des 2003; 3: 159–

165.

[24] Qiao N, Li M, Schlindwein W, et al.

Pharmaceutical cocrystals: An overview. Int J

Pharm 2011; 419: 1–11.

[25] Sarma B, Chen J, Hsi HY, et al. Solid forms

of pharmaceuticals: Polymorphs, salts and

cocrystals. Korean J Chem Eng 2011; 28:

315–322.

[26] Saikia B, Bora P, Khatioda R, et al. Hydrogen

Bond Synthons in the Interplay of Solubility

and Membrane Permeability/Diusion in

Variable Stoichiometry Drug Cocrystals.

Cryst Growth Des 2015; 15: 5593–5603.