*Corresponding Author E-mail: krishna.mbs@manipal.edu
ABSTRACT:
The present study involves the preparation of co-crystal forms of clotrimazole with co-formers namely nicotinic acid and naringenin. Clotrimazole is a BCS class II drug withlow solubility and high permeability. Hence by preparing the co-crystal, an attempt has been made to improve its solubility. Based on thehydrogen bond formation between the API and co-former, two co-formers were selected: nicotinic acid and naringenin. The co-crystals of clotrimazole with nicotinic acid and naringenin were prepared in the molar ratios of 1:1, 1:2, and 2:1 using dry grinding and solvent evaporation. PXRD, DSC and FTIR confirmed the formation of co-crystals. The solubility of co-crystals of clotrimazole with nicotinic acid was increased 2.07 folds for the ratios 1:2 prepared by solvent evaporation method compared to pure clotrimazole. The saturation solubility was also increased for the co-crystals of clotrimazole with naringenin by 2 folds for the ratio 2:1 prepared by solvent evaporation method compared to pure clotrimazole.
KEYWORDS: Co-crystals, Clotrimazole, Nicotinic acid, Naringenin, Co-formers, Hydrogen bonding, Solubility, BCS class II drug.
INTRODUCTION:
Clotrimazole (1-[(2-chlorophenyl) diphenylmethyl]-1H-imidazole) is a member of the azole class of synthetic, broabroad-spectrumi-mycotic drugs that is FDA approves the treatment of oral candidiasis, dermatomycosis, and vaginal candidiasis. It is also used in the treatment of skin infections such as athlete's foot, jock itch, ringworm, pityriasis versicolor, intertrigo, and erythrasma1. Clotrimazole is marketed as a topical cream, ointment (1%, 2%) and lozenge (10mg). It mainly exerts its action by damaging the permeability barrier in the fungal cytoplasmic membrane, thereby inhibiting ergosterol biosynthesis by inhibiting the demethylation of 14 alpha lanosterol in a concentration-dependent manner. As the synthesis of ergosterol is blocked, the cell can no longer build an intact and functional cell membrane.2
Clotrimazole is a BCS (Biopharmaceutical Classification System) Class II drug that has low solubility (0.00049mg/mL) and high permeability (logP 6.30). Less Mucosal surfaces absorb less than 3%, and less than 0.5% is absorbed by the skin3. Following oral or topical application, clotrimazole undergoes rapid biotransformation into inactive metabolites. Hence an attempt has been made to improve its solubility by co-crystallization technology.
Pharmaceutical co-crystals are uniform crystalline materials with a co-crystal former integrated into the crystal lattice with the API in a specific stoichiometric ratio. This method reliably modifies drug properties such as dissolution rate, solubility, partition coefficient, stability, hygroscopicity, and compressibility without altering pharmacological behaviour4. During the last ten years,the design of pharmaceutical co-crystals emerged as an essential approach for improving the bioavailability and processability of drugs with poor physicochemical and biopharmaceutical properties5. Hence, the preparation of co-crystals will serve as a suitable modification to increase the solubility of Clotrimazole.
The current study aims to develop co-crystals of Clotrimazole by using suitable co-formers nicotinic acid and naringenin and confirmation of co-crystal formation by solid-statecharacterisation, which includes FTIR, PXRD, and DSC and analysing the increased solubility of prepared co-crystal by HPLC method.
EXPERIMENTAL:
Materials:
Clotrimazole was obtainedfrom Nulife Pharmaceuticals, Pune.Nicotinic acid was purchased from Loba Chemie, Mumbai, and Naringenin was purchased from Sigma-Aldrich SAFC, Buchs, Switzerland. Methanol (HPLC grade) was provided by Finar chemicals, Ahmedabad. Orthophosphoric acid (85% pure) and Tri-ethylamine were purchased from Merk k specialities Pvt Ltd., Mumbai, India.
Methods:
Selection of co-formers:
One of the main challenges in co-crystal development is selecting a suitable co-former that can be quickly bonded to the API to form a co-crystal. Some approaches like the supramolecular synthon approach, Cambridge structural database and Hansen solubility parameter are used to select the suitable co-former. The supramolecular synthon approach is used for co-former selection in the present study. This approach includes two critical steps: a)Selecting the target API and b) knowledge of hydrogen bonding between the API and the co-former to identify complementary API functional groups capable of forming a hydrogen bond (6).
Clotrimazole has no hydrogen bond donors but has one hydrogen bond acceptor, Nicotinic acid (co-former 1) has 1 hydrogen bond donor and 3 hydrogen bond acceptors, Naringenin (co-former 2)has 3 hydrogen bond donors and 5 hydrogen bond acceptors. Thus, based on this knowledge, there is a high probability of hydrogen bonding of clotrimazole with nicotinic acid and naringenin.
Preparation of Co-crystals:
Two methods prepared co-crystals:thedry grinding method and the solvent evaporation method(7). In this study, co-crystals of clotrimazole with both co-formers were prepared in molar ratios of 1:1,1:2, and 2:1 using both methods.
Dry grinding methods:
Accurately weighed amounts of Clotrimazole (CTZ) and co-formers such as nicotinic acid (NA) and naringenin (NAR) were taken in 1:1, 1:2, 2:1 molar ratio and triturated at constant speed without producing heat for about 10 minutes using a mortar and pestle. The resulting powder mixture was collected and stored.
Solvent evaporation method:
Accurately weighed amounts of Clotrimazole (CTZ) and co-formers such as nicotinic acid (NA) and naringenin (NAR) were taken in 1:1, 1:2, 2:1 molar ratio and were gently mixed with a spatula. This mixture was transferred to previously cleaned and dried petri plates (3 petri plates for each 1:1, 1:2, and 2:1). Ethanol was added to these petri plates till the powder mixture was dissolved completely. The plates were tightly covered with aluminium foil, and small pores were made using a needle to evaporate solvent. Plates were kept without disturbing till all the solvent was evaporated. The petri plates were scraped,and the powder crystals were collected for further analysis.
A. B.
C.
Fig. 1: Chemical structures of A. Nicotinic acid, B. Naringenin and C. Clotrimazole
Solid state characterization:
A. FTIR:
Shimadzu FTIR-8300 system (Kyoto, Japan) was used to obtain the FTIR spectra of all the prepared samples. The spectrum over a range of 4000-500 Cm-1 was collected. Disc is prepared by dispersing the sample in KBr, grinded and pressed (1000 psig) to make pallets.
B. DSC:
Shimadzu TA-60WS thermal analyser (Differential Scanning Calorimetry) characterised pure drug, co-former and prepared crystalline forms. 3-5 mg of samples were weighed and placed in the aluminium cup with a thickness of 0.1 mm and then crimped with an aluminium lid. Samples were kept in a sample holder and then allowed to heat from 25-300° C at the heat flow of 10° C/min under a nitrogen flow (10 cc/min). Powdered alumina (5mg, 100 mesh) was used as a reference to calibrate the heat flow and heat capacity signals.
C. PXRD
X-ray powder diffraction patterns of selected samples are obtained using a Rigaku miniflex 600 X-ray diffractometer (Rigaku Co., Tokyo, Japan). Instrument with a fixed tube current of 15mA and a voltage of 40 kV was operated at 600watts (X-ray tube). Standard scintillation counter is used as detector. They were measured at the speed of 4 scans per minute in the range of 5-80° (2θ).
Saturation solubility studies:
The shake flask method determines the equilibrium dynamic solubility of API, physical mixture, and prepared clotrimazole co-crystals. Ethanol: Water (65:45 %v/v) is used as the solvent system for solubility studies. Excess amounts of each sample were added to 2 mL of selected solvent system to prepare a saturated solution (8). All the samples were then shaken in an orbital shaker at 37°C and 100RPM. After 24hours, samples are collected, centrifugation is performed at 10,000RPM by maintaining a temperature of 4°C. The clear supernatant solution was separated, diluted and then injected into the HPLC system and was analysed using the optimised HPLC method using Photo Diode Array (PDA) as detector.
HPLC method:
Shimadzu LC- 10 series HPLC (Shimadzu Corporation, Kyoto, Japan) was used to analyseclotrimazole physical mixtures and prepare co-crystals. The system consists of an SCL- 10A VP controller unit, an LC- 20AD quaternary gradient pump, a DGU- 20 A5 degasserunit, aSIL20AC HT refrigerated autosampler, and a detector of model SPD- M10 AVP PDA. Buffer solution was filtered using a 0.45µm filter with the help of a glass vacuum-filtration unit. Equitron ultrasonic bath is used for degassing the mobile phase. Kromasil 100 C18(250*4.6mm, 5.0µ) column was used with mobile phase composition of 0.1% Tri-ethylamine in water (pH 3.00±0.05 adjusted by using Ortho-phosphoric acid) and Methanol in ratio of 25:75v/v% at a flow rate of 1 ml/min. The sample was injected (20µl) and allowed to run for 15min. Detection was performed at 215nm using a PDA detector (9). Clotrimazole has a retention time of 9.255 min (Fig 2).
Fig. 2: Chromatogram of Clotrimazole API developed using the above-discussed HPLC method
RESULTS AND DISCUSSION:
DSC results of CTZ:NA cocrystals:
Fig. 3 A shows the DSC results of CTZ, NA, and CTZ:NA Physical mixture and co-crystals of molar ratios 1:1, 1:2, and 2:1 prepared by dry grinding. The difference in melting point was observed when compared to pure clotrimazole (151.37°C). CTZ:NA physical mixture shown endotherm at 129.97°C, CTZ:NA DG1 (1:1 dry grinding) shown endotherm at 126.17°C, CTZ:NA DG2 (1:2 dry grinding) shown endotherm at 125.55°C and CTZ:NA DG3 (2:1 dry grinding) shown endotherm at 127.67°C.
Fig. 3 B shows the DSC results of CTZ, NA, and CTZ:NA Physical mixture and co-crystals of molar ratios 1:1, 1:2, and 2:1 prepared by solvent evaporation. CTZ:NA SE1 (1:1 solvent evaporation) shown endotherm at 127.16°C, CTZ:NA SE2 (1:2 solvent evaporation) shown endotherm at 125.12°C, CTZ:NA SE3 (2:1 solvent evaporation) shown endotherm at 126.42°C.
The endotherm seen in the physical mixture and co-crystals are slightly different from the endotherm of pure clotrimazole and nicotinic acid, 151.37°C and 245.98°C, respectively. Thus, it was concluded that there is a significant interaction between clotrimazole and nicotinic acid in the prepared co-crystals.
A.
B.
Fig. 3: DSC results of A. CTZ:NA co-crystals prepared by dry grinding method, B. CTZ:NA co-crystals prepared using the solvent evaporation method.
Table 1: DSC data of clotrimazole, nicotinic acid and co-crystals of CTZ:NA
|
Sample ID |
Melting Endotherm |
|
CTZ |
151.37° C |
|
NA |
245.98° C |
|
CTZ:NA PM |
129.97° C |
|
CTZ:NA DG1 |
126.17° C |
|
CTZ:NA DG2 |
125.55° C |
|
CTZ:NA DG3 |
127.67°C |
|
CTZ:NA SE1 |
127.16° C |
|
CTZ:NA SE2 |
125.12° C |
|
CTZ:NA SE3 |
126.42° C |
DSC results of CTZ: NAR co-crystals:
Fig. 4 A shows the DSC results of CTZ, NAR, and CTZ:NAR Physical mixture and co-crystals of molar ratios 1:1, 1:2, and 2:1 prepared by dry grinding. The difference in melting point was observed when compared to pure clotrimazole (151.37°C). CTZ:NAR physical mixture showed endotherm at 140.45°C, CTZ:NAR DG1 showedendotherm at 139.03°C, CTZ:NAR DG2 showed endotherm at 138.23°C and CTZ:NAR DG3 showed endotherm at 139.52°C.
Fig. 4 B shows the DSC results of CTZ, NAR, and CTZ:NAR Physical mixture and co-crystals of molar ratios 1:1, 1:2, and 2:1 prepared by solvent evaporation. CTZ:NAR SE1 shows endotherm at 172.65°C while CTZ:NAR SE2 and CTZ:NAR SE3 show no endotherms.
The endotherm seen in the physical mixture and co-crystals are slightly different from the endotherm of pure clotrimazole and naringenin, 151.37°C and 261.99°C, respectively. Thus, it was concluded that there is a significant interaction between clotrimazole and nicotinic acid in the prepared co-crystals.
A.
B.
Fig 4: DSC results of A. CTZ:NAR co-crystals prepared by dry grinding method, B. CTZ:NAR co-crystals prepared using the solvent evaporation method.
Table 2: DSC data of clotrimazole, naringenin and co-crystals of CTZ:NAR
|
Sample ID |
Melting Endotherm |
|
CTZ |
151.37° C |
|
NAR |
261.99° C |
|
CTZ:NAR PM |
140.45° C |
|
CTZ:NAR DG1 |
139.03° C |
|
CTZ:NAR DG2 |
138.23° C |
|
CTZ:NAR DG3 |
139.52° C |
|
CTZ:NAR SE1 |
172.65° C |
|
CTZ:NAR SE2 |
----- |
|
CTZ:NAR SE3 |
----- |
FTIR spectroscopy of CTZ:NA
A.
B.
C.
D.
E.
F.
G.
H.
I.
Fig. 5: FTIR spectra of A. Clotrimazole API, B. Nicotinic acid, C. CTZ:NA PM, D. CTZ:NA DG1, E. CTZ:NA DG2, F. CTZ:NA DG3, G. CTZ:NA SE1, H. CTZ:NA SE2, I. CTZ:NA SE3
FTIR spectroscopy of CTZ:NAR:
The FTIR spectra of CTZ:NAR SE3 revealed a formation of hydrogen bonding between the clotrimazole and the co-former. The broadening of the peak was found at the frequency range of the OH functional group, which suggests hydrogen bonding formation (3411.15-3145.90 cm-1) (Fig 6. I).
A.
B.
C.
D.
E.
F.
G.
H.
I.
Fig 6: FTIR spectra of A. Clotrimazole API, B. Naringenin, C. CTZ:NAR PM, D. CTZ:NAR DG1, E. CTZ:NAR DG2, F. CTZ:NAR DG3, G. CTZ:NAR SE1, H. CTZ:NAR SE2, I. CTZ:NAR SE3
PXRD:
The interaction between the drug and the co-former is indicated by the difference in 2θ values of pure medicine and co-crystals. Clotrimazole showed 100% intensity at 2θ angle of 12.178 whereas nicotinic acid and naringenin showed 100% intensity at 2θ angle of 15.3512 and 22.253.The physical mixture CTZ:NA PM and co-crystal CTZ:NA SE1 showed 100% intensity at 2θ angle of 12.112 and 18.768 respectively, whereas the physical mixture CTZ:NAR PM and co-crystal CTZ:NAR SE3 showed 100% intensity at 2θ angle of 12.155 and 22.527. There was a shift in the 100% intensity for physical mixtures and co-crystals when compared with the pure clotrimazoleand co-formers, indicating a difference in the arrangement of the molecules, which further indicates the development of a new crystalline phase.
Fig 8: PXRD of nicotinic acid
Fig 9: PXRD of CTZ:NA PM
Fig 10: PXRD of CTZ:NA SE1
Fig 11: PXRD of naringenin
Fig 12: PXRD of CTZ:NAR PM
Fig 13: PXRD of CTZ:NAR SE3
Table 3: PXRD results of clotrimazole, co-formers and co-crystals
|
Sample ID |
2θ (degree) |
D (angle) |
Intensity |
|
CTZ |
12.1789 |
7.26138 |
1196.63 |
|
NA |
15.3512 |
5.76724 |
1589.49 |
|
CTZ:NA PM |
12.1129 |
7.3008 |
819.22 |
|
CTZ:NA SE1 |
18.7687 |
4.72411 |
920.19 |
|
NAR |
22.253 |
3.99167 |
1191.18 |
|
CTZ:NAR PM |
12.1556 |
7.27528 |
721.03 |
|
CTZ:NAR SE3 |
22.5275 |
3.94366 |
3321.98 |
Saturation solubility studiesof CTZ:NA co-crystals:
The saturation solubility of pure clotrimazole and co-crystals in Ethanol: Water (65:45 %v/v) at 37°C was 18.63mg/mL after 24hrs. The saturation solubility of DG1 and SE1 was found to be 35.7mg/mL and 38.65 mg/mL, respectively, which shows a 1.9-fold and 2-fold increase in solubility compared to pure clotrimazole.
Fig 14: Solubility chart of CTZ:NA co-crystals
Table 4: Solubility data of CTZ:NA co-crystals
|
Sample ID |
Solubility in Ethanol: Water (65:45 %v/v) at 37° C (µg/mL) |
Fold Increased |
|
CTZ |
18639.17 |
----- |
|
CTZ:NA PM |
29863.86 |
1.60 |
|
CTZ:NA DG1 |
25500.88 |
1.36 |
|
CTZ:NA DG2 |
35702.68 |
1.91 |
|
CTZ:NA DG3 |
27733.13 |
1.48 |
|
CTZ:NA SE1 |
38665.10 |
2.07 |
|
CTZ:NA SE2 |
32039.76 |
1.71 |
|
CTZ:NA SE3 |
28464.54 |
1.52 |
Saturation solubility studies of CTZ:NAR co-crystals:
The saturation solubility of pure clotrimazole and co-crystals in Ethanol: Water (65:45% v/v) at 37°C was found to be 15.03mg/mL after 24 hrs. Whereas the saturation solubility of SE3 was found to be 30.27mg/mL, showing a 2-fold increase in solubility compared to pure clotrimazole.
Fig. 15: Solubility chart of CTZ:NAR co-crystals
Table 5: Solubility data of CTZ:NAR co-crystals
|
Sample ID |
Solubility in Ethanol: Water (65:45 %v/v) at 37°C (µg/mL) |
Fold Increased |
|
CTZ |
15033.79 |
----- |
|
CTZ:NAR PM |
16093.85 |
1.07 |
|
CTZ:NAR DG1 |
17384.67 |
1.16 |
|
CTZ:NAR DG2 |
16775.28 |
1.12 |
|
CTZ:NAR DG3 |
16617.89 |
1.11 |
|
CTZ:NAR SE1 |
17522.95 |
1.17 |
|
CTZ:NAR SE2 |
11222.35 |
---- |
|
CTZ:NAR SE3 |
30276.87 |
2.01 |
CONCLUSION:
Clotrimazole was able to form stable co-crystals with Nicotinic acid and Naringenin. The co-crystals formed were characterised, and the analysis for saturation solubility was carried out. Co-crystals of clotrimazole with nicotinic acid and naringenin were prepared using dry grinding and solvent evaporation. The formation of co-crystals was confirmed by solid state characterisation, which included PXRD, DSC and FTIR, which proved the structural modifications in the co-crystals. The PXRD results showed shifts in peak locations and trends, suggesting the development of a new crystalline phase. The DSC thermogram showed different melting patterns for cocrystals than the pure drug. These observations were also confirmed by the FTIR spectrum, which confirmed the formation of hydrogen bonds.The saturation solubility of co-crystals of clotrimazole with nicotinic acid was increased by 1.91 folds for the ratio 1:2 prepared by dry grinding method, 1.71 and 2.07 folds for the ratios 1:1 and 1:2 prepared by solvent evaporation method when compared to pure clotrimazole. The saturation solubility was also increased for the co-crystals of clotrimazole with naringenin by 2 folds for the ratio 2:1 prepared by solvent evaporation method compared to pure clotrimazole. Thus, the solubility of clotrimazole was improved by the preparation of co-crystals.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 07.02.2023 Modified on 16.11.2023
Accepted on 23.03.2024 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(6):2580-2586.
DOI: 10.52711/0974-360X.2024.00403