INTRODUCTION:

Ranolazine (RAN) is administered for the treatment of chronic angina and is categorized as a BCS Class II drug. Due to poor aqueous solubility, ranolazine exhibits variable pharmacokinetics, leading to poor oral bioavailability (~35 to 50%)¹. RAN also has a short half-life of around 2 to 6 h, fast systemic clearance (>70%), and major hepatic first-pass metabolism via cytochrome P-450 3A (CYP3A) and CYP2D6^2,3. Formerly published works also report that plasma concentration achieved by RAN is therapeutically ineffective and also fluctuates when administered orally. To address this formulation challenge, a unique approach is required to improve the aqueous solubility, and dissolution rate, which can ultimately enhance RAN oral bioavailability^4,5.

Rapid progress in the drug discovery process leading many drug candidates but is associated with limited aqueous solubility. Inadequate solubility is one of the frequently faced formulations and drug delivery challenges that influence the efficacy of many drug substances⁶. However, emerging advancements in the field of crystal engineering support imminent drug discoveries and pipelines to come up with superior and efficacious drugs. Improvement in the physicochemical properties of drugs without modifying molecular structure is one of the key obstacles faced by techniques implemented through crystal engineering⁷.

Molecules carrying complementing functional groups to contribute to supramolecular synthones can be considered suitable candidates for co-crystallization⁸. Cocrystals are neutral crystalline homogenous solid materials composed of two or more different molecular and/or ionic compounds, generally in a fixed stoichiometric ratio. One of the conformers used in cocrystal formation is an API and the second component is a pharmaceutically acceptable substance⁹.Crystal engineering is commonly considered to be the design and growth of crystalline molecular solids, to improve the fundamental physicochemical properties of active pharmaceutical ingredients. Hydrogen bonding plays an important role in co-crystal engineering, as it is responsible for the majority of directed intermolecular interactions, which lead to the self-assembly of different components^10,11. For hydrogen bonding, Etter and co-workers proposed some general rules which should be borne in mind for co-crystal formation:

(1) All proton donors and acceptors participate in hydrogen bonding

(2) Preference to intermolecular hydrogen bonds in the formation of a six-member ring is given over intramolecular hydrogen bonding.

(3) The best proton donors and acceptors remaining after intramolecular hydrogen-bond formation form intermolecular hydrogen bonds with each other¹².

The crystal engineering experiments typically involve the Cambridge Structural Database (CSD) survey, followed by experimental work⁷. Supramolecular synthons are formed using CSD, which facilitates statistical analysis of packing motifs and thereby provides empirical information concerning common functional groups and how they engage in molecular association⁸. The knowledge gained through analysis of the CSD-directed experiments and by selecting appropriate starting components with appropriate molecular properties, which possess a high possibility to engage in precise intermolecular interactions, increases the probability to achieve the co-crystals^13,14. Strong hydrogen bonds may include N–H... O, O–H. . .O, N–H. . .N, and O–H . . .N, while weak hydrogen bonds may include C–H . . .O–N and C–H . . .O=C¹⁵. RAN exhibits pH-dependent solubility which makes its absorption variable throughout the GIT. It shows high solubility in acidic pH and low solubility in basic pH. Additionally, RAN exhibits a shorter half-life which makes it trickier to maintain its concentration in blood^16,17. Low aqueous solubility can frequently cause suboptimal drug delivery and absorption, resulting in ineffective drug efficacy and side effects¹⁸. When the drug absorption process is limited by solubility, the rate of dissolution and bioavailability can be enhanced by using solubilization enhancement methods^19,20. At present many formulation techniques such as size reduction²¹, surface modification²², complexation²³, solid lipid nanoparticles²⁴, and solid dispersion²⁵ are adopted to improve the solubility of active pharmaceutical ingredients²⁶. Among these approaches, the cocrystallization technique is considered an excellent, low cost and feasible approach to improve drug solubility, which allows the transformation of the drug to an amorphous state; thereby/subsequently enhancement of its solubility²⁷.

The development of cocrystals has been shown to boost drug solubility by several orders of magnitude. The solubility of cocrystals in water has been reported to be 1,000 times that of pure-form drugs. A trend in cocrystal solubility advantage (SA=Scocrystal/Sdrug) has also been found with coformer solubility over drug solubility (Scoformer/Sdrug). The cocrystal solubility advantage, or SA, is a dimensionless solubility number that describes a cocrystal's ability to change a drug's solubility at a certain pH, temperature, and concentration of the solubilizing agent, and so on. The solubilization media were chosen based on the administration of conventional dosage forms and the aqueous solubility of pure drugs²⁸.

The present work demonstrates the creation of a cocrystal of RAN, using Nicotinamide (NIC) as a coformer, to enhance its solubility in different buffers. Functional group interactions like OH…..N, OH…NH and OH…CO is the most favored group.

MATERIALS AND METHODS:

Materials:

Ranolazine (Drug; CAS number: 95635-55-5), Nicotinamide (coformer; CAS number: 98-92-0), and methanol (solvent; CAS number: 67-56-1) were provided by Piramal Health Care, Ahmedabad, India as gift samples. All chemicals and solvents used were of analytical grade.

Method for Co-crystal synthesis:

Cocrystals of RAN-NIC were fabricated using distinct molar ratios of two coformers utilizing different methodologies such as solvent-assisted grinding, slurry preparation, and solvent evaporation methods²⁹.

Synthesis via solvent-assisted grinding:

For the preparation of RAN:NIC cocrystals using a solvent-assisted grinding method, equimolar quantities of RAN (100mg, 25mmol) and NIC (15mg, 25mmol) as a coformer were grounded unidirectional in a mortar and pestle with intermittently dropwise addition of approximately 5mL of methanol for 30min³⁰. The grounded mixture was dried in a vacuum drying oven (DZF-6010, Labsnova, China) at 50°C for 30min to get dry powder. The resultant powder was passed through the ASTM sieve no. 40 and 60 and were immediately stored in sealed product bottles.

Synthesis via slurry preparation:

In the case of the slurry method, equimolar quantities of RAN (100mg, 25mmol) and NIC (15mg, 25mmol) were added to 15mL of methanol in a beaker. The slurry was stirred using a magnetic stirrer at 75rpm for 24hrs. The slurry obtained was then filtered and wet powder was vacuum dried at 50°C for 30min to get dry powder. It was subsequently collected in final product bottles and sealed.

Synthesis via solvent evaporation:

For the preparation of RAN-NIC cocrystals using the solvent evaporation method, equimolar quantities of RAN (100mg, 25mmol) and NIC (15mg, 25mmol) were taken with 15mL of methanol in a round bottom flask. The mixture was then stirred at 75rpm and heated at 50°C till the solvent evaporates completely. Subsequently, the drying of the sample was performed in a vacuum drier at 50°C for 2h to obtain a dry powder. The prepared powder mixture was immediately stored in sealed product containers.

RAN:NIC molar ratio screening

Different molar ratios of RAN and NIC were evaluated for cocrystal preparation using the solvent evaporation method. As solvent evaporation technique is faster and more reliable as compared to the solvent-assisted grinding and slurry method. RAN and NIC were taken in different molar ratios as mentioned along with 15mL of methanol in a round bottom flask. The mixture was then stirred at 75rpm and heated at 50°C till the solvent evaporates completely. Additionally, drying of the sample was performed in a vacuum drier at 50°C for 2 h to obtain a dry powder mixture that was later stored in suitable product bottles and immediately sealed.

Characterization:

The powder obtained after drying from three various methods was subjected to in vitro characterization studies using different techniques.

Physical Appearance:

The physical appearance of resultant cocrystals was performed using visual inspection to check the appearance and color of cocrystals.

Melting Point:

The melting point of different samples was measured using the melting point apparatus (Supertek® CH11513B, India). Ranolazine, Nicotinamide, and different cocrystal prepared with different molar concentrations of RAN and NIC were recorded for their characteristic sharp melting points.

Differential scanning calorimetry:

Differential scanning calorimetry (DSC) was performed using the DSC instrument model AQ20 from TA Instruments, USA. Samples (RAN:NIC in various ratios, such as 1:3, 1:2, 2:1, and 3:1) weighing around 2 g were placed in aluminum pans and heated at a rate of 10°C/min under a nitrogen atmosphere with a flow rate of 50ml/min³⁰. DSC was carried out at temperatures ranging from 0 to 400°C. The data was collected by platinum advantage software.

Powder X-ray Diffraction (PXRD):

X’Pert Pro X-ray diffractometer from Malvern PANalytical, UK, equipped with a CU anode, a wavelength of 0.154 nm, maximum 2.2kW, 60kV, long fine focus ceramic tube, type PW3373/00, was used to analyze cocrystal samples. Illumination was done on the 15mm sample size and analyzed from 5° to 40° in 2θ. X'Pert High Score software was used to refine the captured PXRD patterns.

Saturation Solubility:

Saturation solubility of the RAN:NIC cocrystals synthesized in the different molar ratios was performed by dissolving a surplus amount (approx 100mg) of sample in 15mL each of different buffers (pH 1.2, pH 4.5, and pH 6.8) and water. Samples in different buffers and water were placed in a conical flask and shaking was carried out (Innova-2000 portable shaker) at 250 rpm for 24h at room temperature. Resultant samples were filtered through a 0.45μm membrane filter (Millipore, Bedford, MA, USA) and quantitative analysis was done using a UV spectrophotometer (UV-1800 Shimadzu 1800, Japan)³¹. Maximum absorbance value (λmax) for RAN and NIC was observed at 273nm and 261nm, respectively.

Cocrystal stability study:

Cocrystals of RAN:NIC were subjected to accelerated (40°C/75%RH) and long-term (25°C/60%RH) stability conditions as per ICH guidance for 6 months and 12 months respectively. The samples were tested for XRPD and assay at initial, 6-month, and 12-month time points to check crystal lattice intactness after subjecting to stress conditions³².

In-vivo pharmacokinetic study:

The pharmacokinetic characteristics of new cocrystal formed were studied via an In-vivo study conducted on six albino rats weighing 190±20g. Rats were divided into 3 groups, The first group of animals was provided with pure RAN, the second group was provided with cocrystal of RAN:NIC, and the third group is treated as control. Samples were given at a dose of 55mg/kg via a feeding tube³³. The blood samples were collected at 0, 30, 60, 90, 120, 180, and 240, minutes through the tail vein in centrifuge tube³⁴.To separate plasma from blood, collected blood samples were centrifuged at 4000 rpm for 10 minutes. Plasma samples were then analyzed for drug content using a spectrophotometer (UV-1800 Shimadzu 1800, Japan). PKSolver (add-Ins program in Microsoft Excel, MS 2017) is used for pharmacokinetic data analysis.

RESULTS:

Physical Appearance:

The co-crystals prepared using distinct methods resulted in a white-colored powdered form.

Melting point analysis:

RAN exhibited a melting point in the range of 118-120 ºC, while NIC was in the range of 126-128°C. For RAN: NIC samples processed using solvent evaporation and slurry methods, a sharp and distinct melting point was observed. However, for solvent assisted grinding method, no sharp melting point was noticed. Melting of material was started at 119°C and continued till 127°C, which indicates a physical mixture of RAN and NIC.

Furthermore, when different molar ratios were screened using the solvent evaporation method, a narrow melting point in the range of 106-109°C was identified for RAN: NIC prepared at a molar ratio of 1:2. For all other ratios, multiple melting points were detected indicating of the existence of multiple components rather than single crystalline structure (Table 1).

Table 1: Melting point analysis of RAN, NIC, and cocrystal samples

S. No	Samples	Molar ratio	Synthesis methodology	Melting points	Single/narrow melting point observed (Y/N)
1	Ranolazine	-	-	118-120 ºC	Y
2	Nicotinamide	-	-	126-128 ºC	Y
3	RAN:NIC	1:1	Solvent evaporation	104-106 °C, 116-117 °C	N
4	RAN:NIC	1:1	Slurry method	105-109 °C, 119-121°C	N
5	RAN:NIC	1:1	Solvent assisted grinding	107-108 °C, 117-119°C	N
7	RAN:NIC	1:2	Solvent evaporation	106-109 °C	Y
8	RAN:NIC	1:3	Solvent evaporation	105-107 °C, 124-127 °C	N
9	RAN:NIC	2:1	Solvent evaporation	104-106 °C, 117-119°C	N
10	RAN:NIC	3:1	Solvent evaporation	107-109 °C, 117-119°C	N

Table 4. Assay of samples charged on stability for RAN:NIC 1:2 cocrystals

Stability Condition	Initial (T0)	3 month	6 month	12 month
25°C ± 2°C/60% RH ± 5% RH	99.0 %	98.9 %	99.1 %	99.5
40°C ± 2°C/75% RH ± 5% RH	99.0 %	98.8 %	98.9 %	-

In-vivo pharmacokinetic study:

In-vivo pharmacokinetic parameters like peak plasma concentration (Cmax), the area under the plasma concentration-time curve (AUC), and relative bioavailability (%) of RAN and RAN:NIC as cocrystal are reported in Table 5.

Table 5: Pharmacokinetic parameters measured for RAN and RAN:NIC 1:2 cocrystal from In-vivo study

S. No	Pharmacokinetics parameters	RAN	RAN:NIC 1:2 cocrystal
1	Cmax (µg/ml)	1.4	2.5
2	AUC (µg.ml^−1·hr)	91.5	181.5
3	Relative bioavailability (%)	-	198.3

DISCUSSION:

Different synthesis methods and molar ratios to synthesize cocrystal for RAN and NIC ratios evaluated and characterized using different techniques. Out of three synthesis methods applied, solvent evaporation method found to be most effective. Melting point evaluation is one of the preliminary test that can From the melting point evaluation, RAN and NIC exhibited distinct melting points ranging between 118-120°C, and 126-128°C respectively. The equimolar ratio of RAN:NIC cocrystals prepared with different methods displayed multiple melting points, indicating the presence of two distinct crystalline structures. However, melting point ranging from 104°C to 109°C was also observed, which is much lower than the individual melting points of RAC and NIC. This different melting point indicates the presence of different crystalline lattices in the prepared cocrystal system. Further confirmation of the formation of a new crystalline lattice, sensitive analytical techniques like DSC and XRPD were employed.

In the DSC study, the pure NIC displayed a sharp endothermic peak at 129.7°C, which was found similar to the study performed by Sheetal et al., 2015, wherein the NIC displayed a sharp endothermic peak at 128°C which was well within the acceptance range, thereby supporting our study³⁶. This single endotherm peak at 104.3°C of RAN:NIC at a 1:2 molar ratio signifies the formation of a new crystal lattice. Additionally, this isotherm is distinct from RAN and NIC endotherms, indicating an absence of both the starting materials in the cocrystal. On the other hand, molar ratios 1:3, 2:1, and 3:1 exhibits two different endotherms indicating the presence of different crystal lattices in these samples. Furthermore, the thermograph of pure cocrystals displayed only an endothermic peak, however, none of the graphs displayed an exothermic recrystallization peak indicating an interaction of conformer and drug; thereby preventing the conversion of drug to its most stable form¹⁷.

XRPD is one of the most reliable techniques to identify synthesis of new crystal lattice in cocrystals. From the XRPD data, the absence of a peak was detected at 19.0; however, it was present in the pattern of RAN alone and in different molar ratios. Unique XRD pattern of RAN: NIC observed for 1:2 molar ratios confirms the formation of cocrystals. Additionally, the resultant cocrystals displayed a reduction in the intensity of peaks at their characteristic specific angles in comparison to the pure form of the drug indicating the formation of a new cocrystal form³⁷.

To confirm the resultant solubility, saturation solubility was performed on cocrystal samples and compared with individual DS and coformer solubility. From the saturation solubility study, cocrystals displayed 1.08 folds enhancement in solubility as compared to their pure drug form. However, as compared to water, phosphate buffer (pH 6.8), and acetate buffer (pH 4.5), the 0.1 N HCl (pH 1.2) showed 9.09, 6.85- and 1.95-fold enhancement of solubility. Furthermore, the solubility of cocrystals in 0.1 N HCl (pH 1.2) was found to be 152.58 folds higher as compared to the solubility of the pure drug in water. Based on data, more than 10-fold from 0.69 mg/mL to 6.9 mg/mL increase in solubility in phosphate buffer and 17-fold from 0.31 mg/mL to 5.2 mg/mL increase in the water of RAN: NIC 1:2 can be observed as compared to RAN (Figure 4). Thus, it can be concluded that both the pure form of the drug and its cocrystal has solubility in an acidic medium, and as the values shift towards a basic pH environment, the solubility decreases.

Figure 4: Saturation solubility of RAN and RAN:NIC cocrystal in different molar ratios in different buffers

Stability study performed on RAN:NIC 1:2 cocrystals indicates no change in crystal lattice (PXRD) as well as Assay values when subjected to long term stability conditions as compared to initial samples. These results show good stability of cocrystals.

In-vivo study indicates an increase in drug plasma concentration (Cmax) of RAN (1.4µg/mL) compared to RAN:NIC 1:2 cocrystal (3.1µg/mL) by more than 2 folds. Similarly, AUC and relative bioavailability (%) were also found to be increased by 2 folds for cocrystals in comparison to the pure drug (Figure 5).

Figure 5: Comparative In-vivo study for RAN and RAN:NIC 1:2 cocrystal

CONCLUSION:

An attempt has been made to develop a cocrystal of RAN using NIC as a coformer. Different molar ratios RAN and NIC (1:1, 1:2, 1:3, 2:1, and 3:1) were evaluated to find the apt ratio for cocrystal formation. Different methods were evaluated for cocrystal synthesis, out of which solvent evaporation was found to be a better and more convenient approach for synthesis as compared to solvent-assisted grinding and slurry preparation methods. The in- vitro characterization using melting point analysis, DSC, and XRPD was performed on crystalline phases obtained after synthesis. Based on the results, RAN: NIC in the molar ratio of 1:2 was found to be the most appropriate, to achieve the most suitable crystalline arrangement. To evaluate the desired outcome in terms of solubility enhancement, a comparative saturated solubility study of RAN and RAN:NIC 1:2 cocrystals was performed. From the solubility study, an increase in solubility was observed for cocrystals in all the buffers and water. An increase in solubility by more than 10-folds in pH 6.8 phosphate buffer and a 17-fold increase in water solubility has been demonstrated. Results obtained indicate that co-crystalization of RAN using NIC as a conformer significantly helped in improving the solubility in desirable intestinal pH. From the In-vivo study performed in rats, it can be observed that there is approximately 1.8 fold increase in peak plasma concentration (Cmax) and 2 fold increase in area under the curve (AUC) from RAN alone to RAN:NIC 1:2 cocrystal. These pharmacokinetic parameters indicate an increase in the bioavailability of cocrystals from DS alone.

ABBREVIATIONS:

RAN: Ranolazine, NIC: Nicotinamide, BCS: Biopharmaceutical Classification System, CSD: Cambridge Structural Database, GIT: Gastrointestinal tract, ASTM: American Society for Testing and Materials, rpm: Rotations per Minute, DSC: Differential Scanning Calorimetry, PXRD: Powder X-ray Diffraction,

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 20.03.2023 Modified on 15.06.2023

Research J. Pharm. and Tech 2024; 17(1):59-66.

DOI: 10.52711/0974-360X.2024.00010

S. No.	Buffers	Ran Drug dissolved (mg/mL) Average (%RSD)	Ran:NIC 1:1 Drug dissolved (mg/mL) Average (%RSD)	Ran:Nic 1:2 Drug dissolved (mg/mL) Average (%RSD)	RAN:NIC- 1:3 Drug dissolved (mg/mL) Average (%RSD)	RAN:NIC- 3:1 Drug dissolved (mg/mL) Average (%RSD)	RAN:NIC- 2:1 Drug dissolved (mg/mL) Average (%RSD)
1	0.1 N HCl (pH 1.2)	43.5 (1.5)	32.5 (1.8)	47.3 (0.6)	29.7 (1.3)	34.7 (1.8)	38.7 (2.3)
2	pH 4.5 Acetate Buffer	21.7 (3.3)	17.5 (1.5)	24.2 (1.2)	15.1 (1.5)	18.2 (1.1)	19.5 (1.5)
3	pH 6.8 Phosphate Buffer	0.69 (0.2)	4.8 (1.3)	6.9 (2.7)	6.6 (2.3)	4.2 (2.3)	4.4 (1.1)
4	Water	0.31 (0.3)	4.3 (1.0)	5.2 (1.9)	4.9 (2.2)	3.7 (3.2)	3.9 (1.7)

S. No	Sample	Endotherm
1	RAN	119.5°C
2	NIC	129.7°C
3	RAN:NIC 1:2	104.3°C
4	RAN:NIC 1:3	107.9°C and 130.2°C
5	RAN:NIC 2:1	99.2°C and 110.2°C
6	RAN:NIC 3:1	101.3°C and 120.4°C