INTRODUCTION:

Stability studies demonstrate how API’s and formulations vary in quality over a particular time and are affected by environmental conditions such as light, temperature and moisture. These studies are being designed and implemented on various attributes such as API’s which are likely to get modified in terms of their efficacy, safety and quality¹. In case of accelerated stability testing, three time points are being considered and recommended by ICH guidelines, including initial, mid and final time point for a period of 6 months.

The susceptible drug or substance should be stored in an environment which tests its moisture and thermal stability profile for a fixed time period². Cocrystallization technique is widely being applied on various API’s having pharmaceutical and physicochemical properties issues such as lower solubility profile, thermal instability issues, poor flow properties issues etc. however the stability profile of cocrystals with different excipients, various liquid mediums are little investigated. Cocrystal formation take place by interaction of API’s with any pharmaceutically acceptable coformer via hydrogen bonding or any other intermolecular interaction in a fixed stochiometric ratio³. The resultant single phase multicomponent crystalline substance carries different and improved physicochemical properties like better physical stability, different melting point and improvised solubility profile compared to parent components⁴. As drugs are very important objects, so the designing of newer and better crystalline substance through cocrystallization become very significant tool for the variability of characteristics and potential intellectual properties⁵. Cocrystallization technique is applicable on all types of API’s like acidic, basic and nonionizable. There are two important concepts which play key role in designing the newer cocrystals, first one is role of hydrogen bonding which depends upon the availability of good hydrogen donor and acceptor molecules⁶. The interaction of two components via hydrogen bonding leads to generation of a newer synthon system. When two similar functional groups involved in interaction with each other, homosynthon formation occurred while in case of interaction between two different functional groups take place, heterosynthons formation occurred. With better directionality and energy, hydrogen bonding is considered to be the most effective and significant intermolecular interactions. The intermolecular synthon formation is directly governed by the polarity and nature of hydrogen donor and acceptor group and this interaction is facilitated by the charge or resonance present at molecular level⁷. Second key point is to study the robustness or strength of synthons. Various software’s like Cambridge Structural Database (CSD), COSMO-RS etc could be applied for evaluation of structural synthons robustness. With the help of these tools, the variability in physicochemical properties of various single or multicomponent crystalline substance could be accessed and understood⁸.

The dissolution profile of most of API’s in biological systems decided their bioavailability. The physicochemical stability of an API is of great significance as after degradation it becomes inactive. Decomposition of API’s leads to generation of toxic and harmful by-products⁹. Aspirin is an excellent pharmaceutical agent for its pain-relieving, antipyretic and anti-inflammatory effects and is one of the most widely available therapeutic agents. Aspirin is one of the safest "over-the-computer" tranquillizers in treating rheumatism, despite the increased number of new non-steroidal anti-inflammatory drugs (NSAIDs). Aspirin is widely used in a reduced dosage for the prevention of cardiovascular disease, strokes and platelet-aggregation related diseases owing to its anti-thrombotic effects. Aspirin is a BCS class II drug having poor aqueous solubility and high permeability along with stability issue like hydrolysis when comes in contact with moisture leading to its instability in solution form. It instantly converts in to salicylic acid and acetic acid through hydrolysis process¹⁰. In this study, the stability profile of aspirin was evaluated by its cocrystallization with benzoic acid. The cocrystals were prepared by using solvent evaporation technique.

MATERIAL AND METHODS:

Aspirin pure drug was procured from from Loba Chemie Pvt Ltd Mumbai. Benzoic acid was purchased from Sigma-Aldrich Chemical Limited and All other chemicals used were of analytical HPLC grade. For preparation of cocrystals, solvent evaporation method was used.

Preparation of cocrystals:

Aspirin and Benzoic acid were cocrystalized with the help of ethanol as solvent. Both aspirin and benzoic acid were taken in fixed stochiometric ratio viz. 1:2. Cocrystals were procured after complete evaporation of solvent at room temperature¹¹.

Characterization of cocrystals

DSC Analysis:

For DSC analysis of cocrystals DSC Q10 V9.9 Build 303, US instrument was used. The aluminium pan containing 2mg of samples were heated within a range of 20^oC to 160^oC in an atmosphere of Nitrogen gas pursing at a flow of 60ml/min. An empty aluminium pan was taken as reference pan.

FT-IR Analysis:

For FT-IR study of cocrystals KBr disc method was utilised. The spectrum was recorded over the range of 4000- 400 cm^-1. FT-IR Alpha Bruker 1206 0280, Germany instrument was used for analysis.

XRD Analysis:

XRD analysis of cocrystals was performed by using XRD model ‘XPERT PRO’ instrument with continues scanning type at 2θ angle position.

Dissolution profile study:

The dissolution study was performed by using 0.05M, 4.5pH sodium acetate buffer as dissolution medium¹². The USP Type II dissolution apparatus (Lab India DS-8000) was used at 50rpm paddle speed and 37±0.5⁰C temperature for 90 minutes. The drug release profile of pure drug, cocrystals and marketed formulation in equivalent amount was performed. Samples (5ml) were taken at an interval of 15 minutes for 90 minutes and an equivalent amount of dissolution medium was added after every sample withdrawal. Samples were suitable diluted and analysed spectrophotometrically at 267nm for calculation of amount of drug¹².

Stability study:

Accelerated stability studies were conducted as per ICH guidelines¹³ and United State Pharmacopeia (USP) guidelines¹². The samples were kept in small glass beakers covered by aluminium foils in stability chamber at temperature 40⁰C±2⁰C and humidity conditions of 75% RH ± 5% for 6 months. Samples were withdrawn at 0, 3- and 6-months interval and analysed by using HPLC technique for evaluation of drug content¹³. Area Under Curve (AUC) was used to evaluate the drug content.

RESULT AND DISCUSSION:

DSC study:

The DSC thermogram of cocrystals exhibited a sharp endothermic peak at 106.67⁰C. Absence of any other peak and peaks of melting point near to drug and coformer proves generation of newer crystalline substance¹⁴.

FT-IR study:

The homosynthon formation between drug and coformer via hydrogen bonding of corresponding COOH functional group of both components is evident from the FT-IR spectra of cocrystals (figure 1). The Hydrogen bonding was conformed by shifting of characterstic peak of non-bonded Hydrogen of Benzoic acid from 3609-3794 to 3405. The C=O stretch of carbonyl group for Aspirin at 1677 and for benzoic acid at 1684 was shifted to 1624 due to homosynthonic interaction between drug and coformer¹⁴.

Figure 1. FT-IR spectrum of aspirin: benzoic acid cocrystals

XRD Analysis:

XRD spectra of cocrystals exhibited key characteristic peaks at 5.1023, 11.4826, 12.8963, and 19.9027 having relative intensity (in percentage) of 66.48, 34.12, 96.28 and 100.00 respectively (figure 2). Homosynthon formation between drug and coformer occurred through interaction of OH---O and O—OH of COOH functional group of both components. Generation of newer peaks in the spectrum of cocrystals conformed the above interaction¹⁴.

Figure 2. XRD spectrum of aspirin: benzoic acid cocrystals

Dissolution profile study:

The in-vitro drug release study exhibited an increased dissolution profile of cocrystals viz. 87% for cocrystals, 31% for pure drug and 60% for marketed formulation (figure 3) over 90 minutes period respectively¹⁴.

Figure 3. Drug release profile graph of aspirin: benzoic acid cocrystals

Stability Study:

Various concentrations of aspirin (Table 1) were taken as per following mentioned specifications for establishment of calibration curve. The graph was plotted between absorbance and Area Under Curve (AUC)¹².

· Mobile phase: ACN: Water (15:85), pH 3.4 (pH adjustment with glacial acetic acid)

· Standard solution: Aspirin in ACN: Formic acid (99:1)

· Column: 4.0 * 30 cm L1

· Flow rate: 2 ml/ min

· Injection volume: 10μ L .

Table 1. Concentrations of samples and their corresponding AUC for ploting calibration curve

Concentration (mcg/ml)	AUC
2	8753
4	17562
6	25816
8	34072
10	41924
12	52467

Figure 4. Stability study of Pure drug Aspirin and cocrystals at interval of 0, 3 and 6 months

Stability profile of Aspirin and Aspirin: Benzoic acid cocrystals:

Samples were withdrawn at 0, 3- and 6-months interval and analysed by using HPLC technique for evaluation of drug content. Cocrystals exhibited a stability profile of 74% while pure drug exhibited 46% stability profile after 6 months stability study. Area Under Curve (AUC) was used to evaluate the drug content. This conformed that cocrystallization process improvise the stability profiles of API’s along with their solubility issues¹³.

Table 2. Stability data of Pure drug Aspirin at interval of 0, 3 and 6 months

Pure Drug
Time	Concentration (mcg/ml)	AUC	Actual Amount of Drug	%Drug Remaining
0 Month	12	51448	11.98277512	99.85645933
3 months	12	35957	8.367184035	69.72653363
6 months	12	24117	5.603734391	46.6977866

Table 3. Stability data of cocrystals at interval of 0, 3 and 6 months

Cocrystal
Time	Concentration (mcg/ml)	AUC	Actual Amount of Drug	%Drug Remaining
0 Month	12	51243	11.93492823	99.45773525
3 months	12	47871	11.14790524	92.89921033
6 months	12	38451	8.949282297	74.57735247

CONCLUSION:

Herein, aspirin and benzoic acid were cocrystallized in fixed stichiometric ratio of 1:2. Ethanol was used as solvent and solvent evaporation technique was used for preparation of cocrystals. DSC study exhibited a newer sharp endothermic peak other than the drug and coformer which conforms the generation of newer crystalline component through interaction of drug and coformer. FT-IR and XRD study conformed the interaction of drug and coformer through COOH functional group. The dissolution profile of cocrystals exhibited increased drug release profile as compared to pure drug and marketed formulation in equivalent amount. The accelerated stability data concludes that cocrystallization technique helps in improvisation of physicochemical stability of drugs. It also proved that cocrystallization technique not only improvised the solubility or dissolution profile of BCS class II drugs but also effects the physicochemical state of drugs. Further in-vitro and in-vivo correlation studies could be possible for better understanding of this technique.

ACKNOWLEDGEMENT:

Authors wants to thank Department of Pharmaceutical Sciences, Maharishi Dayanand University, Rohtak for providing necessary facilities for conducting this study.

CONFLICT OF INTEREST:

Authors do not have a conflict of interest.

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Received on 12.10.2020 Modified on 11.03.2021

Research J. Pharm.and Tech 2022; 15(2):768-772.

DOI: 10.52711/0974-360X.2022.00128