Preformulation Study of Glimepiride: An Insight for Formulation and Development of Parenteral Formulation

Aim: The objective of preformulation study is to develop the elegant, stable, effective and safe dosage form by establishing kinetic profile, compatibility with other formulation excipients and physico-chemical parameters of new drug substance. This could provide important information for formulation design or support the need for molecular modification. So, in the present study preformulation studies were performed on Glimepiride (GMP) to assess its suitability for parenteral formulation. Glimepiride is the first III rd generation sulphonyl urea used to treat type –II diabetes mellitus. Methods: The authenticity of GMP was established by DSC and FTIR spectra. A UV spectrophotometric method and HPLC method were employed for determination of GMP in bulk Original Research Article Patel et al.; JPRI, 34(15B): 38-49, 2022; Article no.JPRI.83471 39 active pharmaceutical ingredient (API). Results: The UV method was linear in the range of 3-10 μg/ml. The low % CV values of intra-day and inter-day variations revealed that the proposed method is robust. The retention time of GMP in HPLC method was found to be 1.9 min. The method was proven robust by obtaining very high regression coefficient value (0.999). Conclusions: The results of the physicochemical study of drug revealed suitability of GMP for parenteral route. Moreover, the drug was found stable in both solid as well as liquid state at different conditions.


INTRODUCTION
Preformulation is a group of studies that focus on the physicochemical properties of a drug candidate that could affect the drug performance and the development of dosage form [1]. Also, it could provide important information for formulation design or support the need for molecular modification. Every drug has intrinsic chemical and physical properties which have been considered before development of pharmaceutical formulation. This property provides the framework for drugs combination with pharmaceutical ingredients in the fabrication of dosage form [2].
The objective of preformulation study is to develop the elegant, stable, effective and safe dosage form by establishing kinetic profile, compatibility with other formulation excipients and establish physic-chemical parameter of new drug substance. The classic preformulation study requires drug characterization in solid as well as liquid phase. Preformulation can help in cost cutting for effective therapeutic development of the product.
Diabetes mellitus is a condition in which a person has a high blood sugar level, either because the body doesn't produce enough insulin, or because body cells don't properly respond to the insulin that is produced [3,4]. In 2021, Approximately 537 million adults (20-79 years) are living with diabetes. The total number of people living with diabetes is projected to rise to 643 million by 2030 and 783 million by 2045 [5].
Glimepiride is the first III generation sulphonyl urea. Glimepiride is a sulfonyl urea used to treat type -II diabetes mellitus. Molecular formula of glimepiride is C24H34N4O5S with a molecular mass of about 490.617g/mol [6]. It belongs to class-II of Biopharmaceutical classification system. It is completely insoluble in water, acidic media and slightly soluble in various buffers and organic solvents [7]. The mechanism of action of Glimepiride in lowering blood glucose appears to be dependent on stimulation the release of insulin from functioning pancreatic beta cells, and increasing sensitivity of peripheral tissues to insulin [8].
Polymeric microspheres as a parenteral drug delivery system are primarily developed for sustained release of drugs for prolonged systemic therapeutic effects after subcutaneous (SC) or intramuscular (IM) administration. Polymers used for formulation of microspheres are biodegradable and biocompatible. In the present research PLGA is used as a polymer [9,10]. This polymer is usually used for biodegradable controlled release microparticles. In the present research solvent evaporation method is applied to formulate microparticles for injectable controlled release drug delivery system. Microspheres in the finished product are in a dry powder form. Prior to administration, a microsphere product is reconstituted in a liquid diluent which can be supplied in a separate container or in the liquid compartment of a dualchamber prefilled syringe [11,12].
So, in the present study focus was given on preformulation studies of GMP. The main objective of the study was to assess GMP for its suitability to be formulated as SR microspheres for parenteral delivery.

Drug Identification
The drug identification was performed by organoleptic properties, melting point, UV, HPLC, FTIR and DSC.

Determination of Thermodynamic Solubility
Solubility study of drug was performed using different solvents such as methanol, ethanol, dimethyl sulfoxide, ethyl acetate, Dichloromethane (DCM) and N-Methyl-2-Pyrrolidone. Samples were shaken on a rotary shaker at 37ºC for 24 hours. The two phases are then separated by filtration [6]. Amount of solute in supernatant is then determined using UV spectrophotometric analysis at the corresponding λmax of each solvent.

Analysis of GMP by UV spectrophotometry method [13]
Standard stock solutions of GMP was prepared in acetonitrile and scanned spectrophotometrically over the range of 200-400nm with double beam spectrophotometer (Shimadzu UV spectrophotometer, 240 j/PC, Japan), against the respective blank, to determine wave length of maximum absorbance (λmax).
A stock solution containing 1000 µg/ml GMP was prepared by dissolving 25 mg GMP in 5 ml of acetonitrile in a 25 ml of volumetric flask and volume was made upto 25 ml with the acetonitrile. From these stock solutions, suitable aliquots were taken and diluted using appropriate solvent to get dilutions of 3-10 μg/ml. The determinations were conducted in triplicate and studied for three days to check intra and inter day variations.

Calibration
curve was constructed at concentrations range 3-10 μg/ml. Absorbance of each solution was measured at the wavelength of 228 nm. Calibration curve was constructed for GMP by plotting absorbance versus concentration at 228 nm wavelength. The determination was conducted in triplicate.

Analysis of GMP by HPLC method [13]
Glimepiride was quantified using a Shimadzu prominence-iLC2010 high performance liquid chromatography (HPLC) system equipped with isocratic pump, auto sampler, and photodiode array detector. The mobile phase was water: acetonitrile, 50:50 (v/v). The system was equipped with an all-time C18 column (30 x 4.6 mm, 5µ), temperature of column was ambient and the flow rate was set to 1 mL/min. The injection volume for drug loading samples was 10 µl. The chromatographs were analyzed with empower software at 228 nm. Linearity of the method was proved for concentration range of 0.4ppm to 150ppm.

Drug-Polymer Compatibility Study
The physical stability of glimepiride with polymer was evaluated at 25℃ and 60% relative humidity (RH). Additionally, the samples were also closed in vials and stored in refrigerator (2-8℃). The samples were removed after 30 days.

Fourier transform-infrared (FTIR) study [14]
The FTIR analysis was used for qualitative estimation and identification of functional group present in the compound. GMP was mixed with each of the components at an appropriate ratio; equivalent to that used in formulation process. Each mixture was stored in USP type-1 glass vial at 25°C±5°C, 60±5% RH (relative humidity) for one month. FTIR spectroscopy, Shimadzu, Model 8400, Japan, was used to study the compatibility of pure drug and other preparation composites, by KBr pellet method and scanned from 4000 to 400 cm -1 .

Differential Scanning Calorimetry (DSC) [15]
DSC is the thermal analysis method by which we can measure the interaction of drug with polymer. The thermal analysis of Drug, PLGA, physical mixture of Drug and PLGA was performed by using 3-5 mg of samples in a standard thermal aluminum pan with a comparable lid and heated from 0 to 300°C at a 10°C/min heating rate in Mettler toledo DSC (METTLER TOLEDO, Switzerland).

Organoleptic properties and Melting Point
Glimepiride is odourless and almost white powder which is sticky in nature. The melting point of drug was in the range 207-209°C.

Drug identification by UV
The identification of drugs has been increased considerably in recent years by use of maximum absorbance because of their importance in pharmaceutical analysis. The maximum absorbance of GMP in acetonitrile was found at 228 nm as depicted in Fig. 1 which was similar to literature of GMP. This indicates that the received active pharmaceutical ingredient is authentic.

Drug identification by HPLC
Glimepiride was qualified using a Shimadzu prominence-iLC2010 high performance liquid chromatography (HPLC) system equipped with isocratic pump, auto sampler, and photodiode array detector. The mobile phase was water: acetonitrile, 50:50 (v/v). The system was equipped with an all-time C18 column (30 x 4.6 mm, 5µ), temperature of column was ambient and the flow rate was set to 1 mL/min. The injection volume for drug loading samples was 10 µl. Identification of GMP was proved by peak retention time which was found to be 1.9 min at 228 nm (Fig. 2). The result was in accordance with the literature information of GMP. This indicates that the received active pharmaceutical ingredient is authentic.

Results
found in FT-IR spectra and Characteristic peaks of GMP drug founds similar to reference FT-IR spectra of GMP drug. The characteristic absorption peaks of GMP in FT-IR spectra is shown in Fig. 3 and the functional groups responsible for characteristic peaks of GMP are mentioned in Table 1.

Determination of Thermodynamic Solubility
Glimepiride is a practically insoluble in water, soluble in dimethyl formamide and N-Methyl-2-Pyrrolidone, sparingly soluble in dichloromethane, very slightly soluble in methanol, ethanol and ethyl acetate. The solubility of GMP in various solvents is shown in Table 2.

Analysis of GMP by UV spectrophotometry method
The development of spectrophotometry methods for the determination of drugs has been increased considerably in recent years because of their importance in pharmaceutical analysis. Based on the experimental data the standard calibration curves were plotted. The regression analysis showed very good correlation (r 2 =0.9999) in acetonitrile. These solutions obeyed Beer-Lambert's law and the linearity was found in concentration range of 3-10 μg/ml in acetonitrile. The standard curve of GMP is shown in Fig. 5.

Analysis of GMP by HPLC method
The method was developed to quantify Glimepiride for assay and in-vitro release. The chromatography was checked for its linearity and was proved to be linear from 0.40 ppm to 149.87 ppm which covers the linearity requirements for assay and in-vitro release, the chromatograph of the same is depicted in Fig. 6. The quantification of % Assay and/or % in-vitro release was done by estimating area of sample peak with area of standard peak of known concentration using formula Au/As*Cs/Cu*100 after conforming above mentioned system suitability requirements and the results are mentioned in Table 4. Method was found to be suitable for its intended uses.

Drug-Polymer Compatibility Study
The characteristic absorption peaks of GMP in FT-IR spectra as shown in Fig. 3 proves stable and pure drug profile. Further, stability of GMP has been also assessed at various temperatures, moisture, light and oxidation condition. The results obtained from stability study under preformulation exhibited stable characteristics of drug at different storage conditions which are shown in Table 5.

Fourier transform-infrared (FTIR) study
The FTIR spectral analysis showed that there is no appearance or disappearance of any characteristic peaks of pure drug glimepiride and in the physical mixture which confirms the absence of chemical interaction between drug and polymers. The FT-IR spectra of physical mixture in initial condition and after 1 month study are shown in Fig. 7 and 8 respectively and the functional groups responsible for characteristic peaks are mentioned in Table 6.

Differential Scanning Calorimetry (DSC)
DSC thermogram of GMP and polymer mixture showed that there is no change observed in the endothermic peak of drug and polymer in physical mixture at initial condition and after 1 month, which confirms the absence of chemical interaction between drug and polymers as shown in Fig. 9 and 10 respectively.

CONCLUSION
From the results of the different preformulation studies, it can be concluded that GMP is suitable for sustained release microsphere parenteral formulation. The results of UV, HPLC, FT-IR and DSC suggested the drug is authentic. The UV method and HPLC method showed good correlation indicating they can be used for quantification of drug in bulk and in vitro studies. The solubility study of drug suggested that it is soluble in organic media suggesting its suitability for sustained release formulation. Stability study under preformulation studies revealed stable characteristics of drug confirming final stability of formulation.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.