Patra et al. Future Journal of Pharmaceutical Sciences (2021) 7:157 Future Journal of https://doi.org/10.1186/s43094-021-00307-2 Pharmaceutical Sciences RESEARCH Open Access Valsartan in combination with metformin and gliclazide in diabetic rat model using developed RP-HPLC method Rasmita Patra, Yedukondalu Kollati, Sampath Kumar NS and Vijaya R. Dirisala* Abstract Background: Oral administration of biguanides (metformin) and sulfonylureas (gliclazide) are the most common approach of management of type 2 diabetes in humans. Among these diabetic patients, approximately 40–60% suffers from hypertension. Hence, the need of the day is application of polytherapy. A major challenge in polytherapy is the drug-drug interactions that may arise. Hence, this study is focused to develop a reverse phase high-performance liquid chromatography (RP-HPLC) method for concurrent estimation of diabetic drug metformin and hypertension drug valsartan using C18 column and find any possible pharmacokinetic interactions between the two drug combinations strategies, i.e., metformin-valsartan and gliclazide-valsartan in streptozotocin-induced diabetic rats. Result: The bioanalysis of drug-drug interaction pharmacokinetic result showed no significant difference in the tmax of single treatment of gliclazide and single treatment of metformin or upon co-administration with valsartan. Conclusion: Our study has shown that polytherapy of valsartan, a drug administered for hypertension along with hypoglycemic drugs metformin and gliclazide, can be advantageous and safe in patients suffering from both diabetes and hypertension. Keywords: RP-HPLC, Metformin, Valsartan, Gliclazide, Hypertension, Diabetes mellitus Background disease if left undetected or untreated. Such patients re- Diabetes has become a growing epidemic, and the per- quire polytherapy wherein drug-drug interactions may centage of patient population is increasing in leaps and lead to adverse side effects [5, 6]. Besides mortality, the bounds [1]. It is a chronic condition of metabolic dis- therapeutic effect of either drug may lower or enhance order featured by elevated glucose level in blood circula- or may execute new activity which was not observed in tion [2]. This is due to the improper alteration in earlier therapeutic studies [7]. regulation of protein, carbohydrate, and lipid metabol- Metformin, the first known line of therapy for type 2 ism [3], due to reduction in glucose uptake by skeletal diabetes, acts by reducing the rate of gluconeogenesis muscle cells and uncontrolled hepatic glucose output. and enhances sensitivity of insulin [8], reducing free fatty Based on insulin secretion, diabetes can be classified acids (FFA) through adipose tissue inhibition [9]. It also under two major categories: types 1 and 2, where type 1 showed therapeutic effect on obesity metabolic disorder is insulin dependent, whereas type 2 is non-insulin [10]. Women, who suffer from polycystic ovary syn- dependent [4]. The concern among diabetic patients is drome (PCOS), sometimes rely on this drug for ovula- the multi organ damage that happens in course of the tion and regular menstrual cycle [11, 12]. The chemical structure of metformin is shown in Fig. 1. Apart from * Correspondence: [email protected] metformin, another drug, i.e., a sulfonylurea, gliclazide is ’ Department of Biotechnology, Vignan s University (Deemed to be), Guntur, frequently prescribed to control type 2 diabetes. AP 522213, India © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Patra et al. Future Journal of Pharmaceutical Sciences (2021) 7:157 Page 2 of 10 anti-hypertensive drug lisinopril [20, 21]. An in vivo study in diabetic rat and rabbit revealed synergistic effect of gliclazide upon co-administration with anti- hyperlipidemic drug pravastatin [22]. Another study by Patel and Kothari revealed no drug-drug interaction be- tween pitavastatin (cholesterol-lowering drug) and can- desartan (anti-hypertensive drug) [23]. In this study, we have focused on the effects of valsartan with either gli- clazide or metformin by concomitant administration in diabetic rats. Materials Fig. 1 Structure of metformin Ethical approval All experimental procedures and protocols used in the Gliclazide is known to shield pancreatic beta cell apop- animal study were reviewed and approved by the Institu- tosis induced due to hyperglycemia [13–15] and its tional Animal Ethical Committee (IAEC/1657/CMRCP/ therapeutic effect on plaque formation in arteries, T2/PH), Vijaya College of Pharmacy, Hyderabad, Telan- thereby reducing atherogenesis in hyperglycemic pa- gana, India. tients [16]. Torrent Pharmaceutical Ltd., Ahmedabad, supplied The prevalence of hypertension among diabetic pa- valsartan, gliclazide, and metformin. The working stand- tients is very high, accounting to 75% of coexistence ard was kindly provided by Lupin Pharma, India. All [17]. Valsartan, an angiotensin II receptor blocker, is the other reagents were procured from E. Merck, (India) common treatment option for hypertension [18]. The Ltd., and are HPLC grade. The D-glucose GOD-POD chemical structure of valsartan is shown in Fig. 2. Since colorimetric assay kit was supplied by Excel Diagnostic these patients are subjected to polytherapy, there arise Center, Hyderabad. Streptozotocin was obtained from possibilities of occurrences of interactions between Sigma. valsartan-metformin and valsartan-gliclazide. Hence, our study is aimed to look for possible drug interaction ef- Methods fect of antihypertensive drug valsartan along with met- Parameters of chromatography formin. Hence, RP-HPLC method is developed, which The HPLC from Waters Company was integrated with can be applied for routine laboratory analysis, forced XTerra® C18 column of 5 μm (with features 4.6X250 degradation studies, and pharmacokinetic studies (as mm) for separation. Mobile phase was fixed to mixture well as in laboratory analysis, forced degradation stud- of phosphate buffer and methanol in 60:40 ratio, and the − ies). In our previous study, we had developed a method rate of flow was 1 mL.min 1. The filtration of the mobile (RP-HPLC) for concurrent estimation of gliclazide and phase was performed by using a 0.2-μm membrane filter. valsartan [19]. Chromatographic procedure was performed at RT. Clinical studies illuminating drug-drug interaction is an important parameter for polytherapy. Various studies Preparation of standard solutions have thrown light on drug-drug interactions of which To prepare standards, metformin and valsartan were one of the studies has shown reduced hypoglycemic ef- each weighed 100 mg and added into 10 mL of a solvent fect of gliclazide upon prolonged co-administration with which is a mixture of phosphate buffer and methanol in 60:40 ratio. By adding another 15 mL of phosphate buf- fer and methanol solution, the final volume was made up to 25 mL. The stock solution was subjected to sonic- ation for 15 min, and by mixing 75 mL of diluent, the volume was made up to 100 mL to achieve a 1 mg/mL stock solution. Standard stock solution was diluted to − achieve 2 to 20 μg.ml 1 as final concentration for valsar- tan, whereas metformin final concentration was set to − 3.12 to 100 μg.ml 1. Validation of the method The following RP-HPLC parameters were validated as Fig. 2 Structure of valsartan per ICH guidelines [24]. Patra et al. Future Journal of Pharmaceutical Sciences (2021) 7:157 Page 3 of 10 Linearity Table 1 System suitability: metformin-valsartan The standard (metformin and valsartan) stock solutions S. No. Metformin (30 μg.ml−1) Valsartan (10 μg.ml−1) were diluted with mobile phase. The final concentration 1 30.019 9.420 μ −1 of valsartan was in the range of 2 to 20 g.ml whereas 2 29.168 9.159 the final concentration of metformin was 3 to − 3 28.839 9.238 100 μg.ml 1. A volume of 20 μL was injected into C18 column. The calibration graph was achieved by peak 4 29.139 9.550 area on Y-axis versus concentration of valsartan and 5 29.05 9.289 metformin on X-axis. 6 28.939 9.150 Standard deviation 0.423 0.157 Accuracy Mean 29.192 9.301 Accuracy was validated in triplicate by addition of three CV (%) 1.450 1.688 different amounts of valsartan and metformin to previ- ously analyzed samples. The quantities of analytes recov- Accuracy (%) 97.31 93.01 − ered were compared with the quantities of drugs μg.ml 1, microgram per milliliter; % CV, the percentage of coefficient of variation injected. The quantities added were equivalent to 50, 100, and 150% of the amount originally presented. factor, retention time, resolution, area, and % peak area Precision were analyzed. Twenty microliters of the working stand- − To evaluate precision, 6 tests of both the drugs of 10 ard solution of valsartan (10 μg.ml 1) and metformin − − and 30 μg.ml 1 were injected into the column. This pro- (25 μg.ml 1) was injected into the column for 6 times. cedure was performed on a single day (which is also called intraday precision) as well as on three different Analyzed method specificity days (which is also called intermediate precision). It was monitored to achieve clear peaks for both the drugs avoiding interference (from drug impurities or sol- Limit of detection and quantitation vents) by the proposed method.