1. Introduction
Hyperglycemia is a persistent condition characterized by the body’s use of glucose (blood sugar) as fuel. Unlike type 1 diabetes, which is caused by a deficiency of insulin, type 2 diabetes is often the result of insulin resistance, which means the body’s cells become less responsive to insulin’s signals to take up glucose from the bloodstream. It induces elevated blood glucose levels, resulting in various consequences, including coronary heart disease, nerve failure, renal dysfunction, and visual impairment. The prevalence of type 2 diabetes has been steadily increasing worldwide, primarily due to sedentary lifestyles, unhealthy diets, and obesity. Anti-diabetic medications such as pioglitazone (PIO) can treat these disorders [
1]. Chirality refers to molecules with non-superimposable mirror images. In other words, chiral molecules exist in two forms, mimicking each other. However, like hands, they cannot be superimposed over each other. Many drugs are chiral, meaning they exist in two different enantiomers which are mirror-image isomers of the same molecule. PIO is a chiral drug that displays a wide spectrum of bioavailability, distribution, and pharmacodynamic characteristics from a pharmacological and toxicological perspective [
2]. The problem of chirality plagues modern medications. The human body can often distinguish between the enantiomers of a drug, even though they have the same chemical formula and molecular weight. This is because the body contains enzymes and receptors that are themselves chiral and can interact selectively with one enantiomer but not the other. This can lead to differences in the pharmacokinetics and pharmacodynamics of a drug, as well as its potential for toxicity and side effects. In this context, PIO is a chiral drug that is used to treat type 2 diabetes. The two enantiomers of PIO have different pharmacokinetic profiles, meaning that they are absorbed, distributed, metabolized, and eliminated differently by the body. The body reacts differently to each racemic substance, using a chiral selection mechanism to generate an array of biological features. Consequently, to optimize the therapeutic efficacy of chiral drugs, it may be necessary to separate the two enantiomers and administer only the active isomer. This process is known as enantiomeric enrichment or chiral switching. Alternatively, the two enantiomers can be administered together as a racemic mixture, but this can sometimes lead to undesirable side effects due to differences in their pharmacokinetics and pharmacodynamics [
3].
Pioglitazone(PIO) belongs to a class of drugs known as PPAR-gamma activators, which work by activating a receptor in the body that regulates glucose and lipid metabolism. When used as a monotherapy, PIO can improve insulin sensitivity and reduce glucose production in the liver, resulting in lower blood glucose levels. It can also improve lipid profiles and reduce cardiovascular complication risk in people with type 2 diabetes. PIO is often used in combination with other medications such as sulfonylurea, metformin, or insulin to help achieve optimal glycemic control in people with type 2 diabetes who have not been able to achieve adequate blood glucose control with monotherapy alone [
4]. The structure of PIO, with a carbonyl group attached to the thiazolidinedione ring, is depicted in
Figure 1.
In vitro assays are frequently employed as preliminary screening techniques to assess the antidiabetic efficacy of drugs, enabling screening of a wide range of potential therapy contenders. The glucose uptake assay on 3T3L1 cell lines was evaluated using flow cytometry to ascertain its antidiabetic efficacy [
5]. Glucose uptake within adipocytes and insulin’s role in sustaining glucose homeostasis can be studied using adipocytes. 3T3-L1 fibroblasts, which develop into adipocytes during adipogenesis, have been the subject of a comprehensive study on adipogenesis [
6]. Diabetes studies involving glucose uptake by cells show that adipocytes are more responsive to insulin after differentiation than other cells. In response to insulin response, glucose transporter-4 (GLUT4) moves from the intracellular region (localized within vesicles) to the plasma membrane. GLUT4 serves a critical function in maintaining the body’s glucose homeostasis. Consequently, when blood glucose levels are lowered, mature adipocytes can acquire high glucose levels even at lower insulin concentrations [
7]. The fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG), which is transported within cells by GLUT4, the same transporter that regulates glucose transport within cells, has the potential to assess glucose uptake in cells by a non-invasive, simple, and safe method [
8].
In this study, we aim to establish a simpler, more specific, and more accurate technique for distinguishing between the two enantiomers of PIO in rat plasma. Accurately identifying and quantifying Pioglitazone-R (PIO-R) and Pioglitazone-S (PIO-S) is essential for evaluating drug safety and efficacy. This is because enantiomers can have significantly different pharmacological properties, such as potency and toxicity, even though they have the same chemical formula [
9]. Previous research has mostly used chiral high-performance liquid chromatography (HPLC) methods to differentiate between PIO enantiomers [
10]. Our ultimate goal is to determine which PIO enantiomer exhibits significantly enhanced activity over the other. Specifically, we will compare the activity of the dextrorotatory (R) and levorotatory (S) enantiomers to assess which one is more effective. Therefore, this study examined the pharmacokinetic differences between PIO enantiomers in albino Wistar female rats, since hepatic glucose production is faster in the female cerebellum, heart, and brain than in male diabetic rats [
11]. Our findings will help improve our understanding of the pharmacological effects of PIO enantiomers and potentially lead to the development of more effective and safer drugs.
3. Materials and Methods
3.1. Chemicals and Reagents
Aurobindo Pharma, Hyderabad provided PIO racemic form and PIO-R and S for bioanalytical studies. The internal standard, Glimepiride, was obtained from Hetero Labs, Hyderabad. Merck, India supplied Ammonium acetate, Acetonitrile, and methanol HPLC grade for the study, while Millipore, MA, USA provided 0.45 µm pore size filters for filtering the mobile phase and solutions. The in vitro study utilized 3T3-L1-mouse embryo fibroblast cell lines (NCCS, Pune, Maharastra, India) and DMEM glucose-free medium (Himedia, Mumbai, India) for cell culture. The researchers also used adjustable multichannel pipettes and Fetal Bovine Serum (#RM10432, of Himedia, Mumbai, India), D-PBS (#TL1006, of Himedia, Mumbai, India), 2-NBDG (Invitrogen: Cat no. 11046, Cayman Chemical, Rajasthan, India), a 6-well cell culture plate (Biolite—Thermo, Bengaluru, Karnataka, India), 50 mL centrifuge tubes (# 546043 TORSON, Mysuru, Karnataka, India). A glucose reagent kit from AGAPPE (Mumbai, India) was used to assess glucose in rat plasma utilizing a semi-automated biochemistry analyzer (Labmate, Ranchi, India). In the study, all chemicals used were over 95% pure.
3.2. Animals
The study utilized Albino Wistar female rats (weighing 200–250 g) procured from a CPCSEA-approved commercial breeder called the Center for Experimental Pharmacology and Toxicology. The rats were kept in a sterile laboratory environment and permitted at least one week to acclimatize before the study began. The rats received unlimited water access during the experiment and fasted for 12 h before the treatments were administered. All animal care instructions and treatments followed the regulations suggested by the board of CPCSEA. The animal ethical committee of the JSS Academy of Higher Education & Research reviewed and approved the animal use and care protocol (Reg No: 261/PO/ReBi/S/2000/CPCSEA).
3.3. Chromatographic Parameters
A Shimadzu LC-2030C Plus Prominence I High-Performance liquid chromatographic system (HPLC) equipped with a UV detector was used for chromatographic studies. A Phenomenex-manufactured lux i-Amylose-3 (150 mm × 4.6 mm), 5 µm column was utilized for the chromatographic separations. The mobile phase is composed of 10 mM ammonium acetate acetonitrile (60:40, v/v). Through the binary flow pump, isocratic elution was obtained at a flow maintained at 0.6 mL/min and further the wavelength detection by UV at 265 nm. Throughout the study, column temperature was fixed to 35 °C and adjusted with an injection volume of 20 µL. Data acquisition and integration were evaluated using Lab Solutions software version 5.90.
3.4. Standard and Stock Solution Preparations
A 1.0 mg/mL concentration of the primary stock of racemic PIO-R and S, and I.S. (Glimepiride) solutions was obtained by using acetonitrile as a diluting agent. Diluting the principal stock solutions with acetonitrile yielded concentrations ranging from 3.125 to 100 µg/mL for racemic PIO working solutions. Similarly, glimepiride (I.S.) was prepared at a known concentration of 10 µg/mL in acetonitrile. Quality control solutions, including LLOQ at 3.125 µg/mL, LQC at 6.25 µg/mL, MQC at 25 µg/mL, and further HQC at 100 µg/mL, were produced in a way similar to the standard experimental solutions mentioned above.
3.5. Sample Preparation
Liquid–liquid extraction was used for sample preparation. The supernatant, i.e., blank plasma (100 µL), was spiked with PIO (200 µL) solution and IS (100 µL) and vortexed for 45 s. Acetonitrile was used to dilute the solution to 1.5 mL, and the finished product was centrifuged at 10,000 rpm for 10 min at 4 °C. Syringe filters were used to filter the final supernatant liquid before it was injected into the HPLC system for analysis.
3.6. Non-Radioactive Glucose Uptake Assay of 3T3L1 Cell Lines Utilizing Flow Cytometry
The process involved seeding cells in six-well plates and allowing them to incubate overnight. The medium was replaced later with a serum-free, glucose-free DMEM solution that contained insulin and a fluorescent glucose analog called 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) [
14], followed by further testing samples (50 μM). After a 24-h incubation period, the culture medium was extracted, and the cells were thoroughly washed twice with phosphate-buffered saline (PBS). The cells were detached by trypsin, harvested, and kept at 4 °C in FACS tubes. Cells were centrifuged at 2500 RPM for 5 min at 25 °C before being resuspended in 0.5–1 mL of PBS. The cells were examined using a flow cytometer, which collected fluorescence data from 10,000 single-cell events. FITC (Fluorescein isothiocyanate) signals or intensity were detected in the FL1 (Fluorescence 1) channel, which was intended to detect cell absorption of 2-NBDG’s excitation and emission at 465 and 540 nm. The relative amount of fluorescence intensity (FI) was calculated using BD Cell Quest Pro (Version: 6.0) software by subtracting the background FI from the FI of a single cell’s treatment with or without the addition of the 2-NBDG factor.
3.7. Estimation of Glucose in Rat Plasma by Semi Automated Biochemistry Analyzer
The semi-auto biochemistry analyzer used in this study operates on filter photometry. Initially, distilled water was pumped through the machine after turning it on. Each parameter was programmed, and the system was set to flow cell mode with printer mode selected. Estimating glucose levels was carried out using pharmacokinetic plasma samples. Containers were labeled as blanks, standards, and tests. A 1000 µL quantity of standard reagent and 10 µL of distilled water were combined to make a blank analytical tube. A 1000 µL quantity of working reagent and 10 µL of glucose standard were assembled into the sample tube. Finally, 1000 µL of standard reagent and 10 µL of plasma sample were well-mixed in the testing tube. Later, the optical density (T1) was recorded for about 30 s after adding the standard or sample. Similarly, after 60 s of the first reading recording, the second estimation (T2) was measured. The fluorescence intensity of the sample was solely proportional to the concentration of glucose present in the specimen. The concentration of glucose (mg/dL) in the specimen (plasma) was estimated using the fixed time kinetic method, by employing the formula (Absorbance of the sample/Absorbance of the standard) × 100.
3.8. Validation of Bioanalytical Methods
3.8.1. Calibration Curve
The concentrations of both enantiomers ranged from 3.125 to 100 µg/mL and were produced by spiking blank plasma samples with 200 µL of PIO racemic working standard solutions and 10 µL of I.S. Samples for calibration were prepared as mentioned above. To establish the calibration curves, linear regression least squares analysis was applied to plot peak area ratios vs. concentrations of enantiomer spikes in samples by using Microsoft
® Excel (
https://www.microsoft.com/en-us/microsoft-365/excel). Each enantiomer’s detection limit (LOD) and quantification limit (LOQ) were considered.
3.8.2. Recovery
We assessed the peak area ratios of the enantiomer to the internal standard at 3 concentrations (every five runs) based on post-extraction spiked samples to determine enantiomer recovery. Similarly, I.S. recovery was also evaluated at 10 µg/mL [
15]. The analytes’ and I.S.’s absolute recoveries do not have to be 100%, but the recovery limit should be constant, unambiguous, and replicable.
3.8.3. Precision and Accuracy
On five consecutive days, five replicates with similar concentrations were compared to test precision and accuracy. Precision was quantified by determining %CV (Coefficient of variation) for all four QC levels [
16]. While accuracy was represented as a percentage relative error (R.E.%). Except for the LOQ, the acceptable bounds for precision and accuracy were 15% relative standard deviation and 15% relative error, respectively [
17].
3.8.4. Stability
Biomatrix stability was tested for six hours at ambient temperature (25–22 °C) for each enantiomer (bench top). PIO racemate samples were stored at around −20 °C for 30 days to evaluate their freeze stability throughout three stages. Throughout each freeze-thaw cycle, spiked plasma samples were frozen for 24 h at −20 °C and thawed at room temperature. To meet the compliance criteria for stability, the R.S.D. % compared to the recently prepared standard must be within 15%.
3.9. Pharmacokinetic Investigations in Rats: A Preliminary Study
We utilized the validated bioanalytical method to pharmacokinetic studies of PIO in rats. For the study, Wistar albino female rats weighing 200–210 g at 4–6 weeks of age were recruited. Animals were acclimatized for 7 days under laboratory conditions. An oral dose of 30 mg/kg of racemic PIO was administered to each rat after overnight fasting with free access to water. After dosing, blood was collected at 0, 1, 2, 4, 6, 8, 12, 24, and 48 h from the respective animals through a retro-orbital puncture in an anti-coagulant (EDTA) containing vial. The collected blood was centrifuged at 3500 rpm for 10 min at 10 °C. The supernatant was injected into the HPLC system. A graph of plasma concentration v/s time was constructed to obtain various pharmacokinetic parameters using Phoenix WinNonlin 8.1 software. GraphPad Prism 9 software was used to assess the differences in probability values through one-way ANOVA followed by Tukey’s multiple comparison test for the pharmacokinetic parameters of PIO enantiomers.
3.10. Data Analysis
Phoenix WinNonlin 8.1 software was used to analyze pharmacokinetic parameters. Source data analyzed for maximum plasma concentration (Cmax) and maximal plasma concentration-time (Tmax). All statistical calculations were performed using GraphPad Prism 9 software. Using the DAS outcome, a non-compartmental model with pharmacokinetic parameters was derived. Data were displayed as mean values and standard deviation (S.D.). Based on a significance level of p 0.05, the one-way ANOVA followed by Tukey’s multiple comparison test was performed to assess the statistical significance of the variations in pharmacokinetic parameters between the two enantiomers.
4. Conclusions
Pioglitazone (PIO ) is a chiral drug that is used to treat type 2 diabetes. The two enantiomers of PIO have different pharmacokinetic profiles, meaning that they are absorbed, distributed, metabolized, and eliminated differently by the body. To optimize the therapeutic efficacy of chiral drugs, it may be necessary to separate the two enantiomers and administer only the active isomer. Additionally, the HPLC method developed and validated in this study provides a reliable and sensitive tool for measuring PIO enantiomers in plasma. In 3T3 L1 cells, PIO-R exhibited greater glucose uptake by relative fluorescence intensity for 2-NBDG than PIO-S. This method can be used in future studies aimed at investigating the pharmacokinetics and pharmacodynamics of PIO after oral administration of racemic PIO at 30 mg/kg in female albino Wistar rats. In the case of PIO, the two enantiomers, PIO-R and PIO-S, have different pharmacokinetic and pharmacodynamic properties. Our results showed that PIO-R had higher concentrations in plasma and a lower affinity for PPAR-gamma receptors than (S)-PIO. This suggests that PIO-R may be responsible for the majority of the pharmacological activity of the racemic mixture. The AUCisomer (R)/AUCisomer (S) ratio of more than 2.0 indicates that the concentration of PIO-R is more than two times higher than that of PIO-S, which supports the notion that PIO-R plays a more significant role in the pharmacological activity of PIO. The results of this study highlight the importance of considering enantioselectivity in drug development and clinical practice, as the pharmacokinetics and pharmacodynamics of enantiomers can differ significantly. The discovery of discrepancies in the plasma levels of PIO enantiomers in female rats highlights the importance of considering gender differences in drug metabolism and distribution. This finding suggests that factors such as sex hormones, body composition, and liver enzyme activity may play a vital role in the observed stereoselectivity. Further investigations into the underlying mechanisms of these differences may lead to the development of more targeted and effective treatments for female patients and are needed to identify the precise causes of these stereoselectivity variations. Ultimately, this knowledge could lead to the development of more effective therapies for a range of conditions. Overall, our study provides important insights into the pharmacokinetics of PIO enantiomers in rats and their potential impact on drug efficacy.