1. INTRODUCTION:

Diabetes mellitus (DM), commonly referred to as diabetes, is a complex metabolic disorder characterized by hyperglycemia—a pathologically elevated blood glucose level persisting over time¹. First-line hypoglycemic agents for the management of type 2 diabetes primarily include insulin secretagogues, which are subdivided into sulfonylureas and non-sulfonylureas ^2,3.

Glimepiride (GLMP), a third-generation sulfonylurea oral antidiabetic agent, is widely used in the treatment of type II diabetes mellitus⁴.

According to the Biopharmaceutical Classification System (BCS), GLMP is classified as a Class II drug due to its high permeability but poor water solubility⁵. It has a pKa of 6.2 and is only sparingly soluble in water (0.0384mg/mL). Its bioavailability is limited by delayed gastric dissolution and significant inter-individual variability⁶. GLMP exhibits very low solubility at acidic and neutral pH (<0.004mg/mL), with a slight increase (~0.02mg/mL) at pH levels above⁷. These solubility issues contribute to variable bioavailability and suboptimal dissolution. After oral administration, GLMP undergoes rapid hepatic absorption and first-pass metabolism⁷. The drug’s poor aqueous solubility and low wettability hinder formulation development and result in inconsistent oral bioavailability^8,9. Therefore, enhancing its solubility in gastric fluids is essential to improve both the rate and extent of drug absorption¹⁰. Similarly, Gliclazide (GLC), a second-generation sulfonylurea, is an oral hypoglycemic agent that also belongs to BCS Class II^11–13. GLC is a white crystalline powder with low water solubility and a pKa of 5.8. It exhibits modest gastrointestinal absorption and bioavailability that varies among individuals¹⁴. Due to its poor and pH-dependent solubility, GLC displays slow and inconsistent absorption, leading to significant intra- and inter-subject variability in pharmacokinetics following oral administration^15–17.

Permeability and solubility are the two principal factors governing a drug’s absorption rate and extent¹⁸. Approximately 40–70% of therapeutic compounds under clinical development face challenges related to bioavailability, primarily due to poor solubility and limited absorption^19,20. To overcome such pharmacokinetic limitations, nanotechnology-based drug delivery systems have emerged as a promising approach, offering targeted delivery, enhanced drug retention, and improved protection from the biological environment. These systems are particularly effective for both hydrophilic and hydrophobic drugs²¹. Numerous studies have highlighted the efficacy of nanotechnology-based formulations in enhancing pharmacokinetic profiles—particularly in terms of bioavailability and absorption—of poorly soluble drugs^22–24. Nanomedicine, or nano-therapeutics, utilizes nanoparticles (NPs) to encapsulate drugs, thereby enhancing their therapeutic properties while minimizing systemic toxicity. Common NP systems include liposomes, niosomes, solid lipid nanoparticles, nanospheres, and polymeric micelles²⁵.

NPs are gaining popularity in pharmaceutical sciences due to their ability to significantly increase the solubility of poorly water-soluble drugs. Their high surface-area-to-volume ratio not only enhances reactivity but also facilitates improved absorption and transport of various compounds^26,27. As a result, nanoparticles have become pivotal in increasing drug solubility, enhancing bioavailability, and enabling controlled or sustained release. One promising nanotechnological approach is the use of nanosuspensions (NSs)—colloidal dispersions consisting of pure drug particles stabilized by surfactants. These systems can be utilized in both aqueous and organic phases to enhance drug solubility ^28–30. Nanosuspensions, due to their ultra-fine particle size, promote improved dissolution and absorption, thereby increasing therapeutic efficacy^31,32. They are particularly advantageous for drugs with high log P values, high melting points, and poor water solubility but good solubility in oils³³. This technique is widely used to improve the bioavailability of BCS Class II drugs. Various formulation strategies have been explored to enhance the bioavailability and therapeutic performance of GLMP and GLC, including solid dispersions^34,35, inclusion complexes with β-cyclodextrin^36,37, nanocrystals^38,39, solid lipid nanoparticles^40,41, and self-microemulsifying drug delivery systems^42,43. These approaches primarily aim to improve the solubility of GLMP and GLC, which in turn enhances their bioavailability. In the present study, we investigated the In Vivo pharmacokinetics of previously developed GLMP and GLC nanosuspensions to confirm their enhanced bioavailability in comparison to the pure drugs. Prior characterization of these nanosuspensions revealed successful drug incorporation and improved in vitro release profiles, indicating enhanced solubility^44-52.

2. MATERIALS AND METHODS:

2.1 Chemicals and Reagents:

Glimepiride and Gliclazide were received as gift samples from Tiruvision Medicare, Baddi. Acetone was purchased from Ibuychemikals, and methanol was procured from Labogens. Metformin (used as an internal standard, IS) was also obtained from Tiruvision Medicare, Baddi. All other chemicals and reagents used in the study were of analytical grade.

2.2 Animals:

The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC), GJUS&T, during its meeting held on November 3, 2023 (Approval No.: IAEC/2023/28-37). Healthy male and female Wistar rats (250–300g; 8–10 weeks old) were procured from Lala Lajpat Rai University of Veterinary and Animal Sciences. The animals were acclimatized for one week under standard laboratory conditions. All animal experiments were conducted in accordance with the guidelines of the Committee for Control and Supervision of Experiments on Animals (CCSEA).

2.3 Software:

The In Vivo pharmacokinetic analysis was performed using PKSolver, an add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel.

2.4 Formulation of Glimepiride (GLMP-NS) and Gliclazide (GLC-NS) Nanosuspensions:

GLMP-NS was prepared using the precipitation–ultrasonication method and optimized via a 3-factor, 2-level full factorial design. Factors influencing drug release, entrapment efficiency, and particle size included the concentrations of HPC, Kollicoat, and sonication time. The optimized formulation (F2), showing the highest entrapment efficiency, smallest particle size, and maximum drug release, was selected for further studies. Detailed methodology and characterization are discussed in a previously published article^53,54.

Similarly, GLC-NS was formulated using the precipitation–ultrasonication method and optimized using a Box-Behnken design. Soluplus, Kollicoat, and sonication time were the independent variables, while particle size, entrapment efficiency, and drug release were the dependent variables. The F11 formulation, which exhibited the highest entrapment efficiency, smallest particle size, and maximum drug release, was selected for subsequent in vitro and In Vivo investigations⁵⁵.

2.5 In Vivo Experimental Design:

The therapeutic efficacy of GLMP-NS (F2) and GLC-NS (F11) in diabetes management was evaluated using a streptozotocin-nicotinamide (STZ-NA) induced diabetic rat model.

2.5.1 Induction of Diabetes:

Thirty-six rats were acclimatized for seven days. Six rats were maintained as the normal control group on a standard diet. Type 2 diabetes was induced in the remaining 30 rats using a single intraperitoneal injection of STZ (60mg/kg), administered 15 minutes after intraperitoneal administration of nicotinamide (110 mg/kg)^56–58. Diabetes was confirmed 72hours post-induction by measuring fasting blood glucose levels; rats with glucose levels above 250mg/dL were considered diabetic, and those exceeding 280mg/dL were used in the study.

The diabetic rats were randomly divided into five treatment groups (n=6 per group). Oral doses of pure Glimepiride (0.1mg/kg) and pure Gliclazide (10mg/kg), as well as their respective nanosuspensions, were administered once daily based on doses reported in the literature^59,60. Group assignments represented in Table 1. Doses were suspended in 2mL distilled water and vortexed for 10seconds immediately before oral administration.

Table 1: In-vivo experimental design

Group	Treatment	No. of Animals
1	Normal control (vehicle)	6
2	Diabetic control (vehicle)	6
3	Diabetic + Glimepiride	6
4	Diabetic + GLMP-NS	6
5	Diabetic + Gliclazide	6
6	Diabetic + GLC-NS	6

2.6 Blood Glucose Monitoring:

Blood glucose levels were measured at multiple time points to evaluate the antihyperglycemic efficacy of the formulations. Enflurane was applied as a topical anaesthetic, and blood samples (0.25–0.5mL) were collected from the retro-orbital plexus at 0, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24hours post-administration. A drop of blood was analyzed using an Accu-Chek glucometer⁶¹. Plasma was separated from the collected blood (pre-treated with heparin) by centrifugation and stored at −20°C until HPLC analysis.

2.7 Plasma Drug Extraction:

Glimepiride and Gliclazide were extracted from plasma using protein precipitation. Briefly, 100μL of rat plasma was transferred to 1.5mL Eppendorf tubes, mixed with 10μL of metformin (200μg/mL, IS), and vortexed for 1 minute. Then, 100μL of methanol was added, followed by 4 minutes of vertexing. Samples were centrifuged at 10,000rpm for 5 minutes, and 20μL of the supernatant was used for HPLC analysis⁶².

2.8 HPLC Assay:

Plasma concentrations of GLMP, GLC, and their respective nanosuspensions were analyzed using an HPLC system (Shimadzu LC-2050C, Kyoto, Japan) with slight modifications⁶³. The mobile phase consisted of 0.05% formic acid in water (Solvent A) and methanol (Solvent B) in a 42:58 v/v ratio. A C18 column (5µm, 25cm × 4.6mm, Shimadzu) was used, with a flow rate of 1.5mL/min. A 20μL sample was injected, and UV detection was performed at 234nm at room temperature.

2.9 Preparation of Standard and Sample Solutions:

Stock solutions of Glimepiride and Gliclazide (200 μg/mL) were prepared in methanol. Working standard solutions were serially diluted to obtain calibration ranges of 0.05–2.0μg/mL for Glimepiride and 0.1–2.0 μg/mL for Gliclazide. A 200μg/mL stock solution of metformin (IS) was similarly prepared in methanol⁶⁴.

a. Linearity:

Blank plasma was spiked with Glimepiride at concentrations of 0.05, 0.1, 0.5, 1.0, 1.5, and 2.0µg/mL and with Gliclazide at 0.1, 0.5, 1.0, 1.5, and 2.0µg/mL. Each sample was extracted and analyzed to construct calibration curves (peak area vs. concentration) and determine the method’s linearity (R²).

b. LOD and LOQ:

Limit of detection (LOD) and limit of quantification (LOQ) were calculated based on signal-to-noise ratios of 3:1 and 10:1, respectively.

2.10 Pharmacokinetic Parameters:

The area under the plasma concentration-time curve (AUC₀–₂₄) was calculated using the linear trapezoidal method. The maximum plasma concentration (Cmax) and the time to reach Cmax (Tmax) were also determined⁶⁵.

3. RESULT AND DISCUSSION:

3.1 Anti-Diabetic Activity of Glimepiride and Gliclazide:

Glimepiride and Gliclazide are Biopharmaceutics Classification System (BCS) Class II drugs characterized by low aqueous solubility, which limits their oral bioavailability. To address this issue, nanosuspension formulations—GLMP-NS (F2) and GLC-NS (F11)—were developed to enhance their solubility and bioavailability compared to the pure drugs. These optimized formulations were subsequently selected for evaluation of antidiabetic activity and In Vivo pharmacokinetic performance^44,45.

In streptozotocin-induced diabetic rats, GLMP-NS demonstrated significantly improved glycemic control compared to pure GLMP. As shown in Fig. 1, GLMP-NS led to a more rapid and pronounced reduction in blood glucose levels. After 1 hour, GLMP-NS reduced blood glucose to 39±3.52% of baseline, whereas pure GLMP achieved only a 6±4.61% reduction. By 3 hours, GLMP-NS had reduced glucose levels by 73.7±4.42%, while the pure drug achieved a 28±2.68% reduction. This enhanced effect can be attributed to the higher entrapment efficiency (EE) and smaller particle size of the nanosuspension, which improved solubility and systemic absorption. Similar findings have been reported in studies utilizing solid dispersions and microemulsions to enhance Glimepiride’s bioavailability^66,67.

Fig. 1: Mean blood glucose levels following oral administration of pure GLMP and GLMP-NS.

Similarly, GLC-NS also demonstrated improved antidiabetic efficacy over pure GLC in diabetic rats (Fig. 2). One hour post-administration, blood glucose was reduced to ~50±3.17% by GLC-NS and ~40±2.38% by pure GLC. After 3 hours, GLC-NS achieved a 65±2.74% reduction, whereas the pure drug achieved only 34±3.71%. The enhanced performance of GLC-NS is attributed to increased solubility and bioavailability due to nanoparticulate formulation. A similar enhancement in activity was observed in studies involving lipid-based nanosuspensions of Gliclazide⁶⁵.

Fig. 2: Mean blood glucose levels following oral administration of pure GLC and GLC-NS.

3.2 Pharmacokinetic Study:

Pharmacokinetic parameters including the area under the curve (AUC), maximum plasma concentration (Cmax), and time to reach Cmax (Tmax) were evaluated to assess the In Vivo performance of the nanosuspension and pure drug formulations. Plasma drug levels were quantified via HPLC using a mobile phase of 0.05% formic acid and methanol (42:58, v/v) at a flow rate of 0.5mL/min, which provided excellent peak resolution and minimized tailing.

Glimepiride:

For Glimepiride, the calibration curve was linear over a concentration range of 0.05–20μg/mL with an R² of 0.999 (Fig. 3), and sensitivity parameters—LOD and LOQ—were 0.14μg/mL and 0.43μg/mL, respectively (Table 2).

Table 2: HPLC Calibration Curve Data for Glimepiride

S. No.	Concentration (µg/ml)	Area
1.	0.00	0.00
2.	0.05	6569
3.	0.10	10231
4.	0.50	45914
5.	1.00	97068
6.	1.50	140662
7.	2.00	182428

Fig. 3: Calibration curve for Glimepiride.

Retention times for Metformin (internal standard) and Glimepiride were approximately 1.376 and 18.656 minutes, respectively (Fig. 4). Pharmacokinetic profiling revealed a Cmax of 6.46±2.1μg/mL for GLMP-NS compared to 3.26±0.65μg/mL for pure GLMP. Both formulations had similar Tmax (~3hours). The AUC₀₋₂₄ was 71.20±4.56 μg·h/mL for GLMP-NS and 24.34±3.28 μg·h/mL for pure GLMP, indicating a 2.9-fold increase in bioavailability (Table 3).

Table 3: Pharmacokinetic Parameters of GLMP and GLMP-NS (n=6)

Parameter	GLMP	GLMP-NS
C_max (μg/mL)	3.26 ± 0.65	6.46 ± 2.1
T_max (hour)	3 ± 0.15	3 ± 0.12
AUC _0-24h (μg*h/mL)	24.34 ± 3.28	71.20 ± 4.56

Fig. 4: Plasma concentration-time profile of GLMP and GLMP-NS.

The improvement is attributed to increased solubility and surface area due to reduced particle size, as well as surface modifications that improve wettability and prevent aggregation^60,66,67.

Gliclazide:

The calibration curve for Gliclazide was linear between 0.1–2.0μg/mL with an R² of 0.9997 (Fig. 5). LOD and LOQ were 0.09μg/mL and 0.26μg/mL, respectively (Table 4).

Table 4: HPLC Calibration Curve Data for Gliclazide

S. No.	Concentration (µg/ml)	Area
1.	0.1	2968
2.	0.5	14536
3.	1.0	29816
4.	1.5	44029
5.	2.0	57979

Fig. 5: Calibration curve for Gliclazide.

Retention times for Metformin and Gliclazide were 1.643 and 11.339 minutes, respectively. Fig. 6 presents the mean plasma concentration–time profiles. GLC-NS exhibited a Cmax of 8.16±0.13μg/mL, significantly higher than the 1.83±0.36μg/mL for pure GLC. The AUC₀₋₂₄ was 100.33±4.16μg·h/mL for GLC-NS versus 13.64±2.12μg·h/mL for pure GLC, representing a 7.36-fold enhancement (Table 5).

Table 5: Pharmacokinetic Parameters of GLC and GLC-NS (n=6)

Parameter	GLC	GLC-NS
C_max (μg/mL)	1.83 ± 0.36	8.16 ± 0.13
T_max (hour)	2 ± 0.15	3 ± 0.14
AUC _0-24h (μg*h/mL)	13.64 ± 2.12	100.33 ± 4.16

Fig. 6: Plasma concentration-time profile of GLC and GLC-NS.

These results suggest that the nanosuspension strategy significantly enhances the oral bioavailability of Gliclazide, likely due to improved dissolution, greater absorption, and prolonged systemic retention [65,68,69]. Additionally, nanoparticle engineering enables controlled drug release and targeted delivery, further contributing to therapeutic efficacy⁷⁰.

4. CONCLUSION:

Pharmacokinetic parameters—including the area under the plasma concentration-time curve (AUC), maximum plasma concentration (Cmax), and time to reach maximum concentration (Tmax)—were determined using validated analytical techniques to evaluate the performance of both the pure drugs and their corresponding nanosuspension formulations. The results clearly demonstrated that the nanosuspension of Glimepiride (GLMP-NS) enhanced its oral bioavailability by approximately 2.9-fold compared to the raw drug. Similarly, the nanosuspension of Gliclazide (GLC-NS) exhibited a remarkable 7.4-fold increase in oral bioavailability relative to the unprocessed drug. In summary, the nanosuspension approach significantly improved the dissolution and bioavailability of poorly water-soluble antidiabetic drugs, Glimepiride and Gliclazide. These findings support the potential of nanosuspension-based delivery systems as an effective strategy for enhancing the therapeutic performance of BCS Class II drugs.

5. ACKNOWLEDGMENTS:

Authors highly thankful towards the Department of Pharmaceutical Sciences, GJUS&T, Hisar, India for providing research facilities.

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Received on 10.09.2025 Revised on 15.12.2025

Accepted on 05.03.2026 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2207-2213.

DOI: 10.52711/0974-360X.2026.00318

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