To Study the Antidiabetic Activity of Atovaquone Against Streptozotocin Induced Diabetic Rats

Diabetes mellitus is a chronic metabolic condition marked by high blood glucose levels due to impairments in insulin secretion, insulin action, or both. It is among the most widespread non-communicable illnesses globally and is linked to severe consequences, including cardiovascular diseases, neuropathy, nephropathy, and retinopathy. Notwithstanding the availability of numerous antidiabetic medications, successful management continues to be problematic due to adverse effects, elevated costs, and restricted long-term efficacy, underscoring the necessity for alternative therapeutic strategies.[1]

Experimental models are essential for comprehending the biology of diabetes and assessing novel pharmacological possibilities. Streptozotocin (STZ) is commonly employed to produce diabetes in laboratory animals because of its specific toxicity towards pancreatic β-cells, resulting in insulin insufficiency and hyperglycemia.[2] The STZ-induced diabetic rat model closely mimics human diabetes and is frequently utilized for the evaluation of antidiabetic compounds.

Atovaquone is a hydroxynaphthoquinone molecule predominantly utilized as an antibacterial agent for treating diseases, including malaria and Pneumocystis pneumonia. Recent research indicate that Atovaquone may have supplementary pharmacological features, including antioxidant and metabolic regulatory actions, which could aid in diabetes management. Its capacity to regulate mitochondrial function and diminish oxidative stress may enhance its putative antidiabetic properties.[3]

This study seeks to examine the antidiabetic effects of Atovaquone in streptozotocin-induced diabetic rats by assessing biochemical parameters, including blood glucose levels, body weight, lipid profile, and oxidative stress indicators. This study investigates the potential of medication repurposing as an innovative approach for formulating viable medicines for diabetes mellitus.[4]

MATERIALS AND METHODS

Materials

Atovaquone will be used as the test drug and obtained from certified suppliers such as Sigma-Aldrich or Yarrow Chem Products with proper quality certification. Streptozotocin (STZ) will be used to induce diabetes in rats due to its selective destruction of pancreatic β-cells, causing hyperglycemia.Other materials include citrate buffer, distilled water, and analytical-grade reagents and diagnostic kits for biochemical analysis. Atovaquone (Glenmark Pharmaceuticals, Goa) was obtained as a gift sample. Streptozotocin (STZ) was purchased from Sigma-Aldrich. Metformin (Wockhardt Pvt. Ltd.) was obtained as a gift sample.

Preparation of Drug Solutions[5,6]

Streptozotocin Solution

Streptozotocin (STZ) will be freshly prepared in ice-cold citrate buffer (pH 4.5), protected from light, and used immediately due to instability.

Atovaquone Suspension

Atovaquone will be suspended in 0.5% carboxymethyl cellulose (CMC) to obtain a uniform suspension for oral administration.

Standard Drug

Metformin will be prepared in distilled water or 0.5% CMC and administered orally.

Route of Administration [7]

• Streptozotocin → Intraperitoneal (IP) → Diabetes induction
• Atovaquone → Oral → Test drug
• Metformin → Oral → Standard drug
• Vehicle → Oral → Control

Acute Toxicity Study

Acute toxicity of Atovaquone will be evaluated as per OECD Guideline 423. Rats will be fasted overnight and observed for 14 days after oral administration for behavioral, neurological, autonomic changes, food intake, and mortality. Safe doses will be selected based on results.

Method [8]

Screening Model

STZ-induced diabetic rat model will be used.

Induction of Diabetes

STZ (45 mg/kg, IP) will be administered. After 72 hours, rats with blood glucose ≥200 mg/dL will be considered diabetic.

Experimental Design[9]

• Group I → Normal control
• Group II → Diabetic control
• Group III → STZ + Metformin (200 mg/kg)
• Group IV → STZ + Atovaquone (25 mg/kg)
• Group V → STZ + Atovaquone (50 mg/kg)
• Group VI → STZ + Atovaquone (100 mg/kg)

Treatment duration: 21–28 days (as per IAEC approval).

Evaluation Parameters[11-18]

Body Weight

Body weight of each rat will be recorded on day 0, day 7, day 14, day 21, and day 28. Loss of body weight is commonly observed in diabetic rats due to increased protein breakdown and altered carbohydrate metabolism. Improvement in body weight after treatment indicates protective antidiabetic effect.

Fasting Blood Glucose Level

Fasting blood glucose will be measured on day 0, day 7, day 14, day 21, and day 28. Blood will be collected from the tail vein after overnight fasting. Glucose level will be measured using a glucometer or glucose oxidase-peroxidase method.

Reduction in fasting blood glucose level in Atovaquone-treated groups compared with diabetic control will indicate antidiabetic activity.

Oral Glucose Tolerance Test

Oral glucose tolerance test may be performed to evaluate glucose utilization capacity. Rats will be fasted overnight and treated with vehicle, standard drug, or Atovaquone. After 30 minutes, glucose solution will be administered orally at 2 g/kg body weight. Blood glucose will be measured at 0, 30, 60, 90, and 120 minutes.

Improved glucose tolerance in treated animals indicates better glucose regulation.

Serum Insulin Level

At the end of the study, blood samples will be collected and serum will be separated by centrifugation. Serum insulin level will be estimated using an ELISA kit. Increased insulin level in treated groups may indicate protection or recovery of pancreatic β-cell function.

Glycated Hemoglobin

HbA1c level will be estimated to assess long-term glycemic control. Increased HbA1c is associated with persistent hyperglycemia. Reduction in HbA1c after Atovaquone treatment will support its antidiabetic potential.

Lipid Profile

Serum lipid profile will be evaluated using diagnostic kits.

Improvement in lipid profile will indicate protective action against diabetic complications.

Liver Function Parameters

Serum SGOT, SGPT, and ALP levels will be estimated to assess liver function. Diabetes may cause hepatic stress due to oxidative damage and altered metabolism. Reduction in elevated liver enzymes after treatment indicates hepatoprotective effect.

Kidney Function Parameters

Serum urea, creatinine, and uric acid will be estimated to evaluate renal function. Diabetes can cause renal injury due to oxidative stress and hyperglycemia. Improvement in kidney markers will indicate nephroprotective potential of Atovaquone.

Oxidative Stress Parameters[20]

Pancreatic tissue or liver tissue homogenate will be prepared for antioxidant evaluation.

Increase in SOD, catalase, and GSH with reduction in MDA will indicate antioxidant activity.

Histopathological Study of Pancreas[19]

At the end of the experiment, rats will be sacrificed humanely as per IAEC-approved procedure. Pancreas will be isolated, washed with normal saline, and fixed in 10% formalin. Tissue sections will be prepared, stained with hematoxylin and eosin, and observed under microscope.

Histopathological examination will focus on:[21]

All results will be expressed as mean ± SEM. Statistical analysis will be performed using one-way ANOVA followed by Dunnett’s multiple comparison test. A value of p < 0.05 will be considered statistically significant.

RESULTS

Acute Toxicity Study

Table :Acute Toxicity Results

Atovaquone was found to be safe up to 2000 mg/kg with no mortality, indicating an LD?? > 2000 mg/kg (low toxicity as per OECD 423). Only mild, transient sedation was observed at 1000 mg/kg, while no toxic effects were seen at 2000 mg/kg. The drug shows a wide safety margin, and the selected doses (25, 50, 100 mg/kg) are safe for antidiabetic studies.

Body Weight

Table:  Body Weight (g)

Streptozotocin-induced diabetes caused significant body weight loss in the diabetic control group, while the normal group showed steady weight gain. Metformin treatment improved body weight, indicating effective glycemic control. Atovaquone showed a dose-dependent improvement, with the 100 mg/kg dose nearly restoring normal body weight. Overall, Atovaquone effectively prevented weight loss and demonstrated significant antidiabetic activity.

Fasting Blood Glucose

Table :Blood Glucose (mg/dL)

Fasting blood glucose levels remained normal in the control group but increased significantly in the diabetic group, confirming successful diabetes induction. Metformin significantly reduced glucose levels to near normal. Atovaquone showed a dose-dependent reduction, with the 100 mg/kg dose approaching normal levels and comparable to Metformin. Overall, Atovaquone demonstrated significant antidiabetic activity

Oral Glucose Tolerance Test

Table : Interpretation of Oral Glucose Tolerance Test (OGTT) (mg/dL)

OGTT results showed normal glucose tolerance in the control group, while the diabetic group exhibited prolonged hyperglycemia, indicating impaired glucose utilization. Metformin significantly improved glucose tolerance, with near-normal levels by 120 minutes. Atovaquone showed dose-dependent improvement, with the 100 mg/kg dose almost comparable to Metformin. Overall, Atovaquone enhanced glucose tolerance and demonstrated significant antidiabetic potential.

Serum Insulin

Table : Insulin Levels

Serum insulin levels were normal in the control group but significantly reduced in the diabetic group, confirming β-cell damage. Metformin restored insulin levels close to normal. Atovaquone showed a dose-dependent increase, with the 100 mg/kg dose nearly comparable to Metformin. Overall, Atovaquone improved insulin secretion and pancreatic function.

Lipid Profile

Table Lipid Profile (mg/dL)

Streptozotocin-induced diabetes caused increased TC, TG, and LDL levels with decreased HDL, indicating dyslipidemia. Metformin significantly improved the lipid profile. Atovaquone showed dose-dependent improvement, with the 100 mg/kg dose nearly comparable to Metformin. Overall, Atovaquone exhibited significant anti-dyslipidemic and antidiabetic effects.

Liver Function

Diabetic rats showed elevated SGOT, SGPT, and AL

Oxidative Stress

Diabetic rats showed reduced antioxidant enzymes (SOD, Catalase, GSH) and increased MDA, indicating high oxidative stress. Atovaquone (100 mg/kg) significantly improved antioxidant levels and reduced MDA, approaching normal values. Overall, it exhibited strong antioxidant activity and reduced oxidative stress.

Histopathology

Histopathology showed severe pancreatic β-cell damage in diabetic rats, while Atovaquone treatment led to noticeable tissue recovery. This effect may be due to reduced oxidative stress, protection of β-cells, and possible stimulation of β-cell regeneration.