Metformin as Adjunct Therapy in Depression-Obesity Comorbidity: Clinical and Genetic Evaluation

The change in the total PHQ-9 score between baseline and 4-weeks follow-up is the primary outcome measure. This measure is a patient-rated inventory of depressive symptoms. All items are scored on scales ranging from 0-3 and the sum of the scores provides the total score for the measure. On this scale, higher scores indicate poorer outcomes. Scores 9-19 and 20-27 are considered moderate and severe depression respectively.

Change in Body Mass Index (BMI): BMI will be measured at baseline and study completion and calculated as weight in kilograms divided by height in meters squared (kg/m²). The outcome will be assessed as the change in BMI from baseline.

Asian BMI Classification:

Underweight: < 18.5 kg/m² Normal weight: 18.5-22.9 kg/m² Overweight: 23.0-24.9 kg/m² Obesity Class I: 25.0-29.9 kg/m² Obesity Class II: ≥ 30.0 kg/m²

BMI reduction < 1 kg/m²: Minimal change BMI reduction ≥ 1 kg/m²: Clinically meaningful improvement Weight loss ≥ 5% of baseline body weight: Clinically significant weight reduction associated with metabolic health benefits.

A decrease in BMI from baseline and/or movement to a lower BMI category will be considered indicative of improvement in obesity-related outcomes.

Change in Blood Pressure: Blood pressure will be measured in mmHg at baseline, 14 days, 28 days, one month and 3 months.. The outcome will be assessed as the change in systolic and diastolic blood pressure from baseline. Normal blood pressure is <120/80 mmHg, elevated blood pressure is 120-129/<80 mmHg, and high blood pressure is ≥130/80 mmHg. A reduction in blood pressure values from baseline will indicate improvement in cardiovascular health status.

Change in Blood Sugar: Blood glucose levels will be measured at baseline, 14 days, 28 days, one month and 3 months. The outcome will be assessed as the change in blood glucose from baseline. Normal fasting blood glucose is 70-99 mg/dL, prediabetes is 100-125 mg/dL, and diabetes is ≥126 mg/dL. A reduction in blood glucose levels toward the normal range will indicate improvement in glycemic control, whereas increase will be monitored for safety

Change in Renal Function Tests: Renal function will be monitored by measuring serum creatinine and blood urea levels at baseline and study completion. The outcome will be assessed as the change in these parameters from baseline. Normal serum creatinine is approximately 0.6-1.2 mg/dL, while values >1.2 mg/dL may indicate impaired renal function. Normal blood urea is approximately 15-40 mg/dL, while values >40 mg/dL may suggest reduced kidney function or impaired renal clearance. An increase in serum creatinine or blood urea from baseline will be considered indicative of deterioration in renal function, whereas stable values within the normal range will indicate preserved renal function.

Change in Liver Function Tests (LFTs): Liver function will be monitored at baseline and study completion. The outcome will be assessed as the change in liver enzyme levels and bilirubin from baseline.

Alanine Aminotransferase (ALT): Normal 7-56 U/L; elevated >56 U/L may indicate liver injury.

Aspartate Aminotransferase (AST): Normal 10-40 U/L; elevated >40 U/L may indicate hepatic or muscle injury.

Alkaline Phosphatase (ALP): Normal 44-147 U/L; elevated >147 U/L may suggest cholestatic liver disease or biliary obstruction.

Total Bilirubin: Normal 0.2-1.2 mg/dL; elevated >1.2 mg/dL may indicate impaired liver function or biliary obstruction.

Albumin: Normal 3.5-5.0 g/dL; decreased <3.5 g/dL may indicate impaired hepatic synthetic function.

Change in C-Reactive Protein (CRP) Levels: CRP levels will be measured at baseline and study completion to assess systemic inflammation. The outcome will be assessed as the change in CRP levels from baseline.

Normal: < 3 mg/L Mildly elevated: 3-10 mg/L Moderately elevated: 10-50 mg/L Markedly elevated: > 50 mg/L Severely elevated: > 100 mg/L

Decrease in CRP level: Indicates reduction in systemic inflammation. Stable CRP within normal range (< 3 mg/L): Indicates absence of significant inflammation.

Increase in CRP above normal range: Suggests worsening or persistent inflammatory activity.

It will be measured at baseline and study completion to monitor safety of combination therapy.

Total Cholesterol (TC): Desirable: < 200 mg/dL, Borderline high: 200-239 mg/dL, High: ≥ 240 mg/dL Low-Density Lipoprotein Cholesterol (LDL-C): Optimal: < 100 mg/dL, Near optimal: 100-129 mg/dL, Borderline high: 130-159 mg/dL, High: 160-189 mg/dL, Very high: ≥ 190 mg/dL High-Density Lipoprotein Cholesterol (HDL-C): Low: < 40 mg/dL (men), < 50 mg/dL (women), Protective: ≥ 60 mg/dL Triglycerides (TG): Normal: < 150 mg/dL, Borderline high: 150-199 mg/dL, High: 200-499 mg/dL, Very high: ≥ 500 mg/dL

Interpretation:

Decrease in TC, LDL-C, and TG levels indicates improvement in lipid metabolism. Increase in HDL-C levels indicates a favorable cardiovascular risk profile. Lipid values remaining within the normal range throughout the study will be considered indicative of metabolic safety of the combination therapy.

To identify genetic polymorphism in NEGR1 in the Pakistani population, genetic polymorphism in NEGR1 in the Pakistani population will be assessed by ARMS PCR assay.

To identify genetic polymorphism in RPL31P12 in the Pakistani population, genetic polymorphism in RPL31P12 in the Pakistani population will be assessed by ARMS PCR assay.