Study Title:
A randomized, double-blind, single oral dose, two-period, two-treatment, two-sequence crossover comparative pharmacokinetic study of Test product ‘Glutathione loaded in Zeal platform’ of Aurea Biolabs (P) Limited with Reference product ‘Plain glutathione’ of Wonder Ingredient in 16 healthy, adult, human subjects under fasting condition.

Indication

·        Support antioxidant defense and reduce oxidative stress.

·        Promote healthy skin tone and brightness by reducing melanin synthesis.

·        Support liver function and detoxification.

Study Objective

Primary objective: To measure and compare the pharmacokinetics of Test with reference under fasting condition in healthy, adult, human subjects in a randomized cross over study.

Secondary objective: To evaluate Renal Function Test and Liver Function Test.

Screening procedures
Screening procedures will be performed within 21 days prior to check-in of period-I and screening will include registration/identification in biometric system, obtaining written screening consent, demographic parameters, medical history, physical examination, vital signs measurements (blood pressure, pulse rate, temperature and respiratory rate), 12-lead ECG recording, laboratory investigations [hematology, biochemistry, serology and urine analysis etc.] and evaluation of applicable inclusion and exclusion criteria.

Sampling Schedule

In each period, a total of 8 blood samples (04 ml each) will be collected from each subject in vacutainers (containing K₂EDTA as an anticoagulant).

Sampling points: Pre-dose at 0.00 and 01.00, 02.00, 04.00, 06.00, 12.00, 18.00 and 24.00 hours post-dose.

Total Blood loss 
For each male subject, the total volume of blood collection (including pre dose & post study blood sample) will be approximately 83 mL combining all the periods.

Sample Processing
For each time point, blood will be collected using a syringe and immediately transferred into K₂-EDTA vacutainers containing a stabilizer to prevent oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG). Anyone of the below two can be used:

1. N-Ethylmaleimide (NEM): 100 µL of a 200 mM NEM solution in ammonium formate buffer will be pre-aliquoted in the vacutainer. or
2. Metaphosphoric Acid (MPA): 100 µL of a 50% MPA solution will be aliquoted in the vacutainer.

After collection, samples will be gently mixed by inversion. The samples will be stored upright in a deep freezer set at -20 ± 5ºC. Samples will be transferred to the bioanalytical department at the end of clinical phase of the study. Samples in the bioanalytical department will be stored at -20 ± 5ºC deep freezer till analysis and during archival period. Second aliquot (about 02 mL) will be made from the sample and stored.

Analyte(s) and Analytical Method
Total Glutathione in whole blood samples will be quantified using bioanalytical LC-MS/MS method.

Pharmacokinetic Parameters
Primary PK parameters: C_max and AUC_0-t

Secondary PK parameters: T_max, K_el, T_1/2, AUC_extrap, AUC_ratio  and AUC_0-inf 

Pharmacokinetic parameters and descriptive statistics will be evaluated using Phoenix^® WinNonlin^® Software Version 8.4 (Certara Inc., USA) or latest version.

Statistical Analysis
Statistical analysis will be performed by using SAS^® software version 9.4 (or higher version).

The ln-transformed pharmacokinetic parameters [C_max and AUC_0-t] will be analyzed using ANOVA model with the main effect of treatment, period, sequence and subject nested within sequence as fixed effects. The Intra subject CV, Power, Ratio analysis and 90% confidence interval for the ratio of the geometric least squares mean will be computed for ln-transformed pharmacokinetic parameters [C_max and AUC_0-t]. Comparative pharmacokinetic data will be presented for test vs reference