STUDY TITLE:

A randomized, double blind, cross over study to evaluate the amino acid absorption after the intake of whey protein along with probiotic Bacillus coagulans SNZ 1969 in normal, healthy, adult, human subjects.

Project No. : 23-071 Protocol Version No. : 01

Date : 31 Oct 2023

Supersedes Version No. : NA Supersedes Date : NA

CONFIDENTIALITY STATEMENT

The information contained in this protocol, is the proprietary information of the Sanzyme Biologics Private Limited, India and Jeevan Scientific Technology Ltd. (or under its control). This protocol was developed for Sanzyme Biologics Private Limited, India by Jeevan Scientific Technology Ltd. and should not be disclosed to third party without written authorization from the Sponsor with the exception of regulatory agencies and applicable Independent Ethics Committee, study volunteers, study staff and study audit personnel.

PROTOCOL SUMMARY

Study Title	A randomized, double blind, cross over study to evaluate the amino acid absorption after the intake of whey protein along with probiotic Bacillus coagulans SNZ 1969 in normal, healthy, adult, human subjects.
Study aim and Objectives	The purpose of this study is to examine the pharmacokinetic pattern of amino acid absorption after a two-week pattern of whey protein supplementation with and without the addition of Bacillus coagulans SNZ 1969. This study is designed to examine the impact of adding Bacillus coagulans SNZ 1969 to a 25-g dose of whey protein concentrate on post-prandial changes in blood amino acids concentrations. 1. Rate of amino acid absorption: To determine the rate (in minutes) of amino acid absorption into the bloodstream after a two-week pattern of whey protein supplementation with and without the addition of Bacillus coagulans SNZ 1969. 2. Extent of amino acid absorption: To determine the extent (concentration in millimoles/Liter) of amino acid absorption into the bloodstream after a two-week pattern of whey protein supplementation with and without the addition of Bacillus coagulans SNZ 1969. 3. To investigate the acne development after a two-week pattern of whey protein supplementation with and without the addition of Bacillus coagulans SNZ 1969.
Study Design	A randomized, double blind, cross over study to evaluate the amino acid absorption after the intake of whey protein along with probiotic Bacillus coagulans SNZ 1969.
Subjects	A total of 30 normal, healthy, adult, human subjects meeting the inclusion and exclusion criteria will be enrolled in the study.
Investigational Products	Treatment A (Experimental): Whey Protein + Probiotic (n=15) 25 gram dose of whey protein concentrate with Bacillus coagulans SNZ 1969 2 Billion CFU Powder per serving
Investigational Products	Treatment B (Active Comparator): Whey Protein (n=15) 25 gram dose of whey protein concentrate
Randomization	Subjects who meet the eligibility criteria will be randomly assigned to receive the study drugs according to one of the two dosing sequences: AB or BA.
Study Visits	Supplementation Period 01: Subjects should visit the clinical facility on day 0 (For randomization and supply of treatments for 13 days) and day 14 (For administration of 14^th dose, blood sample collection) of Supplementation period 01. Supplementation Period 02: On day 34, Supply of treatments for supplementation period 02 and evaluation of acne development after completion of washout period of at least 21 days, Subjects should

	visit the clinical facility on day 48 (For administration of 14^th dose, For blood sample collection) of Supplementation period 02.
Washout Period	A washout period of at least 21 days will be maintained between consecutive Supplementation periods.
Duration of the study	Total expected duration of the study will be approximately 49 days (Including the washout period will be of at least 21 days).
Methodology	The study will be conducted using a randomized, double-blind, crossover study design. 30 healthy adult human subjects between the ages of 18 - 30 years will be recruited to participate in this study. Prior to beginning the study, all participants will sign an Ethics committee approved informed consent document and complete a healthy history questionnaire to determine study eligibility. The study will be conducted in two supplementation periods with each span of two weeks and separated with a washout period of 21 days. All the subjects will undergo a screening procedure comprising of clinical examination, recording of electrocardiogram and laboratory investigation of blood as well as urine which will be valid for 21 days from the day of screening. Radiological investigations (chest X-ray) will be repeated, if not done in the past 6 months or if clinically indicated at the time of screening. Subjects must be enrolled in the study only after providing written informed consent. Selection of subjects for the study will be done based on assessment against the inclusion and exclusion criteria. On day 0, Clinical examination and vital signs (seated blood pressure, respiratory rate, radial pulse rate and body temperature), well-being and body mass index will be recorded at the time of visit on day 0. The study participants will be randomly assigned to ingest either a single daily 25 grams dose of whey protein concentrate with Bacillus coagulans SNZ 1969 2 Billion CFU Powder (Treatment A) or a single daily 25 grams dose of whey protein concentrate (Treatment B). The sufficient quantities of either Treatment A or Treatment B will be supplied to study participants for 13 days supplementation (Day 1 to Day 13). The study participants will be instructed to ingest the provided dose at same time for day 1 to day 13. The powder of either Treatment A or Treatment B will be mixed thoroughly with approximately 300 mL of water at ambient temperature and ingest between 08:00 to 10:00 hours at least 1 hour prior to the breakfast. All participants will be required to complete a supplementation log to document when the each dose of their assigned treatment was consumed.

On day 14 and day 48, After consuming 13 consecutive doses, the study participants will report to the clinical facility between 06:00 to 08:00 hours, after observing an 8.00 to 10.00 hours fasting.

Vital signs (seated blood pressure, respiratory rate, radial pulse rate and body temperature), well-being and body mass index will be recorded prior to ingestion of their 14^th and final assigned supplementation dose.

The venous blood sample of 10 mL (2x5mL in sodium heparin vacutainer) will be collected at prior to ingest the 14^th and final dose of their assigned supplement.

The study participants will be instructed to ingest the 14^th or final dose of their final assigned supplementation dose. Upon ingestion of their final assigned supplementation dose, the subsequent venous blood samples of 10 mL each will be collected at 00.50 (30 minutes),

01.00 (60 minutes), 01.50 (90 minutes), 02.00 (120 minutes), 03.00

(180 minutes), and 04.00 (240 minutes) hour after ingestion.

The participants will be provided 200 mL of water to ingest after each blood collection.

The study participants will be discharged after 05.00 hours post ingest of powder. The clinical examination and vital signs during discharge of each visit may be started 120 minutes prior to the scheduled time of discharge of each subject, well-being of the subject will be confirmed at the time of actual discharge in each visit.

On day 34, Vital signs (seated blood pressure, respiratory rate, radial pulse rate and body temperature), well-being will be recorded.

Study participants will be provided an additional 13 days (Day 35 to 47) supply of the alternative treatment to begin after observing a three-week washout. After 13 days of supplementation, study participants will return to the clinical facility for their remaining testing visit. All subsequent study visits will be completed in a randomized fashion to minimize any order effects from testing.

Acne grading system will be done at baseline, end of 2 weeks of protein supplementation Period 01 (day 14) and prior to start of protein supplementation Period 01 (Day 34) and end of 2 weeks of protein supplementation Period 02 (day 48). To investigate If subject experience any acne development or that worsens during the study.

Acne grading will be evaluated by a dermatologist. Photographs will be taken at each time of acne grade assessment

No. of Blood samples in supplementation period 01	07
No. of Blood samples in supplementation period 02	07
Total blood samples for both supplementation periods	14
Volume of each sample	10 mL

	The below Acne grading system will be used to know the acne development.
		Table: Grading severity of acne
		Grade	Severity	Clinical findings
		I	Mild	Open and closed comedones with few inflammatory papules and pustules
		II	Moderate	Papules and pustules, mainly on face
		III	Moderately severe	Numerous papules and pustules, and occasional inflamed nodules, also on chest and back
		IV	Severe	Many large, painful nodules and pustules
Overview of Study design
Blood Sampling Schedule	The venous blood sample of 10 mL (2x5mL in sodium heparin vacutainer) will be collected at prior to ingest the 14^th and final dose of their assigned supplement at each supplementation period. The subsequent venous blood samples of 10 mL each will be collected at 00.50 (30 minutes), 01.00 (60 minutes), 01.50 (90 minutes), 02.00 (120 minutes), 03.00 (180 minutes), and 04.00 (240

	minutes) hour after ingestion in Sodium heparin vacutainer during each supplementation period. Total blood loss will not exceed 167.00 mL for each male subject and 176.00 mL for each female subject.
Clinical Safety Measures	On day 0, Clinical examination and vital signs (seated blood pressure, respiratory rate, radial pulse rate and body temperature), well-being and body mass index will be recorded at the time of visit on day 0. On day 14 and day 48, Vital signs (seated blood pressure, respiratory rate, radial pulse rate and body temperature), well-being and body mass index will be recorded prior to ingest of their 14^th and final assigned supplementation dose. The study participants will be discharged after 05.00 hours post ingest of powder. The clinical examination and vital signs (seated blood pressure, respiratory rate, radial pulse rate and body temperature) during discharge of each visit may be started 120 minutes prior to the scheduled time of discharge of each subject, well-being of the subject will be confirmed at the time of actual discharge in each visit. On day 34, Vital signs (seated blood pressure, respiratory rate, radial pulse rate and body temperature), well-being will be recorded. Clinical examination and measurement of vital signs may also be carried out at any time during the conduct of the study if the attending physician feels it necessary. In case of abnormality vital signs during pre-ingestion, medical opinion will be taken to dose the subject.
Bioanalytical Methodology	Plasma samples will be assayed for concentration of different amino acids (1-Methyl-histidine, 3-Methyl- histidine, Alpha Aminoadipic acid, Alpha Amino butyric acid, Alanine, Anserine, Arginine, Argino succinic acid, Asparagine, Aspartic acid, Beta -Amino- isobutyric acid, Beta- Alanine, Carnosine, Citrulline, Cystathionine, Cystine, Ethanolamine, Gamma Amino isobutyric acid, Glutamine, Glutamic acid, Glycine, Homocitrulline, Homocystine, Histidine, Hydroxylysine, Hydroxyproline, Isoleucine, Leucine, Lysine, Methionine, Methionine sulfoxide, Norleucine, Norvaline, Ornithine, Phosphoethanolamine, Phenyl alanine, Proline, Phosphoserine, Sarcosine, Serine, Taurine, Threonine, Tryptophan, Tyrosine, Valine) using a LC-MS/MS in Dr. Lal Path Labs., National Reference Laboratory, B7 Road, Rohini Sector 18, Delhi 110085 (Near Astha Kunj Apartment & Sector 18 Metro Station, Beside Petrol Pump, Block E)
Pharmacokinetic Parameters	The following pharmacokinetic parameters will be estimated using Phoenix^Â® WinNonlin^Â® Version 8.4 or higher.

AUC, Cmax and Tmax for different amino acids (1-Methyl-histidine, 3-Methyl- histidine, Alpha Aminoadipic acid, Alpha Amino butyric acid, Alanine, Anserine, Arginine, Argino succinic acid, Asparagine, Aspartic acid, Beta -Amino-isobutyric acid, Beta- Alanine, Carnosine, Citrulline, Cystathionine, Cystine, Ethanolamine, Gamma Amino isobutyric acid, Glutamine, Glutamic acid, Glycine, Homocitrulline, Homocystine, Histidine, Hydroxylysine, Hydroxyproline, Isoleucine, Leucine, Lysine, Methionine, Methionine sulfoxide, Norleucine, Norvaline, Ornithine, Phosphoethanolamine, Phenyl alanine, Proline, Phosphoserine, Sarcosine, Serine, Taurine, Threonine, Tryptophan, Tyrosine, Valine)

Primary outcomes for this trial will be considered to be the area under the curve (AUC) data for the measured amino acids.

Secondary outcomes will be considered to the maximum concentrations (Cmax) identified for the measured amino acids.

Study assessment

For all dependent measures, descriptive statistics will be presented as mean Â± standard deviations. Before any statistical tests, the normality will be assessed for all dependent variables. All non- normal data will be log transformed and then analyzed using both parametric and non-parametric approaches. All reported p values will be computed using parametric approaches. Paired sample t-tests will be used to determine between-group differences for the AUC, Cmax, and Tmax values for all individually measured amino acids

Statistical analysis will be performed using SAS^Â®, Version 9.4 or higher.

The appropriate statistical approach will be followed for analysis of acne development data.

A randomized, double blind, cross over study to evaluate the amino acid absorption after the intake of whey protein along with probiotic Bacillus coagulans SNZ 1969 in normal, healthy, adult, human subjects.

Contract Research Organization

Sponsor

CONFIDENTIALITY STATEMENT

PROTOCOL SUMMARY