An open label single centre, single dose oral bioavailability and biodistribution study (through pharmaco-scintigraphy) to compare and evaluate orally administered Calciol® formulations (granules and soft gelatin capsule) manufactured by cadila pharmaceuticals limited., Ahmedabad, India in twelve healthy human volunteers (A CALSCI study: CALcirol SCIntigraphy study)

Cholecalciferol, also known as vitamin D3 and cholecalciferol, is a type of vitamin D which is made by the skin when exposed to sunlight; it is also found in some foods and can be taken as a dietary supplement.   Cholecalciferol is made in the skin following UVB light exposure. It is converted in the liver to calcifediol (25-hydroxyvitamin D) which is then converted in the kidney to calcitriol (1,25-dihydroxyvitamin D). One of its actions is to increase the uptake of calcium by the intestines. It is found in food such as some fish, beef liver, eggs, and cheese. Certain foods such as milk, fruit juice, yogurt, and margarine also may have cholecalciferol added to them in some countries including the United States.   Cholecalciferol can be taken as an oral dietary supplement to prevent vitamin D deficiency or as a medication to treat associated diseases, including rickets. It is also used for familial hypophosphatemia, hypoparathyroidism that is causing low blood calcium, and Fanconi syndrome. Vitamin-D supplements may not be effective in people with severe kidney disease. Excessive doses in humans can result in vomiting, constipation, weakness, and confusion. Other risks include kidney stones. Doses greater than 40,000 IU (1,000 μg) per day are generally required before high blood calcium occurs. Normal doses, 800–2000 IU per day, are safe in pregnancy.   Cholecalciferol was first described in 1936. It is on the World Health Organization’s List of Essential Medicines. In 2019, it was the 84th most commonly prescribed medication in the United States, with more than 9 million prescriptions. Cholecalciferol is available as a generic medication and over the counter.[8] Cholecalciferol is also used at much higher doses to kill rodents. Medical uses Vitamin D deficiency Cholecalciferol is a form of vitamin D which is naturally synthesized in skin and functions as a pro-hormone, being converted to calcitriol. This is important for maintaining calcium levels and promoting bone health and development. As a medication, cholecalciferol may be taken as a dietary supplement to prevent or to treat vitamin D deficiency. One gram is 40,000,000 (40x106) IU, equivalently 1 IU is 0.025 µg. Dietary reference intake values for vitamin D (cholecalciferol and/or ergocalciferol) have been established and recommendations vary depending on the country:   In the US: 15 µg/d (600 IU per day) for all individuals (males, females, pregnant/lactating women) between the ages of 1 and 70 years old, inclusive. For all individuals older than 70 years, 20 µg/d (800 IU per day) is recommended. In the EU: 20 µg/d (800 IU per day) In France: 25 µg/d (1000 IU per day) Low levels of vitamin D are more commonly found in individuals living in northern latitudes, or with other reasons for a lack of regular sun exposure, including being housebound, frail, elderly, obese, having darker skin, or wearing clothes that cover most of the skin. Supplements are recommended for these groups of people.  

Objectives:

Primary Objective

·        To evaluate the bioavailability and biodistribution pattern of orally administered formulations, transit time and gastrointestinal (GI) clearance of Calcirol® formulation manufactured by Cadila Pharmaceuticals Limited, Ahmedabad, India in 12 healthy adult human subjects (either male or female).

·        To estimate the vitamin D level in plasma sample of healthy human volunteer on different days.

Secondary Objective

·        To monitor the safety of the subjects.

|Study Design:

A single centre, two arm, two treatment, non-randomized, single oral dose, bioavailability and biodistribution control study to compare and investigate the In-vivo distribution pattern, gastrointestinal (GI) clearance and bioavailability of orally administered Calcirol®  formulations.

|No. of subjects**:**

A sufficient number of subjects will be enrolled to ensure dosing of 12 healthy adult human subjects.

|Housing:

At least 2 hours prior to dosing until at least 12 hours post dose and an ambulatory visit for post study safety sample at 24 Hrs of dosing

|Study duration**:**

Expected study duration of clinical part is 01 day from the day of dosing

|Investigational Product

Product

Calcirol® Sachet

Cap Calcirol®

|Dosage Form

Granules

Soft gelatin capsule

|Strength

60K IU

60K IU

|Drug Name

Cholecalciferol

Cholecalciferol

|Manufactured By

Cadila Pharmaceuticals Ltd., Ahmedabad

|Radioactive Element

Technetium Pertechnetate - 99mTcO4

|Radiation Dose

99mTc- labelled –API for both capsule and granules will contain 500-700 µCi of Technetium pertechnetate

|Inclusion Criteria

i.     Subject who are able to understand and ready to provide written informed consent.

ii.     Subject must be healthy subject (male/female) human beings within 18-45 years of age (both inclusive).

iii.     Subject should be having Body Mass Index (BMI) in the range 18.5-24.9 kg/m2 and weighing at least 50 kg.

iv.     Subject will be declared healthy on the basis of medical history and physical examination, ECG and laboratory tests performed within 21 days prior to the commencement of the study.

v.     Subject whose screening laboratory values are within normal limits or considered by the physician / Principal Investigator to be of no clinical significance.

|Exclusion Criteria

i.      Subjects who have deficient Vitamin D level.

ii.      Subject with significant history of hypersensitivity to Study Drug or any ingredients of the formulation or any related products as well as severe hypersensitivity reactions (like angioedema) to any drugs.

iii.   Subject who have suffered any illness or who have been hospitalized within the last 4 weeks preceding the start of the study.

iv.   Subject who have taken over the counter or prescribed medications, including any enzyme modifying drugs within the last 14 days prior to the study.

v.   Subjects who are already taking pain killers or took pain killer within 24 hours of prior to initiation of study.

vi.   Subject with a history of drug abuse or alcoholism i.e. alcohol consumption > 2 units / day or 10 units / week (one unit of alcohol = 50 ml spirit or 200 ml wine or 500 ml beer).

vii.   Subject with smoking history of > 10 Cigarettes / day or Tobacco consumption > 4 packets / day.

viii.   Subject who has participated in any other clinical trial requiring repeated blood sampling or a blood donation program or blood loss of more than 450 ml, in the past three months (approx. 90 days) (This 450 mL includes the total blood loss that will occur during the study).

ix.   Subject with positive Breath Alcohol Analysis before admission.

|Pre-study Screening

Demographic data, medical and medication histories, general and systemic physical examination, hematology, biochemistry, serology.

|Tests at the time of Admission

Breath alcohol analysis (BAA) will be done at the time of admission

|Admission and stay

After signing informed consent document, a unique ID will be allotted to study subjects to maintain their identity confidential. Subject will be housed at least 2.00 hours before drug administration to 12.00 hours after drug administration.

|Administration of Investigational products:

12 Subjects will be grouped into 2 groups each group will receive different product i.e. granule and soft gel capsule. A single oral dose of all the investigational products will be labeled with Technetium -99mTc & will be administered to the subjects in a sitting posture with about 240 mL of milk at ambient temperature during the study under supervision of trained study personnel. The clinical staff (dosing personnel) will ensure that the study participant has swallowed medication by performing mouth check using torchlight and spatula to assess compliance to dosing. Investigational products must be swallowed whole and must not be chewed, crushed or divided.

|Gamma Imaging Schedule

A sequential static imaging- shall be performed at baseline (Within 05 Minutes of dosing), 0 min, 30 min, 1 h, 2h, 4h, 6h, 12h and 24h post dose for two minutes. Gamma Imaging can be stopped, as soon as the radioactive granules reach to large intestine, gastric clearance from GI is observed for its bio distribution. Additional images can be taken as per investigator discretion without discomfort to subject. For post dose Gamma Imaging, will be performed with a window period of ± 05 Minutes.

|Food and fluid restrictions:

Subjects will be advised to take food before consuming the medicine and fast at least 04 hours post-dose. Water will be restricted from at least 01 hour prior to dosing until at least 01-hour post-dose in each period (no fluid, except milk given with dosing).

Subjects should not consume alcohol and smoke 48 hours before drug administration and throughout study period. Subject should not consume grapefruit containing products for 48 hours before the drug administration and throughout the study.

|Posture restrictions**:**

Subject will remain in supine position for initial 60 min post-dose and only necessary movement will be allowed during this period. Thereafter subjects will be allowed to ambulate freely during the remainder of the study.

|Collection of blood samples for screening and post study safety:

In this study, total 2 (10mL) blood samples will be collected for Screening of volunteer to declare him/her healthy and to compliance with the study protocol and collection of samples after the completion of study to observe any changes in hematology and biochemistry.

The pre-dose blood sample for screening shall be taken within 120 minutes prior to dosing within ±2 minutes of the scheduled time.

The post-dose blood sample for ascertain the safety of subject shall be taken on 30th day of post dosing for safety of participants.

|Blood samples for drug concentration estimation

In this study, total 17 venous blood samples (04 mL each, except pre-dose 10 mL) will be collected at pre-dose (0.0 hour) and at 0, 1h, 3h, 6h, 12 h, 24h, 48h, 3rd day, 6th day, 9th day, 12th day, 15th day, 18th day, 21th day, 24th day, 27th day and 30th day post dose in labeled K2EDTA vacutainers through forearm vein/dorsal aspect of hand of the subjects. If required, a sample may alternatively be collected through a fresh venipuncture.

|Handling of blood samples:

After collection, the blood samples will be placed in an ice bath or other chilling device until centrifugation. Blood samples will be placed in a refrigerated centrifuge within 45 minutes of blood sample collection, and then will be spun at 3000 rpm at 5°C ± 3ºC for 15 minutes. The plasma will then be separated, transferred to labeled polypropylene tubes in duplicate (primary and secondary aliquots with equal volume) and stored in a freezer at –20ºC ± 10ºC at the clinical facility until shipment to the analytical facility. The samples will be stored in a freezer at –20ºC ± 10ºC at the analytical facility until analyzed.

Note: Transfer of plasma samples into the freezer shall take place as soon as possible so the total elapsed time from blood collection to placement of plasma samples in the freezer does not exceed 90 minutes.

|Safety Assessment:

Vital signs:

·   Sitting blood pressure, pulse rate, body temperature and SPO2 will be measured at the time of check-in and prior to check-out. Subjects must have vital signs clinically acceptable prior to check-in of each period.

·   Sitting blood pressure, pulse rate, body temperature and SPO2  will be measured prior to dosing in each period and during the visit for the last study sample.

·   Sitting/ Supine blood pressure, pulse rate and SPO2 will be measured at 2, 6 & 10 hours (± 40 minutes) post dose and as needed in each period.

Clinical examination: At the time of check-in and prior to check-out in each period and during the visit for the last study sample.

Well Being assessment:

Subjects will be advised to report any AE and will be specifically asked by trained study personnel in a non-leading manner about any AE at the time of clinical examinations, during vital signs recording, at about 12.0 & 24.0 hours post dose, Necessary treatment of an AE will be performed by the Investigator or physician and recorded.

For female volunteers of childbearing potential, a UPT examination will be performed prior to check-in and at the time of the end of study laboratory assessment. Only volunteers with a negative pregnancy test result (prior to check-in of each period) will be dosed.

All dosed study subjects will be assessed for clinical examination including vital sign (sitting blood pressure, pulse rate and body temperature) and laboratory tests at the end of the study or as applicable (for details refer to section: Safety Assessment).

|Analytical methodology: Parameters by Scintigraphy

Parameters will be evaluated by imaging captured by Gamma camera (99mTc based Scintigraphy)- A sequential static imaging- shall be performed at baseline (Within 05 Minutes of dosing), 0 min, 30 min, 1 h, 2h, 4h, 6h, 12h and 24h post dose for two minutes.

·         Location of radiolabeled formulations and in GIT at different time interval

·         Gastric residence time

·         Disintegration time

·         Disintegration pattern

·         Rate of erosion of radiolabeled tablets

|Analytical methodology for drug estimation using RP-HPLC**:**

Method development for Vitamin D analysis in blood plasma.

Followed by estimation of cholecalciferol concentration in blood plasma at 0, 1h, 3h, 6h, 12 h, 24h & 48h by RP-HPLC method. Further samples will be estimated for full vitamin D profiling using Chemiluminescent immunoassay (CLIA) method which is specific for the determination of study drug on 3rd day, 6 day, 9th day, 12th day, 15th day, 18th day, 21th day, 24th day, 27th day and 30th day post dose.

| Pharmacokinetic Parameters:

Pharmacokinetic parameters Cmax, AUCt, AUCi, Tmax, Kel, AUCt/AUCi and Thalfwill be calculated using Win Nonlin® professional software.

|Statistical Analysis:

Statistical analysis will be performed on the pharmacokinetic parameters using SAS® statistical software.

|Compensation

As approved by the IEC.

|Study Report

The report will be prepared after completion of the study as per norms.