This was a double-blind, randomized, comparator controlled, three-phase, cross-over clinical trial conducted at KGK Science (London, ON, Canada) between 18th December 2019 and 26th January 2021. The study compared the bioavailability of three calcium products (Ca-SC, Ca-LAB, and calcium citrate) in healthy postmenopausal women. Each participant took part in three single dose administrations with two washout periods of 7 days between interventions, as shown in Figure 1. The study protocol was reviewed by Advarra Institutional review board (Aurora, ON, Canada) and Health Canada’s Natural and Non-prescription Health Products Directorate, and an unconditional approval was received. IRB protocol # Pro00040124, Health Canada Notice of Authorization # 246340. The study was conducted in compliance with the international council for harmonization of technical requirements for pharmaceuticals for human use (ICH) Guideline for good clinical practice (GCP) and in accordance with the Declaration of Helsinki guidelines and its subsequent amendments and followed the CONSORT guidelines for randomized controlled trials (33, 34). All participants provided written informed consent prior to any study procedures and were told that they could withdraw from the study at any time (35).

Participants were included if: 45–65 years of age with a body mass index (BMI) between 19.0 to 29.9 kg/m²; postmenopausal as defined by no menstrual period via natural processes for at least 12 months; had normal electrocardiogram (ECG); parathyroid hormone (1–84 PTH) between 1.6 and 6.9 pmol/L; 25-hydroxyvitamin D [25(OH)D] level ≥ 20 ng/mL; and had a calcium intake of >450 mg per day from diet and supplementation (36–39). Participants were excluded if they had: an allergy, sensitivity, or intolerance to the investigational product’s active, inactive ingredients or the ingredients of the standardized meals; any disease that affected Calcium or vitamin D metabolism; or had any other condition that in the medical director’s opinion would affect the participants ability to complete the study or its measures or pose a significant risk to the participant. Detailed inclusion and exclusion criteria are specified in Supplementary Tables S1, S2.

Each participant completed three intervention visits, consuming a single serving of two calcium formulations and a reference product. Ca-SC (Re-Natured® calcium) and Ca-LAB (Biogurt™ calcium) are both derived from enriched calcium cultures of S. cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus-87, respectively, (Grow Company, Ridgefield, NJ, USA) (40). The cells were grown in the presence of calcium, heat-killed at the end of production process and spray dried into a dried powder, delivering inactive cells. Subsequently, vitamin D3 (Xiamen Kingdomway group Company, Xiamen, China) was added to the dried powders, and the mixed formulations were encapsulated in identical capsules, providing each 630 mg calcium and 400 IU vitamin D3 per serving. The reference product was calcium citrate tablets (Bayer, Leverkusen, and Germany), containing 315 mg calcium and 200 IU vitamin D3 per tablet. The tablets were grinded into a powder form and encapsulated in the same capsule format as the experimental products, providing 630 mg Calcium and 400 IU vitamin D3 per serving. Encapsulation of the powders was conducted under good manufacturing practice (GMP) by an independent company. After encapsulation, the three products were analyzed to assure standardized and identical quantities per serving. Calcium content was analyzed by inductively coupled plasma-optical emission spectrometry (ICP-OES), and vitamin D content was analyzed by high performance liquid chromatography (HPLC). All products were stored at room temperature, protected from moisture and direct light until administration.

Participants who were found eligible were assigned a randomization code on day 1 and randomly allocated into one of three intervention sequence arms: A➔B➔C, B➔C➔A, or C➔A➔B according to the randomization list: (A) A Single dose of Ca-LAB (investigational product), (B) A Single dose of calcium citrate (reference product), or (C) A single dose of Ca-SC (investigational product). Each arm comprised eight participants. The randomization schedule was generated using www.randomization.com.

Study products were labeled with an individual randomization and visit number and were identical in appearance, texture, and taste. Labeling was done by designated personnel not involved in other aspects of the study to maintain blinding. Intervention allocation was concealed by using opaque sealed envelopes, each labelled with a randomization number. Each envelope contained information regarding the intervention associated with each randomization number. Investigators and study staff remained blinded to the assigned sequence until the database was locked, and the data were analyzed.

Participants underwent screening procedures within 37 days prior to the study run-in period (7 days) to assess their eligibility for participation. At the screening, written consent was obtained from each participant prior to the conduct of any study-related activity. Once consent was obtained, medical history, concomitant medications and current health status were reviewed, seated systolic and diastolic blood pressure (SBP and DBP), heart rate (HR), oral temperature, height, weight, and BMI were measured, and an ECG was recorded. In addition, fasted blood and urine samples were collected for analysis. Blood test included: complete blood count (CBC), electrolytes (sodium, potassium, chloride, phosphate), total calcium, hemoglobin A1c (HbA1c), fasting glucose, thyroid stimulating hormone (TSH), estimated glomerular filtration rate (eGFR), creatinine, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin, total bilirubin, 1–84 PTH, 25(OH)D, low-density lipoprotein cholesterol (LDL-C), and triglycerides. Urine test included: total calcium, creatinine, and albumin. Nutritional counselling and nutritional guidelines were provided. From 1 week prior to baseline (day 1) to the end of the study, participants were instructed to restrict their calcium and sodium dietary intake to 400 and 2,300 mg/day, respectively. These dietary instructions were intended to provide a high level of standardization in terms of the level of calcium concentration/metabolism (39). Prior to the intervention days, i.e., day’s −1, 8, and 16, participants fasted for 14 h and were only instructed to drink 600 mL distilled water 12 h prior to administration and 300 mL distilled water 9 h prior to administration. These activities were recorded in the participants’ study diary.

On the intervention days, i.e., days 1, 9, and 17, participants were instructed to empty their bladder (including the first urine of the morning) 2 h prior to administration time and consume 600 mL distilled water. During this time, participants also began collecting all their pre-intervention urinary output. Once attending the study site, participants were under continuous medical supervision until 10 h post dose. At the study site, seven-day food record was reviewed and concomitant therapies, health status and vitals (BP, HR, and oral temperature) were assessed. Under fasting conditions, a pre-intervention blood sample was collected, and participants emptied their bladder again and the overall urinary output was collected over a time window of 2 h prior administration. Then, participants consumed a standardized low-calcium breakfast (scrambled egg made of three egg whites cooked with one teaspoon olive oil, one slice sourdough bread with one teaspoon butter, and one cup of raw watermelon; overall calcium content of 39.2 mg) with 600 mL distilled water and half-way through consumed the investigational product. Participants drank 300 mL of distilled water every 2 h post dose and were given a standard lunch low-calcium meal (90 grams roasted beef, 80 grams banana, and three cups of salad containing: iceberg lettuce, tomato, cucumber, and onion with one tablespoon vinaigrette; overall calcium content of 50 mg) 6 h after the standard breakfast. The main purpose for the standardized low-calcium was to reduce the risk of confounding outcomes. Vital signs (BP, HR, and oral temperature) were collected every 2 h up to 10 h post-dose. Blood samples were collected at 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7 and 8 h. Urine samples were collected for time frames 0–2, 2–4, 4–6, 6–8, 8–10 and 10–24 h. At 10 h post dose, participants drank 600 mL of distilled water at home and returned to the study site the next morning to submit their 10–24 h urine container. Before each intervention day, there was a 7 ± 2 days washout period where participants completed a seven-day food record to monitor compliance with the restricted calcium and sodium dietary intake between visits. The completed food records were reviewed by clinic staff prior to starting assessments on each intervention day to ensure participants complied with the dietary instructions.

A sample size of 20 participants, with a total of 24 assuming 20% attrition rate, was estimated to detect a difference in pharmacokinetic parameters with an effect size of d = 0.66 and 80% power using a paired t-test with 0.05 two-sided significance level (41). Head-to-head comparisons between the calcium products were made with respect to pharmacokinetic parameters based on the intent-to-treat (ITT) population, using a mixed model Analysis of Variance (ANOVA), with product, sequence and period as fixed effects, and participant as the random effect. Pharmacokinetic parameters that were not distributed normally by the Wilk Shapiro test were log transformed prior to conducting statistical analysis. Differences in time parameters (t_max) between treatments were assessed by Non-parametric Wilcoxon–Mann–Whitney Rank sum test. The difference between study products in calcium concentration at a respective timepoint was assessed by paired t-test. Categorical variables were assessed using Poisson Generalized Linear Model. In the statistical analysis, value of p less than 0.05 was considered statistically significant. The data were analyzed using the SAS ® version 9.4 (SAS Institute, Cary, North Carolina).

Twenty-four healthy postmenopausal females who met the inclusion and exclusion criteria described in the study protocol were enrolled in the clinical study. A total of 22 completed the study, with two participants withdrawing consent following the first intervention (Figure 2). At baseline, the mean age of the women was 56.2 years, 23 were of European descent and one of southeast Asian descent. Participants were normal weight, with a mean BMI of 24 kg/m² (Table 1).

The change in serum calcium concentration from pre-intervention over 8 h after administration is shown in Figure 3. Increment in serum calcium concentration was significantly higher following Ca-LAB administration compared to calcium citrate and Ca-SC, from 2 to 5 h post dose and from 2 to 6 h post dose, respectively. Following calcium citrate administration, there was no significant difference in serum calcium concentration compared to Ca-SC post dose up to 5 h but increment serum calcium was significantly higher at 6 h post dose. Comparison of serum calcium pharmacokinetic parameters among the three products are presented in Table 2. Ca-LAB had higher iAUC_0-8h compared to calcium citrate (p = 0.006) and Ca-SC (p = 0.004). For the other pharmacokinetic parameters, the AUC_T0-8h of Ca-LAB, was significantly higher than that of Ca-SC (p = 0.001) and showed trend toward significance compared to calcium citrate (p = 0.060). C_max was higher for Ca-LAB compared to Ca-SC (p < 0.001). t_max was not significantly different between the three products. There was no difference in pharmacokinetic parameters between calcium citrate and Ca-SC except for C_max that was substantially higher for calcium citrate (p = 0.028).

The change in urinary calcium over the 24-h collection period for the three test products is represented in Figure 4A. Urinary calcium significantly increased following Ca-LAB administration compared to calcium citrate and Ca-SC administrations for urine collected in intervals 2–, 4–, and 6–8 h. Urinary calcium profiles were similar following calcium citrate and Ca-SC administration, with only one significantly higher calcium value for calcium citrate compared to Ca-SC in interval 10–24 h. Total calcium excreted up to 10 h post dose was higher following Ca-LAB administration compared to calcium citrate (p = 0.002) and Ca-SC (p = 0.004; Figure 4B). Furthermore, urine calcium excretion over 24 h was higher following Ca-LAB administration compared to calcium citrate administration (p = 0.013; Figure 4B). Urine calcium pharmacokinetic parameters between the three products are presented in Table 2. AUC_T0-10h, AUC_T0-24h, and C_max were significantly higher for Ca-LAB compared to calcium citrate (p < 0.001, p = 0.006, and p < 0.001, respectively) and Ca-SC (p < 0.001, p = 0.003, and p < 0.001, respectively). No differences were observed between calcium citrate and Ca-SC.

A total of 26 AEs was reported in 11 participants throughout the study, four participants following Ca-LAB administration, seven participants following calcium citrate administration, and six participants following Ca-SC administration (Supplementary Table S3). There was neither a statistically significant difference in the proportion of participants reporting AEs, nor in the rate of AE incidences between the study products (data not shown). Out of the 26 AEs, only four gastrointestinal related AEs reported by one participant following the Ca-LAB administration classified as possibly product related (data not shown). The majority of the AEs (73.1%) were mild, and the rest were moderate (Supplementary Table S3). Headache was the most common AE, reported eight times during the study by six participants. No clinically significant changes in clinical chemistry or hematology, anthropometrics or vital signs were observed (data not shown). Both Ca-LAB and Ca-SC were well tolerated by the study participants.