Evaluation of Cholecalciferol and Glycerol Supplementation on Inflammatory Markers in Obese Elderly Patients with Vitamin D Deficiency: A Randomized Double-Blind Study

The primary objective of this study is to evaluate the effect of **cholecalciferol** supplementation on inflammation in obese patients with vitamin D deficiency. This is clinically relevant as inflammation is a common comorbidity in obesity, and vitamin D deficiency may exacerbate inflammatory processes, potentially impacting overall health and disease progression in this population.

Secondary objectives include:

• Evaluating the inflammatory processes of muscle and adipose tissue following supplementation with cholecalciferol.
• Assessing the effects of cholecalciferol supplementation on the modulation of the WNT pathway in bone, muscle, and adipose tissue in obese subjects.
• Evaluating the safety of cholecalciferol supplementation.
• Exploratorily determining whether cholecalciferol supplementation can reduce bone marrow fat.

The clinical trial focuses on **obese elderly people with vitamin D deficiency**, specifically targeting post-menopausal women and men aged between 55 and 75 years. The study population includes individuals scheduled for hip or knee replacement surgery, with a **Body Mass Index (BMI)** of 30 kg/m² or higher and 25(OH)D levels below 20 ng/ml. Both male and female participants are included, and the trial does not involve a vulnerable population. The sponsor has not provided information regarding the total number of participants. Participants are expected to adhere to the study procedures as outlined in the informed consent. The trial does not specify any particular lifestyle considerations such as diet or physical activity. Key inclusion criteria include obtaining written informed consent and a willingness to comply with study procedures.

The clinical trial is designed to evaluate the effect of **cholecalciferol** supplementation on inflammation in obese elderly individuals with vitamin D deficiency. This study is a randomized, double-blind, controlled trial, categorized as a Phase IV clinical trial. The trial is expected to commence recruitment on February 28, 2025, and conclude by February 28, 2027. Participants will be randomly assigned to receive either the active treatment, **colecalciferol**, or a placebo, which is **refined olive oil**. Both interventions will be administered orally over a maximum treatment period of 12 months.

The study will include several key visits: an initial screening visit, regular follow-up visits, and an end-of-study visit. During the screening visit, eligibility will be confirmed based on criteria such as age, BMI, and vitamin D levels. Participants must be post-menopausal women or men aged 55 to 75, with a BMI of 30 kg/m² or higher, and 25(OH)D levels below 20 ng/ml. Follow-up visits will occur at predetermined intervals to monitor safety and efficacy, including assessments of serum cytokine levels and other biomarkers. The end-of-study visit will involve final evaluations and data collection.

Participant involvement is expected to last up to 12 months, with conditions for early termination including non-compliance with study procedures or adverse events. Primary endpoints include changes in serum pro-inflammatory cytokine TNF-alpha levels, assessed via ELISA assays. Secondary endpoints involve comprehensive analyses of muscle, adipose, and bone tissues, as well as synovial fluid and peripheral blood mononuclear cells (PBMCs), using techniques such as RT-PCR, Western Blot, and flow cytometry. Safety monitoring will include regular assessments of blood calcium levels to ensure the safe administration of **cholecalciferol**.

The clinical trial involves the administration of **colecalciferol**, also known as **vitamin D3**, as the experimental medication. The pharmaceutical form of colecalciferol is identified as PHF00165MIG, and it is administered orally. The dosage is measured in international units (IU), with a maximum daily dose of 7000 IU and a maximum total dose of 50000 IU over the treatment period. The treatment duration is set for a maximum of 12 months. Colecalciferol is a chemical substance, and its administration is intended to evaluate its effects on inflammation in obese patients with vitamin D deficiency. Participant compliance with the dosing schedule will be monitored throughout the study.

The study also includes the use of **glycerol** as a component of the experimental medication. Glycerol, also known as glycerine or glycerin, is a chemical substance that is administered orally in combination with colecalciferol. The role of glycerol in the formulation is to aid in the delivery and absorption of the active ingredient, colecalciferol. The dosing schedule and administration route for glycerol are consistent with those of colecalciferol, ensuring a standardized approach to treatment.

In addition to the experimental treatment, the study employs **olive oil, refined** as a placebo comparator. Olive oil is administered orally in an oil form, with a maximum daily dose of 2.50 milliliters and a maximum total dose of 17.5 milliliters over the 12-month treatment period. The use of olive oil as a placebo is intended to provide a control for evaluating the effects of colecalciferol supplementation. The administration of olive oil will follow the same oral route as the experimental medication, ensuring consistency in the study design.

Efficacy in this clinical trial will be assessed through a series of primary and secondary endpoints. The primary endpoint involves evaluating changes in the pro-inflammatory cytokine **TNF-alpha** in serum, which will be measured using an ELISA assay. This will provide insight into the inflammatory response to cholecalciferol supplementation in obese patients with vitamin D deficiency.

Secondary endpoints encompass a comprehensive analysis of various tissues and biological markers. Muscle tissue will be assessed using nuclear magnetic resonance imaging (MRN) of the ilio-psoas muscle, and gene expression of inflammatory cytokines and myogenic proteins will be evaluated using RT-PCR. Adipose tissue analysis will include gene and protein expression of cytokines and markers related to the WNT pathway, using RT-PCR and Western-Blot techniques. Bone tissue will be examined for gene expression and immunohistochemical analysis to identify specific cell types. Synovial fluid will be analyzed for pro- and anti-inflammatory molecules using the Luminex assay.

Serum analysis will include the evaluation of cytokines, adipokines, and bone turnover markers through ELISA assays, as well as the serum concentration of 25-hydroxy-vitamin D using radioimmunoassays. Peripheral blood mononuclear cells (PBMCs) will be extracted to study T cell phenotypes via multiparametric flow cytometry, and intramedullary fat will be quantified using magnetic resonance spectroscopic (MRS). These assessments will be conducted at specified time points throughout the study to monitor the efficacy of the intervention.