Prevention of Glucocorticoid Induced Impairment of Bone Metabolism

By advertising in newspapers, in shops, health clubs, in public places, and on social media platforms, and by contacting interested persons (by mail and phone) identified publicly available websites (e.g. www.ratsit.se), 46 young, healthy men and women will be recruited, to participate in the study. Study subjects will be contacted by phone and receive more information regarding the study and undergo a preliminary evaluation about eligibility to participate. Detailed study information will be sent by mail to study subjects. At the screening visit, participants will receive information about the study verbally, and if participants sign the informed consent form, participants will undergo a test of fasting blood glucose, HbA1c, and an oral glucose tolerance test (OGTT). A standardized self-reported questionnaire will be used to collect information at the screening visit about smoking habits, calcium intake, medical history, medications, previous fractures, physical activity, and diet habits. Eligible participants are then invited to the randomization visit. At the randomization visit, the study subjects will be asked to refrain from physical exercise, other than walking and keep an unchanged normal diet from study day 14. Randomization will be carried out by the supplier of LR (BioGaiaAB). The investigators will not have access to the randomization code and blinding will be maintained until study end and database lock. After randomization, study participants will consume either LR or placebo for 30 days. After the 14-day run-in period, all participants will start taking 25 mg of oral prednisolone every morning for 7 days. At the 14-day visit, a 24-hour glucose monitoring for the following 10 days will be started. At the next visit on day 16, fasting blood samples will be drawn in the morning (7-9 am), feces samples provided (within 24 hours of collection) and participants will start the oral GC treatment on that visit after blood samples have been drawn. At the 19-, 23- and 30-day visits, blood samples will be drawn in the morning (7-9 am for all visits, prior to taking the GC dose). Feces samples will also be collected at these visits. Included participants will be asked to come to the clinic and report compliance and possible adverse events during the duration of the study and at all visits.

Analyses.

Questionnaires:

A standardized questionnaire will be used to collect information at the screening visit about smoking habits, calcium intake, medical history (e.g. stroke, rheumatoid arthritis, and diabetes), medications, and previous fractures. Current physical activity and diet habits will be assessed by self-reported questionnaires (IPAQ and FFQ) at baseline. Daily intake of calcium will be calculated from the questionnaires about calcium-containing foods (e.g. dairy products, vegetables etc). Gastrointestinal symptoms will be analyzed using the GSRS questionnaire.

Serum, plasma, and stool analyses:

Serum and plasma will be collected in the morning (fasting), frozen, and stored at -80° C until further analyses. Samples from day 16 will be drawn prior to oral GC treatment start. A maximum of 30 ml of blood will be collected at each visit. Fasting blood glucose will be analyzed using fresh blood at screening (with the addition of HbA1c at screening), and days 16 (GC start), 19, 23, and 30. Serum and plasma samples will be frozen immediately and kept until analysis. Analyses of serum or plasma and bone markers (osteocalcin, P1NP, and CTX) will be performed using commercially available immunosorbent assays. Stool samples will be collected within 24 hours since produced and stored at -80oC until further analysis.

Oral glucose tolerance test (OGTT):

At the screening visit, a morning fasting blood glucose will be measured, followed by ingestion of 24 cl of glucose syrup, containing 75 grams of sugar. After 2 hours another blood glucose measurement will be performed. Inclusion in the study will depend on having a normal fasting blood glucose (≤6 mmol/l) and OGTT (<7.8 mmol/l).

Markers of intestinal permeability and intestinal inflammation:

Analyses of markers for intestinal permeability (endotoxin analysis, PyroGeneTM Recombinant Factor C Endpoint Fluorescent Assay) will be performed using a fluorescent assay at day 16, day 19, and day 23. Markers of intestinal inflammation (lipocalin-2 and calprotectin) will also be analyzed in feces and serum.

Analyses of gut microbiota - exploratory analyses:

DNA from feces samples will be isolated using a well-validated protocol and perform PCR to amplify the variable regions (V4) of the bacterial 16S rRNA gene using barcoded primers. Metagenome analyses of all samples will also be performed.

Continuous glucose monitoring:

On study day 14, continuous glucose monitoring (CGM) will be started, allowing a 48-hour observation period prior to commencement of oral prednisolone dosing. CGM will be used from day 14 until prednisolone treatment cessation on day 23. The Dexcom G6 system will be used for CGM on all participants.

Inclusion criteria

• Healthy men and women, 18-45 years old.
• Stated availability throughout the entire study period.
• Ability to understand study instructions and willingness to adhere to the protocol.
• Signed informed consent.
• Vaccinated for Covid-19

Exclusion criteria

• History of diabetes or glucose intolerance, defined as an abnormal oral glucose tolerance test (OGTT).
• Obesity, BMI>30 kg/m2
• History of adrenal disease or impairment.
• Previous (within the last 5 years) or current use of antiresorptive therapy, including systemic hormone therapy (estrogen), bisphosphonates, strontium ranelate or denosumab.
• Participation in other clinical trials.
• Current antibiotics treatment or within the last 2 months prior to inclusion.
• Current and within the past 2 months use of probiotic supplement.
• Untreated hyperthyroidism or hyperthyroidism within the last 5 years.
• Known untreated hyperparathyroidism.
• Rheumatoid arthritis.
• Diagnosed with disease causing secondary osteoporosis, including chronic obstructive pulmonary disease, inflammatory bowel disease, celiac disease, or diabetes mellitus.
• Recently diagnosed malignancy (within the last 5 years).
• Systemic skeletal disease (including e.g. Paget's disease and osteogenesis imperfecta).
• Any systemic disease that could affect bone loss, as judged by the investigator.
• Oral corticosteroid use.
• History of peptic ulcer.
• Diagnosed osteoporosis.
• Current smoking or other use of nicotine containing products.
• Pregnancy.
• History of any psychiatric disorder, including psychosis, depression, anxiety disorder and bipolar disorder.