Prevention of Glucocorticoid Induced Impairment of Bone Metabolism
Condition: Bone Loss
Study type: Interventional

Summary: 
      Glucocorticoid (GC) therapy is used to treat a variety of inflammatory conditions such as
      rheumatoid arthritis, inflammatory bowel disease, bronchial asthma, allergies, ankylosing
      spondylitis and some forms of cancers. Despite the well-known side-effects, GC treatment is
      widely used. Oral GC therapy leads to a rapid and profound effects on bone metabolism, with
      increased osteoblast apoptosis and prolonged osteoclast survival, which increases bone
      resorption, resulting in bone loss, and a subsequent increased fracture risk. Within days of
      high dose oral GC, glucose tolerance decreases and bone turnover is shifted in favour of less
      bone formation and increased bone resorption. Bone formation and bone resorption can be
      estimated by measuring serum bone turnover markers.

      The gut microbiota is involved in regulating bone metabolism and recently it was demonstrated
      that Lactobacillus reuteri 6475 (LR) could reduce bone loss over 12 months by half in older
      women. In a recent experimental study, it was discovered that mice treated either with broad
      spectrum antibiotics, eradicating gut microbiota, or with LR did not experience GC induced
      bone loss in the spine and femur. L. reuteri has been widely studied in clinical trials and
      has been shown to have probiotic, health-promoting effects in both adults and children.

      The aim of this planned randomized, double-blind, placebo-controlled trial is to investigate
      if daily supplementation with LR, compared to placebo, can prevent the negative effects of
      oral glucocorticoid (GC) on bone turnover and on blood glucose regulation in healthy young
      adult men and women.

      If LR is able to prevent deleterious side effects, such as bone loss and impaired glucose
      tolerance, of oral GC treatment, the probiotic could potentially be recommended and used to
      improve health in a substantial yearly number of patients treated with GC.
    

Detailed description: 
      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.
    

Intervention name(s): Lactobacillus reuteri ATCC PTA 6475 (L. reuteri 6475); Prednisolone
Intervention type(s): Dietary Supplement; Drug
Intervention description(s): L.reuteri with cholecalciferol compared to cholecalciferol only.; Oral glucocorticoid 25 mg daily for 7 days.
Intervention MeSH terms: Prednisolone
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Treatment