OsteoPreP: Food Supplements for Postmenopausal Bone Health

It is well established that bone loss occurs throughout life after the attainment of peak bone mass which is usually reached by the end of the second decade of life. During the first 5-8 years following menopause, women experience an accelerated bone loss, which is then followed by a slower phase of decline in bone mineral density (BMD). As over 40% of all women in Australia will suffer an osteoporotic (fragility) fracture in their lifetime, investigating interventions that can prevent bone loss in postmenopausal women is a critical focus. The menopausal transition is also associated with an increased risk of cardiovascular disease, diabetes, and cognitive decline. These conditions and their treatments can also affect bone health.

Previous research has indicated a potential link between the gut microbiome and bone health. Animal studies indicate that interventions affecting the gut microbiome may be successful in reducing bone loss, but human data is limited. Moreover, there is an emerging body of evidence linking the gut microbiome to cognitive, muscle and cardiometabolic function. Such studies indicate that probiotics (healthy gut bacteria) or prebiotics (food for healthy bacteria, e.g. fibre) can increase the amount of short chain fatty acids - such as butyrate -produced by the bacteria in the gut which may mediate the beneficial effects of improving gut health.

The proposed study is a double-blind, placebo-controlled randomised trial, which will investigate whether consuming a probiotic supplement containing inulin (a prebiotic soluble fibre) twice daily for 12 months will improve bone health in postmenopausal women. In addition, secondary outcomes will measure the effect of the intervention on immune system modulation and cognition as well as musculoskeletal and metabolic function as potential mediators.

One hundred and sixty postmenopausal women residing in Melbourne (Victoria, Australia) who are at least one year from their final menses will be recruited from the community via a mail out, advertisements in newspapers, social media, flyers as well as a landing page on an ACU managed website. The investigators have also applied for assisted mail outs through Services Australia for recruitment purposes. Services Australia is able to extract names and addresses of some target study demographics (females living in Melbourne aged between 40 and 65) from the Medicare database and mail them the study details on our behalf. Those wishing to participate will undergo a series of baseline assessments inclusive of bone mineral density scans, blood and stool sampling and physical activity and lifestyle questionnaires. They will then be randomised, in a blinded fashion, to consume one of the following two supplements:

• Placebo control group (n = 80): Placebo capsule - 2 capsules per day
• Probiotics group (n=80): Probiotic capsule - 2 capsules per day

Study outcomes will be measured at baseline, 6 months, and 12 months. In addition, participants will be contacted via the telephone and email at three-month and nine-month timepoints to report any adverse responses to the supplementation. At the 12-month time point, participants will cease supplementation and immediately (within 24 hours) attend a post intervention assessment and will complete identical assessment measures to those they did at baseline. Finally, at 2 weeks post supplementation cessation participants will be asked to attend an appointment to provide a final stool sample.

If it can be confirmed that long term consumption of a probiotic supplement can have beneficial effects on bone health, muscle health, and metabolic health in postmenopausal women, this intervention could be recommended in the prevention of osteoporosis and associated musculoskeletal and metabolic conditions.

Pendulum WBF-038, a proprietary formulation of the following strains: Akkermansia muciniphila, Clostridium butyricum, Clostridium beijerinckii, Anaerobutyricum hallii, Bifidobacterium infantis, plus chicory inulin and magnesium stearate - 1 capsule with the morning meal and 1 capsule with the evening meal for 12 months.

Dietary Supplement: Pendulum WBF-038

Pendulum Placebo containing Magnesium stearate - 1 capsule with the morning meal and 1 capsule with the evening meal for 12 months.

Dietary Supplement: Pendulum Placebo

All bacteria contained in Pendulum WBF-038 are commensal organisms that have been repeatedly documented to inhabit the human gastrointestinal tract under normal circumstances. Pendulum's WBF-038 is a proprietary formulation of the following strains: Akkermansia muciniphila, Clostridium butyricum, Clostridium beijerinckii, Anaerobutyricum hallii, Bifidobacterium infantis, plus chicory inulin and magnesium stearate. The organisms were grown under controlled conditions consistent with Good Manufacturing Practices (GMP) and employ no animal-derived products. All ingredients utilized during manufacturing were food grade and qualified as generally recognized as safe (GRAS).

The product is provided as acid-resistant capsules in bottles that are to be stored refrigerated at 4℃.

Pendulum placebo capsules containing magnesium stearate

Plazo: 12 months

Percentage change in total volumetric bone density measured using high resolution peripheral quantitative computed tomography (HR-pQCT) of the distal tibia.

Plazo: 12 months

Relative change in lumbar spine (L1-L4) bone mineral density (BMD) measured using dual energy X-ray absorptiometry (DXA)

Plazo: 12 months

Relative change in total hip BMD measured using DXA

Plazo: 12 months

Relative change in tibia and radius trabecular bone volume fraction measured using HR-pQCT

Plazo: 12 months

Relative change in tibia and radius cortical area measured using HR-pQCT

Plazo: 12 months

Relative change in tibia and radius cortical volumetric BMD measured using HR-pQCT

Plazo: 12 months

Relative change in total volumetric BMD of the distal radius measured using HRpQCT

Plazo: 6 months and 12 months

Relative change in ßCTX-I in serum

Plazo: 6 months and 12 months

Relative change in P1NP levels in serum

Plazo: 6 months and 12 months

Relative change in OC in serum

Plazo: 6 months, 12 months, and 2 weeks post intervention

Relative change in SCFA levels (including butyrate) in stool

Plazo: 6 months, 12 months, and 2 weeks post intervention

Relative change in species-level gut microbiota composition in stool using 16s rRNA sequencing

Plazo: 6 months and 12 months

Relative change in fasted blood glucose levels in circulating blood

Plazo: 6 months and 12 months

Relative change in HbA1c levels in circulating blood

Plazo: 12 months

Relative change in lower leg muscle area measured using HR-pQCT

Plazo: 12 months

Relative change in appendicular lean body mass measured using DXA

Plazo: 6 months and 12 months

Relative change in left and right hand grip strength measured using a hand dynamometer

Plazo: 6 months and 12 months

Relative change in hs-CRP levels in serum

Plazo: 12 months

Relative change in circulating number of Tregs in blood

Plazo: 6 months and 12 months

Relative change in oral glucose tolerance in circulating blood

Plazo: 6 months and 12 months

Relative change in muscle tissue glycogen content obtained from a muscle biopsy

Plazo: 6 months and 12 months

Relative change in muscle tissue triglyceride content obtained from a muscle biopsy

Plazo: 6 months and 12 months

Relative change in muscle tissue type 1 fibre proportion obtained from a muscle biopsy

Plazo: 6 months, 12 months, and 2 weeks post intervention

Relative change in Lipocalin2 (intestinal inflammation) in stool

Plazo: 6 months and 12 months

Relative change in speed of performance and number of errors in the One Back Test using the Cogstate cognitive assessment tool. Lower score = better performance

Plazo: 6 months and 12 months

Relative change in number of errors in the Groton Maze Learning Test using the Cogstate cognitive assessment tool. Lower score = better performance

Plazo: 6 months and 12 months

Relative change in accuracy of performance and number of errors in the Continuous Paired Associate Learning Test using the Cogstate cognitive assessment tool. Lower score = better performance

Plazo: 6 months and 12 months

Relative change in accuracy of performance in the Social Emotional Cognition Test using the Cogstate cognitive assessment tool. Higher score = better performance

Plazo: 6 months and 12 months

Relative change in depression, anxiety and stress measured using the Depression Anxiety and Stress Scale 21 (DASS-21) questionnaire. The DASS-21 has a minimum score of 0 and a maximum score of 63 with higher scores indicating a worse outcome.

Plazo: 6 months and 12 months

Relative change in gastrointestinal symptoms measured using the Gastrointestinal Symptom Rating Scale (GSRS) questionnaire. The GSRS has a minimum score of 5 and a maximum score of 45 with higher scores indicating a worse outcome.

Plazo: 6 months and 12 months

Relative change in gut hormone glucagon-like peptide 1 \[GLP-1\] in plasma

Plazo: 6 months and 12 months

Relative change in gut hormone peptide tyrosine-tyrosine \[PYY\] in plasma

Plazo: 6 months and 12 months

Relative change adiponectin in plasma

Plazo: 6 months and 12 months

Relative change in quality of life measured using the EuroQol Five Dimensions (EQ-5D) questionnaire. The EQ-5D has a minimum score of Level 1 and a maximum score of Level 5 with higher levels indicating a worse outcome

Plazo: 6 months and 12 months

Relative change in social anxiety measured using the Social Interaction Anxiety Scale (SIAS) questionnaire. The SIAS has a minimum score of 0 and a maximum score of 80 with higher scores indicating a worse outcome.

Plazo: 6 months and 12 months

Relative change in mental wellbeing measured using the Warwick-Edinburgh Mental Wellbeing Scale (WEMWBS) questionnaire. The WEMWBS has a minimum score of 14 and a maximum score of 70 with higher scores indicating a better outcome.

Plazo: 6 months and 12 months

Relative change in blood glucose (area under the curve) over 10 days measured using a continuous glucose monitoring device

Plazo: 6 months and 12 months

Relative change in fasted blood insulin levels in circulating blood

Plazo: 12 months

Relative change in total body fat mass measured using DXA

Plazo: 12 months

Relative change in blood metabolites using liquid chromatography-mass spectrometry

Plazo: 6 months and 12 months

Relative change in Systolic and Diastolic blood pressure measured using a sphygmomanometer

Plazo: 6 months and 12 months

Relative change in current pain intensity measured using a visual analogue scale (VAS) pain intensity scale. The VAS has a minimum score of 0 and a maximum score of 10 with higher scores indicating a worse outcome.

Plazo: 6 months, 12 months, and 2 weeks post intervention

Relative change in calprotectin (intestinal inflammation) in stool

Plazo: 12 months

Relative change in total body muscle mass measured using DXA

OsteoPreP: The Effect of Probiotic Supplementation on Bone, Muscle, and Glucose Metabolism in Postmenopausal Women: A Randomised Placebo-controlled Trial