Periodic Reporting for period 1 - TAMYOCAL (Tamoxifen mediated protection on X-linked centronuclear myopathy: a mechanistic and pre-clinical study)
Reporting period: 2018-05-01 to 2020-04-30
Tamoxifen (TAM) is a selective estrogen receptor modulator that mimics estrogen signalling in skeletal muscle. Pilot study and previous results from the hosting group, have shown that TAM improve muscle symptoms and survival rate in Mtm1-/y mice, a validate model of XLMTM. Results strongly support the idea that TAM protects skeletal muscle via enhancement of estrogenic signalling positively modulating multiple pathways linked to muscle function. However, the exact mechanism(s) is not yet understood. This project aims at elucidating the mechanism(s) of action of TAM and evaluate further its efficacy in Mtm1-/y mice. Two specific aims are proposed. First, to determine how TAM acts on pathways and key players involved in XLCNM. Estrogen signalling, phosphoinositide balance, autophagy and mTORC1 signalling were targeted in Mtm1+/y (WT from now on) and Mtm1-/y (KO from now on) mice treated and non-treated by TAM. Second, to investigate the pre-clinical efficacy of TAM combined with other compounds in XLMTM mice. TAM extends the lifespan of XLMTM mice according to the pilot study but it prevents only partly leg muscle atrophy, restricting complete functional rescue. Thus, TAM is combined with other approved drugs that target muscle atrophy (L-Citrullin and IGF).
These parallel approaches will provide knowledge about not-yet explained events related to XLMTM and TAM effects, from molecular to in-vivo level, and instrumental information for other myopathies and rare diseases where lipid phosphatases are missing and phosphoinositide are unbalanced. This knowledge might provide the rationale for new therapeutic options for these dramatic conditions, being highly relevant to patients and families.
The overarching objective of the project was to understand the mechanisms of action of tamoxifen and to evaluate further its efficacy in XLMTM. Two specific aims were determined to accomplish this main objective. First aim was the determination of the mechanisms underlying tamoxifen protection on XLMTM muscles. Second aim was the pre-clinical study of the combination of tamoxifen with other compounds in order to achieve a complete rescue of the XLMTM phenotype, targeting the observed muscle atrophy.
The second aim of the project was the pre-clinical study of tamoxifen treatment with other compounds. After the good results of tamoxifen extending lifespan, combination therapy seemed to be the next step to prevent leg muscle atrophy and achieve complete functional rescue. The selected compounds were L-citrullin and IGF. L-citrullin is used by body-builders to increase muscle mass. To determine the optimal dose for mice, a pilot study was carried out and 3 mg/kg/day was choses as the minimum dose having beneficial effects on muscle mass. This compound can be added to the food and taken orally without manipulation of the mice. A modified version of IGF-1, growth factor similar to insulin, was the second compound. It stimulates proliferation and differentiation of satellite cells during muscle regeneration. Increasing IGF-1 levels on animal models affected by loose and degradation of muscle fibers has shown beneficial effects. For the moment, only a pilot study with few animals per group has been carried out. Summarizing, animals were followed-up during the whole treatment (from 21 to 45/90 day-old depending on the group). Mice weight and clinical state were determined twice a week and grid test, once a week. When arriving at the stablished time point, final measurements of respiratory function (plethysmography), body composition (echo-MRI), bone density and kyphosis (micro-CT) were performed. In general, no differences were observed between obtained results from mice treated with tamoxifen and those treated with a combined treatment. Tamoxifen combined with L-citrullin seemed to increase muscle mass in comparison with the other treatments at 45-days-old. This effect was only noticeable after muscle dissection and sample weighting. The results for bone density and the kyphose are still being analyzed."
Often, rare diseases like XLMTM share symptoms, pathogenic mechanisms of affected pathways. Thus, all the obtained results could be useful to understand or to increase the knowledge of other myopathies.