BIO103 a drug candidate for the treatment of muscle wasting disorders

Maria Serova1, Blaise Didry-Barca1, Sissi On1, Anne-Sophie Foucault1, Sophie Raynal1, Stanislas Veillet1,

Pierre Dilda1, René Lafont2

1 Biophytis, UMPC – BC9, 4 place Jussieu, 75005 Paris, France2 Sorbonne Universités, UPMC Univ Paris 06, CNRS - Institut de Biologie Paris Seine (BIOSIPE), 75005 Paris, France

Introduction

Muscle wasting disorders, including cachexia andsarcopenia, are multifactorial diseases whichcontribute to overall physical frailty. Theyrepresent a worldwide health challenge withlimited therapeutic options. Many cellular factorswere identified to maintain normal musclefunction. Cell metabolism influenced byAKT/mTOR and mitochondrial biogenesis inparallel with physical activity contribute tomaintaining of muscle mass and strength. Age-related deregulation of these mechanisms leads tomuscle wasting.About ecdysteroids: The steroid hormone (20E)plays a key role in insect development throughnuclear ecdysone receptors (EcRs) and at leastone membrane receptor (DopEcR). 20E alsodisplays pharmacological effects on mammals,where it stimulates protein synthesis although EcRwere not found in mammals.About BIO103: BIO103, ecdysteroid derivative, isthe product of hemisynthesis associated with thescreening of more than 100 derived molecules.This screening had the object of selecting amolecule with improved activity and bioavailability.

The aim of this study was to characterize a newsmall molecule, BIO103, in vitro on myocytes andin vivo on a mouse model designed to analyze theeffects of aging and muscle disuse.

Proposed mechanism of action

Results

• In vitro

Effects of BIO103 on C2C12 myotube diameter

→ 3-day treatment of differentiated C2C12 myocytes withBIO103 (1, 5, 10 µM), induced significant increase of myotubediameters

Effect of BIO103 on myostatin gene expression

→ BIO103 6 hour-treatment of differentiated C2C12 cellssignificantly inhibits myostatin gene expression

Effects of BIO103 on intracellular signaling

o PI3K/AKT/mTOR

o MAPK

o AMPK/ACC

→ BIO103 rapidely activates the major kinases ofPI3K/AKT/mTOR, MAPK and AMPK signaling pathways indifferentiated C2C12 cells

Effects of BIO103 on mitochondrial respiration and

glycolysis of differentiated C2C12

→ BIO103 increases both basal and maximal oxygenconsumption rates (OCR) in muscle C2C12 cells after 72 hexposure to differentiated cells

• In vivo

Old (22 months) C57Bl6/J female mice were treated orally for14 weeks with either vehicle or or BIO103 (50 mg/kg/day).

Running velocity

IGF-1 Myostatin

AMPK

Methods

Cell line: C2C12 murine myoblasts were induced for differentiationfor 5 days. Appropriate treatment was administered for 6h (for geneexpression analysis) or 4 days (for fluorescent microscopy).Gene expression: Total mRNA was extracted and purified usingTrizol method. mRNAs were reverse-transcribed into cDNAs andMyostatin gene expression was analyzed by quantitative RT-PCR.HPRT was used as housekeeping gene.Immunofluorescence: Cells were grown, differentiated and treatedon 8 well chamber slides. Then the cells were fixed withglutaraldehyde 2.5%/triton 0.1%, covered by DAPI-containingmounting medium. After 24h in the dark, myotubes were observedunder fluorescent microscope.Western blot: Cells were lysed, equal amounts of proteins wereelectrophoresed on 4-12% SDS-PAGE and transferred tonitrocellulose membranes. Membranes were blocked with 5% non-fat milk and incubated with specific antibodies overnight.Immunostaining was visualized using ECL. Bands intensity wasquantified using ImageJ software.OCR measurements: Oxygen consumption was recorded using aSeahorse XF Analyzer.

In vivo studies. Adult (12 months) and old (22 months) C57Bl6/Jfemale mice were used. The old animals were treated orally for 14weeks with either vehicle or BIO103 (50 mg/kg). After 13 weeks oftreatment), the animals were tested for functional activity in toto(Vmax). Plasma and muscles were collected after sacrifice forfurther analysis

Conclusions

→ Treatment of oldanimals by BIO103compensate for thesignificant loss ofrunning velocity as aconsequence ofaging.

• BIO103 displayed in vitro and in vivo anaboliceffects on myofibers. These effects wereaccompanied with Myostatin inhibition.

• Anabolic properties of BIO103 result from anactivation of AKT/mTOR, MAPK and AMPKpathways.

• BIO103 warrant further studies towards itsdevelopment as a drug candidate for the treatmentof muscle wasting disorders.

→ BIO103 treatment tends to increase AMPKphosphorylation in gastrocnemius muscles of old mice

→ Chronic oral administration of BIO103 was responsiblefor a significant increase in animal IGF-1 plasma level anddecreased myostatin expression in gastrocnemiusmuscles.

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