RWXEH GLDPHWHUV - Biophytis€¦ · treatment), the animals were tested for functional activity in...
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BIO103 a drug candidate for the treatment of muscle wasting disorders Maria Serova 1 , Blaise Didry-Barca 1 , Sissi On 1 , Anne-Sophie Foucault 1 , Sophie Raynal 1 , Stanislas Veillet 1 , Pierre Dilda 1 , René Lafont 2 1 Biophytis, UMPC – BC9, 4 place Jussieu, 75005 Paris, France 2 Sorbonne Universités, UPMC Univ Paris 06, CNRS - Institut de Biologie Paris Seine (BIOSIPE), 75005 Paris, France Introduction Muscle wasting disorders, including cachexia and sarcopenia, are multifactorial diseases which contribute to overall physical frailty. They represent a worldwide health challenge with limited therapeutic options. Many cellular factors were identified to maintain normal muscle function. Cell metabolism influenced by AKT/mTOR and mitochondrial biogenesis in parallel with physical activity contribute to maintaining of muscle mass and strength. Age- related deregulation of these mechanisms leads to muscle wasting. About ecdysteroids: The steroid hormone (20E) plays a key role in insect development through nuclear ecdysone receptors (EcRs) and at least one membrane receptor (DopEcR). 20E also displays pharmacological effects on mammals, where it stimulates protein synthesis although EcR were not found in mammals. About BIO103: BIO103, ecdysteroid derivative, is the product of hemisynthesis associated with the screening of more than 100 derived molecules. This screening had the object of selecting a molecule with improved activity and bioavailability. The aim of this study was to characterize a new small molecule, BIO103, in vitro on myocytes and in vivo on a mouse model designed to analyze the effects 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 with BIO103 (1, 5, 10 µM), induced significant increase of myotube diameters Effect of BIO103 on myostatin gene expression → BIO103 6 hour-treatment of differentiated C2C12 cells significantly 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 of PI3K/AKT/mTOR, MAPK and AMPK signaling pathways in differentiated C2C12 cells Effects of BIO103 on mitochondrial respiration and glycolysis of differentiated C2C12 → BIO103 increases both basal and maximal oxygen consumption rates (OCR) in muscle C2C12 cells after 72 h exposure to differentiated cells • In vivo Old (22 months) C57Bl6/J female mice were treated orally for 14 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 differentiation for 5 days. Appropriate treatment was administered for 6h (for gene expression analysis) or 4 days (for fluorescent microscopy). Gene expression: Total mRNA was extracted and purified using Trizol method. mRNAs were reverse-transcribed into cDNAs and Myostatin gene expression was analyzed by quantitative RT-PCR. HPRT was used as housekeeping gene. Immunofluorescence: Cells were grown, differentiated and treated on 8 well chamber slides. Then the cells were fixed with glutaraldehyde 2.5%/triton 0.1%, covered by DAPI-containing mounting medium. After 24h in the dark, myotubes were observed under fluorescent microscope. Western blot: Cells were lysed, equal amounts of proteins were electrophoresed on 4-12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% non- fat milk and incubated with specific antibodies overnight. Immunostaining was visualized using ECL. Bands intensity was quantified using ImageJ software. OCR measurements: Oxygen consumption was recorded using a Seahorse XF Analyzer. In vivo studies. Adult (12 months) and old (22 months) C57Bl6/J female mice were used. The old animals were treated orally for 14 weeks with either vehicle or BIO103 (50 mg/kg). After 13 weeks of treatment), the animals were tested for functional activity in toto (Vmax). Plasma and muscles were collected after sacrifice for further analysis Conclusions → Treatment of old animals by BIO103 compensate for the significant loss of running velocity as a consequence of aging. • BIO103 displayed in vitro and in vivo anabolic effects on myofibers. These effects were accompanied with Myostatin inhibition. • Anabolic properties of BIO103 result from an activation of AKT/mTOR, MAPK and AMPK pathways. • BIO103 warrant further studies towards its development as a drug candidate for the treatment of muscle wasting disorders. → BIO103 treatment tends to increase AMPK phosphorylation in gastrocnemius muscles of old mice → Chronic oral administration of BIO103 was responsible for a significant increase in animal IGF-1 plasma level and decreased myostatin expression in gastrocnemius muscles. CTL 1 µM 5 µM 10 µM IG F -1 Dex 0 50 100 150 200 Myotube diam eters (% ) 100% 117% ** 134% *** 118% ** 117% * 78% *** BIO103 CTL 1h 2h 5h 24h 0 1 2 3 4 5 pAKT relative protein level ** ** BIO103 10µM CTL 1h 2h 5h 24h 0.0 0.5 1.0 1.5 2.0 pP70S6K relative protein level * BIO103 10µM CTL 1h 2h 5h 24h 0.0 0.5 1.0 1.5 2.0 2.5 pS6 relative protein level BIO103 10µM CTL 1h 2h 5h 24h 0.0 0.5 1.0 1.5 2.0 pERK1/2 relative protein level ** BIO103 10µM CTL 1h 2h 5h 24h 0 1 2 3 4 pP38 relative protein level BIO103 10µM * CTL 1h 2h 5h 24h 0 1 2 3 pJNK relative protein level BIO103 10µM CTL 1h 2h 5h 24h 0 2 4 6 8 10 pAMPK Relative protein level **** **** *** ** BIO103 10µM CTL 1h 2h 5h 24h 0 1 2 3 4 5 pAMPK relative protein level **** **** ** * * BIO103 10µM CTL 1h 2h 5h 24h 0 1 2 3 4 pACC relative protein level ** *** * BIO103 10µM CTL BIO103 CTL BIO103 0 50 100 150 200 OCR (% ) 100% 130% *** 100% 140% ** Stress Basal CTL BIO103 CTL BIO103 0 50 100 150 ECAR (%) Basal Stress 100% 108% 100% 108% Adult O ld Old+BIO103 0.3 0.4 0.5 0.6 0.7 Vm ax (m /s) *** * Adult O ld Old+BIO103 0 100 200 300 IGF (ng/m l) ** * Placebo BIO103 0 2 4 6 8 pAMPK Relative protein level Placebo BIO103 0 1 2 3 4 pAMPK Relative protein level Placebo BIO103 0.0 0.5 1.0 1.5 100% 67% * 2 - CT
Transcript of RWXEH GLDPHWHUV - Biophytis€¦ · treatment), the animals were tested for functional activity in...
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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