A NEW STRATEGY TO ESCAPE FROM EXPRESSION LINES V.0
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1 A NEW STRATEGY TO ESCAPE FROM EXPRESSION LINES V.0
2 WRINKLES: THE HALLMARK OF AGING The face is the most visible part of our body and increased wrinkling is one of the most striking signs of aging Repeated facial movements caused by the contraction of the muscles of facial expression originate EXPRESSION WRINKLES Wrinkles are oriented according to the forces imposed by facial muscles. The dynamic expression lines progressively become permanent p The Neuromuscular Junction is essential for muscle contraction to take place
3 THE NEUROMUSCULAR JUNCTION THE NEUROMUSCULAR JUNCTION (NMJ) IS A SYNAPSE THAT ACTS AS A SPECIALISED CONNECTION BETWEEN A MOTOR NEURON AND A MUSCLE FIBER Effective SYNAPTIC TRANSMISSION Regulated neurotransmitter (ACh) exocytosis in the pre-synaptic membrane Receptors in the postsynaptic membrane Activation of receptors by neurotransmitters results in electrical activation and MUSCLE CONTRACTION
4 TARGETING MUSCLE CONTRACTION: TWO MAIN STRATEGIES PRE-SYNAPTIC STRATEGY: restrain or inhibit the release of the neurotransmitter (acetylcholine) from the motor neuron SNAP-8 SNAP-7 ARGIRELINE LEUPHASYL Botulinum Toxin NEUROMUSCULAR JUNCTION POST-SYNAPTIC STRATEGY: avoid or diminish the ligand (ACh) receptor (AChR) binding responsible for the formation of the action potential
5 AChR CLUSTERING IN THE POST-SYNAPTIC MEMBRANE Is a hallmark of the NMJ Ensures reliable communication from motor neurons to muscle fibres Is critical for synaptic function Is required to generate a synaptic potential of sufficient magnitude to initiate an action potential in the myofibre Is regulated by the agrin/musk pathway
6 THE AGRIN/MuSK PATHWAY The post-synaptic process of AChR WHO IS WHO? MuSK: a receptor tyrosine kinase essential for a functional post-synaptic apparatus clustering requires these actors Rapsyn: presents MuSK activated kinase to the AChRs RATL: associates with rapsyn and MuSK agrin MuSK ACh Agrin: activates MuSK and orchestrates the post-synaptic ti function MASC: a coreceptor for agrin to activate MuSK ACh: the neurotransmitter
7 MUSCLE CONTRACTION Nerve stimulation Release of ACh and other factors (like agrin) from the presynaptic terminal to the NMJ a b Agrin binds with MuSK at the post-synaptic membrane MuSK activation Agrin-MASC-MuSK-RATL-rapsyn cascade triggers AChR clustering c d AChR cluster formation
8 MUSCLE CONTRACTION d e ACh released from the nerve reaches the AChR cluster in the post-synaptic apparatus ACh binds with AChR A synaptic potential of sufficient magnitude is created MUSCLE CONTRACTION The action potential propagates through the muscle Contraction The modulation of muscle contraction is the most reliable approach to fight the unpleasant feature of expression wrinkles FORMATION OF EXPRESSION WRINKLES
9 A NOVEL POST-SYNAPTIC APPROACH INYLINE Is a new Molecular Cosmetics hexapeptide Has been specifically designed to avoid agrin binding to MuSK and block the agrin/musk pathway Targets muscle contraction using a post-synaptic strategy Is anovel active ingredient for the cosmetic treatmentt t of expression wrinkles
10 A NOVEL POST-SYNAPTIC APPROACH: AGRIN/MuSK PATHWAY BLOCKAGE If INYLINE avoids agrin binding to MuSK, two events are expected: No agrin-mediated activation of MuSK No stimulation of AChR clustering No triggering of contraction signal along the NMJ No beta-achr subunit phosphorylation No functional AChR clustering Anti-wrinkle i effect Anti-wrinkle effect Agrin/MuSK pathway blockage turns out in muscle relaxation
11 A NOVEL POST-SYNAPTIC APPROACH: MECHANISM OF ACTION Agrin/MuSK pathway Agrin/MuSK pathway blockage by INYLINE MUSCLE CONTRACTION MUSCLE RELAXATION MODULATION OF MuSK ACTIVATION LEADS TO MODULATION OF MUSCLE CONTRACTION
12 PEPTIDE DESIGN: MOLECULAR MODELLING BIOLOGICAL ACTIVITY IS DEPENDENT ON THE 3-DIMENSIONAL PLACEMENT OF SPECIFIC FUNCTIONAL GROUPS OR DOMAINS INYLINE : Has been specifically designed using the molecular modelling technique Was modelled according to the predicted structure of agrin s binding domain of MuSK s tyrosine kinase domain Such a specific peptide design allows specific agrin/musk pathway blockage and modulation of muscle contraction Attenuation of expression wrinkles
13 EFFICACY IN VITRO: INHIBITION OF AChR CLUSTERING Efficacy of INYLINE was evaluated in an assay of competition with the C-terminal fragment of agrin for the induction of the aggregation of AChRs in cultured differentitated t d myoblasts Differentiation of myotubes was induced Treatments: INYLINE (10 µm, 100 µm) / positive control (LiCl) / negative control (NaCl) Incubation with agrin Treatment t with α-bungarotoxin b t i (AChR irreversibleibl antagonist) t) conjugated to tetramethylrhodamine (fluorophore) Examination of the treated myoblasts on a fluorescence microscope Pictures were taken and values were calculated and normalised regarding the negative control
14 EFFICACY IN VITRO: INHIBITION OF AChR CLUSTERING INYLINE highly reduces AChR clustering induced by agrin The inhibition induced by INYLINE 100 µm is comparable to that of the positive control (LiCl) NaCl LiCl INYLINE 10 µm INYLINE 100 µm Negative control Positive control This in vitro assay undoubtedly proves that INYLINE specifically avoids AChR clustering, a key step in the post-synaptic functionality of the NMJ Negative control: NaCl 50 mm Positive control: LiCl 50 mm
15 EFFICACY IN VITRO: INHIBITION OF AChR CLUSTERING INYLINE proved to be able to reduce the formation of AChR clusters induced by the addition of the C-terminal region of agrin with a doseresponse efficacy
16 EFFICACY IN VIVO: ANTI-WRINKLE ACTIVITY An anti-wrinkle efficacy test was conducted on a panel of 20 female volunteers aged Volunteers applied a cream containing i % 0025% INYLINE (equivalent to 5% of INYLINE SOLUTION) on the test area (crow s feet) twice daily during 28 days Wrinkle depth was measured using PRIMOS 3D technique Direct in vivo measurements were taken before the treatment and after 28 days Images taken were processed to obtain the data. Statistical analysis of the data yielded the final results
17 EFFICACY IN VIVO: ANTI-WRINKLE ACTIVITY INYLINE significantly decreased wrinkle depth: mean decrease of 14.9% after 28 days 0 days % 28 days Volunteer 13
18 CONCLUSIONS Is a molecular cosmetics active ingredient specifically designed by molecular modelling to target the agrin/musk post-synaptic pathway, a novel approach in the cosmetic treatment of expression wrinkles Acts as a competitive antagonist of MuSK at the agrin binding site and its mechanism of action causes modulation of muscle contraction leading to muscle relaxation Markedly reduces the formation of AChR clusters in vitro with a dose-response efficacy thus preventing ACh from triggering the contraction signal Significantly decreased wrinkle depth in vivo, which demonstrates its ability to attenuate expression wrinkles Disclaimer: While the claims and supporting data provided in this publication are believed to be reliable and they are presented free and for guidance only, there are no warranties of any kind. All expressed and implied warranties are disclaimed. The recipient is solely responsible for ensuring that products marketed to consumers comply with all relevant laws and regulations. LIPOTEC is the exclusive holder of the both industrial and intellectual property rights identified herein. Recipient of this publication agrees to indemnify and hold harmless each entity of the LIPOTEC organization for any and all regulatory action arising from recipient s use of any claims or information in this publication, including, but not limited to, use in advertising and finished product label claims, and not present this publication as evidence of finished product claim substantiation to any regulatory authority.
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