PENTAPHARM SYN®°-AKE Substantiation File OSM Nutritional Products Ltd. Branch Pentapharm, Engelgasse 109, P.O.Box, CH-4002 Basel/Switzerland Phone: +41 61 706 48 48, Fax: +41 61 319 96 19, wnww.pentapharm.comSubstantiation File The following study reports are parts of this document and demonstrate the efficacy of SYN®-AKE: 1. Biological activity of SYN®-AKE on the contraction frequency of muscle cells cocultured with spinal cord explant The aim of this study was to analyze, in a coculture of muscle cells and spinal cord explants, the effects of SYN®-AKE on the frequency contraction of muscle cells after 1 minute, 2 hours, 48 hours and 96 hours of incubation. 2. _ Biometrological evaluation of the anti-wrinkle properties of SYN®-AKE The aim of this study was to determine, on volunteers, the anti-wrinkle effect of three tested products after 28 days of twice-daily use. > Results of the cream containing SYN®-AKE: A significant anti-wrinkle effect was measured on the “horizontal wrinkles’ of the forehead. Moreover, an improvement of the cutaneous relief of the crow’s feet was. observed for a majority of volunteers. Conclusion SYN®-AKE peptide reduces muscle cell contraction. Its action is reversible, SYN®-AKE, up to 52% decrease in wrinkle size (measured on the forehead). SYN®-AKE, anti-wrinkle effect measured on 73% of the volunteers. SYN®-AKE, smoothing effect measured on 80% of the volunteers. 5705 bwayue|dxe puod jeulds uyM peinyjno09 s]]@9 ajosnw Jo Aouenbe) uoioe11U09 84} UO FYV-gNAS Jo AuAnoe jeoibojoig *|BlOalternatives Proposal n°: RS040801 Study n°: RSO40801V2 BIOLOGICAL ACTIVITY OF COMPOUND 184-37B ON THE CONTRACTION FREQUENCY OF MUSCLE CELLS COCULTURED WITH SPINAL CORD EXPLANT Botox-like effect Study promoter: PENTAPHARM Dr Dominik IMFELD Engelgasse 109 4002 BASEL SWITZERLAND Téléphone: (41) 61 706 48 77 Télécopie: (41) 61 719 96 19 E-mail: dominik.imfeld@pentapharm.com, date: December 31°, 2004Study n *RSO40801v2 Biological activity of compound 184-37B on the contraction frequency of muscle cells PENTAPHARM ‘cocultured with spinal cord explant. Botox-like effect BlOatternatives AVL The investigators and the author of this report hereby certify the validity of the data presented and attest their full agreement with the conclusions presented at the end of the report. Certified by: Name: R. STEINSCHNEIDER Position: Manager, pharmaceutical division Date: December 31°, 2004 Signature BlOalternatives‘Suidy n °RSO40801v2 Biological activity of compound 184-37B on the contraction frequency of muscle cells PENTAPHARM. cocultured with spinal cord explant. Botox-like effect BlOalternatives 2 2-MATERIALS AND METHODS... 2.1. BIOLOGICAL MODEL. 2.1.1 CYTOTOXICITY. 2.1.2. ASSAY. 2.2, TEST COMPOUND: 23. CYTOTOXICITY... suns 2.4. COCULTURE OF HUMAN MUSCLE CELLS AND RAT SPINAL CORD EXPLANTS... 2.5, CULTURE TREATMENT AND ANALYSIS OF CONTRACTION FREQ 3 RESULTS AND CONCLUSIO’ 3.1. PRELI YTOXICITY DETERMINATION 10. 3.2, MODULATION OF FIBER MUSCLE. NCY CONTRACTION 3.2.1 CONTROL MEDIUM ... 3.2.2 CARISOPRODOL 3.2.5 COMPOUND 184-37B. 4- EK AND TABLI BlOalternativesStudy n °RS040801v2 Biological activity of compound 184-37B on the contraction frequency of muscle cells. PENTAPHARM ‘cocultured with spinal cord explant. Botox-like effect BlOalternatives 3/1 1 INTRODUCTION PENTAPHARM has the compound “184-37B” that could have Botox-like effect. ‘The aim of this study was to analyze, in a coculture of muscle cells and spinal cord explants, the effects of these compounds on the frequency contraction of muscle cells after 1 minute, 2 hours, 48 hours and 96 hours of incubation, Ina first culture of human muscle cells, the cytotoxicity of the products was evaluated in order to determine the nontoxic maximum concentration to use for the continuation of the tests. 2 - MATERIALS AND METHODS 2.1. Biological model 2.1.1 Cytotoxicity - Type: Normal human muscle cells (myoblastes : Mla, 3“ passage). = Culture medium: Mix of 2/3 MEM (Invitrogen 21090-022) and 1/3 M199 (nvitrogen 31153-026) L-glutamine 2mM (Invitrogen 25050024) Penicillin SO Ubml- Streptomycin S0 ue/m! (Invitrogen 15070063) Feral calf serum 5 % (W/, Invitrogen 10270098) = Culture : 37° Cand 5% CO2 2,12. Assay - Type: ‘Normal human muscle cells (myoblastes : Mla, 3" passage). Explants of 13-day-old rat embryo spinal cord with dorsal root ganglia attached = Culture medium: Mix of 2/3 MEM (Invitrogen 21090-022) and 1/3 M199 (Invitrogen 31153-026) L-glutamine 2mM (Invitrogen 25030024) Penicillin $0 UL'ml- Streptomycin 50 ig/ml (Invitrogen 15070063) Fetal calf serum 5 % (viv, Invitrogen 10270098) - Culture: 37° C and 5% CO2 2.2. Test compounds, reference Test compounds | Stock solution Dilution Final tested concentrations 184-378 | Powder supplied by the study |In culture [0.5 mM and 0.1 mM (RS040801/3, | promoter and stored at room | medium MW 496) | temperature. Solution ‘prepared at 10 mM in culture ‘medium. BlOalternativesStudy n °RS040801v2 Biological activity of compound 184-37B on the contraction frequency of muscle cells PENTAPHARM cocultured with spinal cord explant. Botox-like effect BlOalternatives ai Reference | Stock-solution Dilution |~ Final tested concentration Carisoprodol _|1 Min ethanol in culture ‘TmM and 0.1 mM (Sigma C-8759) medism 2.3. Cytotoxicity Preliminary cytotoxicity assay was performed by the MTT assay for each compound to determine the non-cytotoxic limit of the compound to be assayed. - plates: 96-well plates - pre-culture: Rh - cells/well: 4700 ~ tested concentrations: 8 (see table 1) - replicates: 6 - cellsicompound contact: 48.h - Evaluation parameter: MIT hydrolysis assay 2.4, Coculture of human muscle cells and rat spinal cord explants Human muscle cultures were cultured in gelatin-coated well plates. After fusion of muscle cells showing myo-fibers without contractile activity, one 13-day-old rat embryo spinal cord explant with dorsal root ganglia attached was placed on each muscle monolayer. After one day of co-culture, neurites were seen growing out of the explant. Reaching their target, they made contact with the myotubes and induced the first contractions after 5 days. After 3 weeks in co-culture, innervated muscle fibers became cross-striated, had well-differentiated neuromuscular junctions and displayed other criteria such as biochemical and pharmacological maturation, ‘The model was used after 21 days of co-culture when the muscle fibers had a high level of mature neuromuscular junctions. 2.5. Culture treatment and analysis of contraction frequency Cultures were observed with an inverted microscope (Nikon Diaphot 300) equipped with a camera (DMX 1200 Nikon) for video sequence record and with a motor stage driven by analysis software (Lucia 6.0). In this way, the position of noticeable fields can be recorded and fields can be automatically positioned under lens. For each experimental point, one well with continuous contraction frequency muscle fiber ‘was selected and the contraction frequency was counted for 30 seconds. The tested product ‘was then added and the contraction frequency was counted for 30 seconds after | minute and 2 hours. BlOalternativesStudy n *RSO40801v2 Biological activity of compound 184-37B on the contraction frequency of muscle cells PENTAPHARM ‘cocultured with spinal cord explant. Botox-like effect BlOalternatives SUL After 48 and 96 hours of compound incubation, a visual analysis of cell culture was made in order to verify cell viability. To eliminate a possible lack of glucose in the medium, a concentrated glucose solution (5 g/l) containing the tested compound was added to the ‘medium with a final glucose concentration of 1g/L. This solution was made at the start of the incubation and was kept at 37°C in the incubator in other to have the same condition as that of the compound in supernatant culture. In such conditions the contraction frequency was counted for 30 seconds. After 96 hours of product incubation, only for the blocked muscle fibers, supernatant was changed by control medium and incubated during 48 h. At the end of this last incubation and after recharge of glucose, the contraction frequency was counted for 30 seconds, The results are given as a number of fiber contractions during 30 seconds and as a percentage of the contraction frequency compared to the contraction frequency before incubation. To analyze the results, the following parameters were considered: - A 25 % decrease in contraction frequency is not significant - A decrease of frequency contraction comprise of 25% and 75 % is classified as a slowing down - A decrease higher than 75 % is considered as a blockage of contraction. Results are illustrated in a representative video recording of muscle contraction (joined CD- Rom). BlOatternatives