138 ANTI-AGEING


bridge and sliding (Fig 1) In vivo, Munapsys (now referred to as ‘the botulinum toxin-like peptide’) obtained similar or even better results at a lower concentration than a combination of benchmarks covering both pathways.


Munc18-1/Syntaxin-1 binding assay The capacity of the botulinum toxin-like peptide to bind to syntaxin competing for the Munc18-1 binding position was assessed in tubo.


Munc18-1 was bound to a Nickel-coated 96-well plate and incubated for 90 minutes. Syntaxin-1 with or without being previously pre-incubated with the botulinum toxin-like peptide was added. The amount of binding of Syntaxin-1 to Munc18-1 was evaluated through an ELISA assay.


The botulinum toxin-like peptide bound to syntaxin preventing its interaction with Munc18-1 in a dose-dependent manner, which should modulate SNARE complex assembly. Munc18-1/syntaxin binding was significantly reduced by 37% with 0.8 mg/mL of peptide (p<0.001 vs SNARE proteins).


Pre-synaptic neurotransmitter release The effect of the botulinum toxin-like peptide on modulating ACh release in the differentiated human neuroblastoma cell line LAN-2 was evaluated compared to a benchmark (Acetyl-Hexapeptide-8). After the treatments, a depolarisation was induced with KCl to stimulate ACh release which was measured with Amplex Red Acetylcholine Assay Kit.


The botulinum toxin-like peptide


decreased ACh release by 35% with respect to control, and by 12% versus 0.01 mg/mL benchmark with 0.001 mg/mL of peptide. Hence, there was less neurotransmitter available to reach the clustered AChR and activate the post-synaptic pathway.


Post-synaptic gene expression modulation


Primary human skeletal myoblasts differentiated into mature myocytes were


Roughness Improvement (14 days, %)


 Placebo  3% MunapsysTM  3% MunapsysTM


-7% vs placebo


-4% -11% #


* ** 67% vol Average


roughness (Ra)


-10% vs placebo 72% vol


Average relief (Rz)


PERSONAL CARE EUROPE 67% vol


Max relief height (Rt)


Figure 4: Roughness improvement in a panel of volunteers that tested 3% Munapsys and a placebo.


-6% -12% *


** * ** (average) (responder)


-7% -14% *


*


Human neuron-muscle contraction Human motor neurons derived from fibroblast cells reprogrammed into pluripotent (hiPS) cells and human muscle cells were co-cultured to allow to form NMJ. Spontaneous contractions were observed after 5 days. The botulinum toxin- like peptide was added and let incubate for 24 hours. Then it was washed out. Movies of co-cultures were recorded before and after the incubation and after the removal of the peptide. Acetyl-Hexapeptide-8 was used as a benchmark and α-bungarotoxin


April 2019 Modulation of muscle contraction  Basal  0.1mg/ml MunapsysTM 100% 100 -18% vs benchmark 75 50 35% 25 18% 18% 0 T0 1% T30 min 3% 17% T2h 10% 0% T24h Recovery 1 Benchmark: Acetyl Hexapeptide-8


Figure 3: Frequency of contraction in a co-culture of human motor neurons and human muscle cells compared with a benchmark.


treated with the botulinum toxin-like peptide. The RNA was extracted, purified and quantified. Then, it was retrotranscripted to cDNA and it was measured by RT-qPCR (Real Time Quantitative Polymerase Chain Reaction). The botulinum toxin-like peptide down-


regulated the gene expression of LRP4, Rapsyn and Utrophin by 1.3-fold change, which are involved in the formation and stabilisation of the AChR clustering. Moreover, the botulinum toxin-like peptide down-regulated the expression of Sodium Voltage-Gated Channel Alpha Subunit 3 (SCN3A, -1.3-fold) gene, modulating the Na+


influx that creates the


depolarisation. It also regulated genes related with Ca2+


mobilisation and


availability such as Calcium Voltage-Gated Channel Auxiliary Subunit Beta 1 (CACNB1, -1.6-fold), which may decrease the Ca2+ released to the sarcoplasm, and the Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase (ATP2A, +2.0-fold), which may enhance the encoding of the Ca2+ ATPase SERCA which returns the Ca2+ sarcoplasmic reticulum relaxing the muscle.


- to the In addition, the peptide down-regulated


troponin (TNNC1, 1.3-fold), actin (ACTA1, 1.4-fold) and myosin (MYH1, 2.1-fold) genes, which could lead to a modulation of the actomyosin cross-bridge and sliding, and hence muscle contraction.


Calcium mobilisation in the sarcoplasm


A depolarisation was induced to mature skeletal myocytes treated with the botulinum toxin-like peptide and the released calcium was measured by fluorescence.


The botulinum toxin-like peptide significantly reduced Ca2+


mobilisation


dose-dependently, reaching a diminution of 30% with 0.1 mg/mL of peptide (p<0.001 vs basal).


Myosin quantification Primary human skeletal myoblasts differentiated into mature skeletal myocytes were treated with the botulinum toxin-like peptide. Myosin was determined by immunofluorescence. The levels of myosin were significantly and dose-dependently lowered by the botulinum toxin-like peptide (Fig 2), reaching a reduction of 38% when treated at 0.5 mg/mL (p<0.01 vs basal).


6% 45% 35% 100% 100% 100%


 0.5 mg/ml Benchmark1  1µm α-bunarotox


100% 100%


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