SKIN MICROBIOME 71


In vitro 1: Characterisation of the bacterial in situ postbiotic To better understand the mechanism of action of the microbial in situ postbiotic, screening analyses have been performed on the bacterial SuperNatant (bSN). The composition of those bSN was evaluated with the aim to link this specific metabolic profile with this activation of ‘pleasure’ neurochemicals. The active was added to separate


cultures of skin commensal microbiota (Cutibacterium acnes and Staphylococcus epidermidis). The resulting bacterial culture supernatants were combined in a 1:1 ratio (bSN) (Fig 2). The bacterial supernatant (bSN) treated


with the active produced less total proteolytic content and biofilm forming exopolysaccharides and had lower antioxidant activity (data not shown). Compared to placebo, the results of the


postbiotic generated after the application of Kannabia SensePLF


were statistically significant


by achieving the modulation of microbial stress and virulence related metabolites. Thanks to these positive environmental conditions, the consequently secreted in situpostbiotic will be full of balanced healthy and beneficial molecules for our skin.


In vitro 2: Analysis of the microbiota-skin- brain axis networks A series of sophisticated and originally designed studies were performed in order to evaluate the active’s ability to activate directly and/or indirectly the neurochemical networks for ‘feeling good’ through the microbiota- skin-brain axis (Fig 2). The active was added to separate cultures


of commensal microbiota (Cutibacterium acnes& Staphylococcus epidermidis).1


The


resulting bacterial culture supernatants were combined in a 1:1 ratio (bSN)2


and added to


a human keratinocyte culture (NHEK).3 Oxytocin levels produced by the NHEK were analysed and compared. This way we can study if the active stimulates the microbiome to produce less cellular stress-related markers, and then if the bSN (low in cellular stress markers) can stimulate the NHEK to synthesise more relaxing molecules, such as oxytocin.4 After that, the supernatant of the treated


NHEK (kSN) was added to sensory neurons and the levels of oxytocin were analysed.5 The production levels of oxytocin of untreated sensory neurons were compared with those treated with the active and with those treated with bSN or with kSN.6 In the 6 steps of this cell-to-cell


communication chain (active ingredient , microbiota, keratinocytes, sensory neurons), the effect of the active was compared with that of the supernatant/s in order to analyse the direct effect at the same time as the indirect activity:


November 2020 log(r=0) Poor outcome (mRS≤2) Figure 6: 3D brain model and correlation coefficient matrices. The oxytocin levels were analysed in


both keratinocytes and sensory neurons after applying the active: l Oxytocin levels in Keratinocytes (NHEK - Step 4): The active increased the oxytocin synthesis on NHEK by up to 1.5-fold versus untreated control and the bacterial supernatant (bSN) increased even more the oxytocin synthesis on NHEK, by up to 3-fold versus control (Fig 3). The active directly activates the oxytocin


production in keratinocytes, but its effects were higher by activating it indirectly through skin microbiome stimulation. The bSN indirect effect was significantly


higher than the direct effect of the active: l Oxytocin levels in sensory neurons (Step 6): The active increased the oxytocin synthesis on sensory neurons by up to 9-fold versus untreated control. The keratinocytes’ supernatant also increased the oxytocin synthesis on sensory neurons NHEK, by up to 8-fold versus control (Fig 4). Therefore, the active directly activates


the oxytocin production in sensory neurons, and indirectly through skin microbiota- mediated keratinocyte stimulation. The active centres its mechanism of


action in activating the cascade of reactions from the skin microbiota to the brain, even having a direct effect.


In vivo efficacy The clinical evaluation was a solid scientific argument to demonstrate the efficacy of the active on several clinical trials performed.


In vivo 1: Evaluation of skin well-ageing effect The first in vivo test was performed on a 40- volunteer panel aged between 46 and 64 years old. The study was a double blind intra-individual vs placebo, with 2 daily applications for 28 days.


l Mood wrinkles (marionette & frown lines): The anti-wrinkle effect of the active was evaluated on a subgroup of 30 volunteers by Bio3D Structured-light Scanner, a refined 3D digitalising system developed by Bionos Biotech, S.L. This unique software is based on structured light projection which uses 290 pictures per second to prevent movement effects and allow very high- resolution images. The measured parameters were area


and length of the so-called emotional wrinkles after 28 days of treatment (Fig 5). The results showed a reduction of up to


44% and 28% in both area and length of the wrinkles in marionette lines and up to 27% and 32% of both area and length of wrinkles in frown lines at 1% dosage. l Skin hydration Compared to placebo, the application of a cream containing 1% dosage of the active increased the skin hydration levels of the 40 volunteers at 8% more than at the beginning of the study and by 1.7-fold at 28 days of treatment. l Skin radiance Finally, there was another 30-volunteer group who applied a cream containing 2% active, being demonstrated that their skin gloss and radiance increased by 1.2-fold versus placebo at 28 days and up to 50% in glow intensity versus initial time.


In vivo 2: Evaluation of the emotional modulation For this in vivo assay, there was a panel compound of 30 volunteers (46 -69 years old) who were applied a 2% active dosage in a double-blind study vs placebo on 2 daily applications. l Analysis of the brain activation by functional Magnetic Resonance Imaging (fMRI) In order to test the efficacy of the active,


PERSONAL CARE ASIA PACIFIC log(r=1) ROI-TO-ROI effect -4.69 4.69


log(r=0) Good outcome (mRS≤2) log(r=1)


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