NeuroScientific Biopharmaceuticals Ltd. noted that the following announcement serves as a clarification to the previously released announcement on the 15 June 2022, to provide further information regarding the specifics of the study mentioned within the announcement. The objective of the study was to assess the effect of EmtinBTM in the myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE) mouse model of Multiple sclerosis across a dose range (5mg/kg, 10mg/kg, 20mg/kg and 40mg/kg) during a treatment period of 30-days (Table 1). The results from this study will be used to inform the selection of the two most effective doses for a larger study to be undertaken during the 2H 2022. The control group involved MOG-EAE mice that were not treated with EmtinBTM. These mice received a solution (vehicle) that was identical to formulated EmtinBTM except that it did not contain any EmtinBTM active pharmaceutical ingredient. The endpoints of the study included the following (a) The daily assessment of clinical symptoms for the duration of the study (30 days) using a standardised scoring system (Table 2). Higher scores mean more severe symptoms. (b) Analysis of concentrations of neurofilament light polypeptide (NfL) in cerebral spinal fluid (CSF) and plasma at the terminal time point following the 30-day treatment period. (c) Quantitative assessment of the expression of myelin basic protein (detected using the antibody MBP) in tissue sections from spinal cords at the terminal time point following the 30-day treatment period (d) Quantitative assessment of activated T cells (detected using the antibody CD3) in tissue sections from spinal cords at the terminal time point following the 30-day treatment period. (e) Quantitative assessment of microglia and macrophages (detected using the antibody Iba-1) in tissue sections from spinal cords at the terminal time point following the 30-day treatment period. (f) Quantitative assessment of astrocytes (detected using the antibody GFAP) in tissue sections from spinal cords at the terminal time point following the 30-day treatment period. Mice treated with 10mg/kg and 20mg/kg EmtinBTM consistently achieved lower clinical scores, indicating reduced disease severity, from the onset of symptoms and through to the peak of the disease in comparison to the untreated controls. The clinical scores for mice treated with 5mg/kg and 40mg/kg EmtinBTM exhibited more variability and were considere less reliable in comparison to 10mg/kg and 20mg/kg EmtinBTM dose groups. Neurofilament light polypeptide (NfL) is a biomarker associated with damaged neurons that can be detected in CSF and blood plasma samples. Mice treated with 5mg/kg, 10mg/kg, 20mg, and 40mg/kg EmtinBTM had lower concentrations of NfL in cerebral spinal fluid (CSF) and plasma samples in comparison to the untreated control group at the conclusion of the 30-day treatment period. Myelin is important for the efficient function of nerve cells. The destruction of myelin contributes to the onset of neurological dysfunction associated with MS. Mice treated with 10mg/kg and 20mg/kg EmtinBTM consistently exhibited higher levels of myelin in comparison to the untreated control group. Results were more variable for mice treated with 5mg/kg and 40mg/kg of EmtinBTM and considered less reliable. The study assessed a number of markers associated with chronic inflammatory responses of MS. Fundamental to the dysfunctional immune responses of MS in humans, activated T cells (CD3) penetrate the blood brain barrier and stimulate inflammatory responses of the CNS,
activating resident immune defence cells such as microglia and macrophages (Iba-1), and astrocytes (GFAP). 1 Mice treated with 5mg/kg, 10mg/kg, 20mg/kg and 40mg/kg EmtinBTM exhibited lower levels of CD3 expressing T cells in comparison to the untreated control group following the 30-day treatment period (Figure 1A). Mice treated with 5mg/kg, 10mg/kg, 20mg/kg and 40mg/kg EmtinBTM exhibited lower Iba-1 expression in tissue samples, indicating less microglia and macrophages, in comparison to the untreated control group following the 30-day treatment period (Figure 1B). Mice treated with 5mg/kg, 10mg/kg, 20mg/kg and 40mg/kg EmtinBTM exhibited lower GFAP expression in tissue samples, indicating less activated astrocytes, in comparison to the untreated control group following the 30-day treatment period (Figure 1C).