<p class="ADMETabstracttext">Peptide therapeutics are new modalities offering several challenges to drug discovery. They are generally less stable and permeable in vivo. The characterization of their lipophilicity cannot be carried out using the traditional in silico or wet octanol/water partition coefficients. The prediction of their in vivo distribution and permeability is also challenging. In this paper, it is demonstrated that the biomimetic properties such as lipophilicity, protein and phospholipid binding can be easily assessed by HPLC using chemically bonded protein and immobilized artificial membrane (IAM) stationary phases. The obtained properties for a set of potential therapeutic peptides with 3 to 33 amino acids have been analysed and it was found that similar characteristics of the properties could be observed as for small molecule drugs. The albumin binding showed correlation with their measured lipophilicity on the C-18 stationary phase with acidic peptides showing stronger than expected albumin binding. The (IAM) chromatography revealed peptide membrane affinity, which was stronger for positively charged peptides (containing arginine) and showed correlation to the alpha-1-acid glycoprotein (AGP) binding, which was also stronger for positively charged compounds. The in vivo volume of distribution and drug efficiency of the peptides have been estimated using the models developed for small molecules. One of the candidate linear peptides has been assessed in various cellular and in vivo assays and the results have confirmed the estimated cell partition and brain to plasma ratio. It can be demonstrated, that up to 21 amino acids, the peaks of the peptides obtained on the protein phase were symmetrical and narrow. The interaction of larger peptides with the protein stationary phases resulted in wide peaks showing multiple equilibrium processes with slow kinetics during chromatography. The larger peptides showed narrow and symmetrical peaks on the IAM column enabling the quantification of peptide - cell membrane interactions.</p>

Motoneuronotrophic factor (MNTF) is an endogenous neurotrophin that is highly specific for the human nervous system, and some of the observed effects of MNTF include motoneuron differentiation, maintenance, survival, and reinnervation of target muscles and organs. MNTF is a neuro-signaling molecule that binds to specific receptors. Using In Silico Analysis, one of the active sites of MNTF was identified as an analog of six amino acids (GM6). The effect of chemically synthesized GM6 on ischemic stroke was studied in the middle cerebral artery occlusion (MCAo) mouse model. Mice were subjected to 1 hour of ischemia followed by 24 hours of reperfusion. Mice were injected intravenously with a bolus of GM6, at various doses (1 and 5 mg/kg) immediately after the start of reperfusion and examined for changes in physiological parameters, neurological deficits and infarct volume. GM6 was able to penetrate the blood brain barrier, and at both 1 and 5 mg/kg showed a significant protection from infarct damage, which translated to improvement of neurological deficits. Administration of GM6 demonstrated no changes in HR, BP, pO2, pCO2, or pH. A significant increase over the control group in CBF after reperfusion was observed with GM6 administration, which helped to mitigate the ischemic effect caused by the blockage of blood flow. The time window of treatment was assessed at various times following cerebral ischemia with GM6 demonstrating a significant protective effect up to 6–12 hours post ischemia. In addition, GM6 increased neurogenesis, and decreased apoptosis and inflammation in the mouse brain following cerebral ischemic injury. These data suggest that GM6 is neuroprotective to the brain following IV injection in the mouse model of MCAo.

<p class="ADMETabstracttext">Amyotrophic lateral sclerosis (ALS) is an idiopathic, fatal neurodegenerative disease of the human motor system. Subunits of the 33-amino acid containing motorneurontrophic factor (MNTF) have been investigated and GM6 has been found as a potential peptide therapeutic for ALS. This linear peptide drug candidate has been characterized by HPLC based physicochemical and biomimetic measurements to estimate its in vivo distribution behavior and to estimate its cell penetration and brain to plasma concentration ratio. The free tissue concentration vs time profile has been estimated using the measured physicochemical and biomimetic properties of the intact GM6 molecules and its microsomal stability. The in vitro and in vivo measurements supported the estimated in vivo distribution behavior of GM6.</p>

BackgroundAmyotrophic lateral sclerosis (ALS) is currently an incurable disease without highly effective pharmacological treatments. The peptide drug GM604 (GM6 or Alirinetide) was developed as a candidate ALS therapy, which has demonstrated safety and good drug-like properties with a favorable pharmacokinetic profile. GM6 is hypothesized to bolster neuron survival through the multi-target regulation of developmental pathways, but mechanisms of action are not fully understood.MethodsThis study used RNA-seq to evaluate transcriptome responses in SH-SY5Y neuroblastoma cells following GM6 treatment (6, 24 and 48 h).ResultsWe identified 2867 protein-coding genes with expression significantly altered by GM6 (FDR < 0.10). Early (6 h) responses included up-regulation of Notch and hedgehog signaling components, with increased expression of developmental genes mediating neurogenesis and axon growth. Prolonged GM6 treatment (24 and 48 h) altered the expression of genes contributing to cell adhesion and the extracellular matrix. GM6 further down-regulated the expression of genes associated with mitochondria, inflammatory responses, mRNA processing and chromatin organization. GM6-increased genes were located near GC-rich motifs interacting with C2H2 zinc finger transcription factors, whereas GM6-decreased genes were located near AT-rich motifs associated with helix-turn-helix homeodomain factors. Such motifs interacted with a diverse network of transcription factors encoded by GM6-regulated genes (STAT3, HOXD11, HES7, GLI1). We identified 77 ALS-associated genes with expression significantly altered by GM6 treatment (FDR < 0.10), which were known to function in neurogenesis, axon guidance and the intrinsic apoptosis pathway.ConclusionsOur findings support the hypothesis that GM6 acts through developmental-stage pathways to influence neuron survival. Gene expression responses were consistent with neurotrophic effects, ECM modulation, and activation of the Notch and hedgehog neurodevelopmental pathways. This multifaceted mechanism of action is unique among existing ALS drug candidates and may be applicable to multiple neurodegenerative diseases.Electronic supplementary materialThe online version of this article (10.1186/s40035-018-0135-7) contains supplementary material, which is available to authorized users.

Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that lacks effective treatment options. Genervon has discovered and developed GM604 (GM6) as a potential ALS therapy. GM6 has been modeled upon an insulin receptor tyrosine kinase binding motoneuronotrophic factor within the developing central nervous system. Methods This was a 2-center phase 2A, randomized, double-blind, placebo-controlled pilot trial with 12 definite ALS patients diagnosed within 2 years of disease onset. Patients received 6 doses of GM604 or placebo, administered as slow IV bolus injections (3x/week, 2 consecutive weeks). Objectives were to assess the safety and efficacy of GM604 based on ALSFRS-R, FVC and selected biomarkers (TDP-43, Tau and SOD1, pNFH). This report also includes results of compassionate treatment protocol GALS-C for an advanced ALS patient. Results Definite ALS patients were randomized to one of two treatment groups (GM604, n = 8; placebo, n = 4). 2 of 8 GM604-treated patients exhibited mild rash, but otherwise adverse event frequency was similar in treated and placebo groups. GM604 slowed functional decline (ALSFRS-R) when compared to a historical control (P = 0.005). At one study site, a statistically significant difference between treatment and control groups was found when comparing changes in respiratory function (FVC) between baseline and week 12 (P = 0.027). GM604 decreased plasma levels of key ALS biomarkers relative to the placebo group (TDP-43, P = 0.008; Tau, P = 0.037; SOD1, P = 0.009). The advanced ALS patient in compassionate treatment demonstrated improved speech, oral fluid consumption, mouth suction with GM604 treatment and biomarker improvements. Conclusions We observed favorable shifts in ALS biomarkers and improved functional measures during the Phase 2A study as well as in an advanced ALS patient. Although a larger trial is needed to confirm these findings, the present data are encouraging and support GM604 as an ALS drug candidate.