Summary of research evaluating GM6 Mechanisms of Action and Efficacy in ALS 

Executive Summary

Amyotrophic lateral sclerosis is an incurable disease characterized by motor neuron death leading to muscle paralysis. Genervon Biopharmaceuticals has developed a peptide drug (GM6) based upon a novel concept and approach to ALS treatment. In contrast to existing ALS drug candidates, GM6 does not selectively interact with a single receptor target, but rather interacts with multiple receptors linked to diverse pathways. These receptors include insulin receptor, Notch receptors 1 – 4, patched 1, and smoothened frizzled class receptor. Stimulation of these and other receptors by GM6 leads to activation of the insulin signaling cascade, Notch Intracellular domain and mitogen-activated protein kinase signaling. These signal transduction pathways converge upon a core network of transcription factors such as hes family bHLH transcription factor 7 (HES7), GLI family zinc finger 1 (GLI1), homeobox D11 (HOXD11), and signal transducer and activator of transcription 3 (STAT3). These transcription factors together modulate expression of thousands of genes. In neurons, GM6-regulated genes are associated with axon guidance, intrinsic apoptosis, microtubule stability, synaptic transmission and glutamate clearance. In T lymphocytes, GM6 appears to blunt expression of genes associated with acute phase responses, lymphocyte proliferation, leukocyte migration, and production of pro-inflammatory cytokines such as TNF and IL-1. Gene expression responses to GM6 are thus linked to both neurogenesis and anti-inflammatory effects in distinct cell types. From a pharmacological standpoint, we have demonstrated that GM6 has good drug-like properties and is able to cross the blood-brain barrier, with an estimated volume of distribution of 7.8 L/kg and good penetration into neuronal tissues. To demonstrate in vivo efficacy of GM6 as an ALS treatment, we evaluated effects in a well-validated mouse model (SOD1-G93A transgenic mice). GM6 increases survival in this model and confers dose-dependent improvements in physical performance measures of disease progression and biomarkers in CSF and spinal cord sections. To translate these findings, we performed a double-blind, randomized, placebo-controlled phase 2A clinical trial to evaluate effects of GM6 in ALS patients (NCT01854294). The phase 2A study confirmed safety of GM6 in humans, with no serious drug-related treatment-emergent adverse effects. We additionally observed favorable trends in GM6-treated patients, including slowed decline in forced vital capacity and ALS Functional Rating Scale, along with decreased plasma abundance of ALS biomarkers (e.g., TDP-43, Tau and SOD1). These findings have supported the hypothesis that GM6 favors neuron survival in ALS patients, slowing symptom progression and promoting a biomarker profile consistent with less severe disease. These studies collectively show that, from decades of research on GM6, we have reached a critical point for bench-to-bedside translation. A cellular mechanism of action for GM6 has emerged from preclinical studies, demonstrating unique effects not replicated by existing pharmacological treatments. This mechanism is bolstered by in vivo findings from a well-validated ALS mouse model and a phase 2A placebo-controlled clinical trial with ALS patients. This work has demonstrated proof-of-concept and the considerable promise offered by GM6 for treatment of this devastating disease.

(1) Introduction

Amyotrophic lateral sclerosis (ALS or “Lou Gehrig” disease) is an incurable disease of unknown etiology characterized by motor neuron death leading to muscle paralysis. The disease most commonly occurs between the ages of 50 and 70, and is twice as common in men compared to women. It is a unique disorder with deficits impacting both lower and upper motor neurons, although either lower or upper motor neuron dysfunction may be dominant for any one patient. There is no concomitant loss of sensory function, oculomotor capacity, or bowel/bladder control. The disease frequently presents with asymmetric limb weakness, muscular denervation, and progressive symmetric muscular atrophy, with fasciculations and progressive bulbar palsy characteristic of lower motor neuron dysfunction. These symptoms are accompanied by degeneration of lateral corticospinal tracts and anterior motor neurons within the spinal cord. Key manifestations of upper motor neuron dysfunction include pseudobulbar palsy and/or primary lateral sclerosis, including hyperreflexia, spasticity and pathologic reflexes. The disease is ultimately fatal and death is typically due to respiratory failure within 1 to 6 years.

Only two drugs have been approved by FDA for treating ALS in the United States, including riluzole (Rilutek/Teglutik) and edaravone (Radicava/Radicut). Riluzole acts by decreasing presynaptic glutamate release, whereas edaravone is an anti-oxidant and believed to prevent neuronal loss secondary to oxidative stress accumulation. Unfortunately, both treatments have only marginal benefits for ALS and provide only modest extension of survival. The mainstay and bulk of ALS treatment therefore has been to provide supportive measures (e.g., muscle relaxants) and patient education (e.g., speech therapy). Overall, more than 50 ALS randomized controlled trials have been performed in recent decades, but the majority of these have failed to provide evidence of efficacy for the investigational product. Several factors have contributed to these clinical failures, including a lack of adequate animal models, inability of drugs and cross the blood-brain barrier, and a paucity of quality disease biomarkers.

GM6 is a peptide drug consisting of 6 amino acids (Phe-Ser-Arg-Tyr-Ala-Arg) that has been developed by Genervon Biopharmaceuticals (Pasadena, CA). The drug is modeled upon an endogenous developmental-stage neurotrophic factor identified from the developing human nervous system (previously identified as MNTF1). Through 20 years of preclinical and clinical research, Genervon has characterized pro-survival and neurotrophic effects of GM6 within the central and peripheral nervous systems. This work has utilized a diverse range of model systems, including human studies, and has laid the groundwork for understanding cellular mechanisms by which GM6 may be effective as an ALS treatment.

The purpose of this document is to summarize mechanisms of action for GM6 as an ALS therapy, starting at the level of extracellular receptors engaged by GM6, with further characterization of signal transduction mechanisms linked to transcriptional responses. These downstream transcriptional responses have now been comprehensively characterized using genome-wide profiling technologies, and we have demonstrated that this response involves modulation of genes linked to key disease-associated processes, such as intrinsic apoptosis, microtubule stability, synaptic transmission, glutamate clearance, acute-phase inflammatory responses and cytokine production. Finally, this report describes how we have extended our foundational preclinical investigations to vertebrate systems, including studies of a validated ALS mouse model and a phase 2A clinical trial. The totality of these studies have demonstrated that GM6 has unique effects compared to existing ALS treatments, and have provided a “big picture” view of core mechanisms by which GM6 appears to act upon diverse cell types to influence disease progression.

(2) Receptors

Extracellular and intracellular receptors represent interaction points between a drug and individual target cells. These upstream pathway elements are the essential gatekeepers that ultimately control and initiate the cascade of biochemical effects leading to drug responses at the cellular level. GM6 was not designed to interact exclusively as an agonist or antagonist of any one receptor, but is instead hypothesized to act upon multiple receptors. We used enzyme fragment complementation (EFC) with β‐galactosidase assays to screen GPCR receptor libraries and have shown that GM6 has antagonist activity with respect to melanocortin 5 receptor (MC5R), neuropeptide FF receptor 1 (NPFFR1), opioid related nociceptin receptor 1 (OPRL1) and domatostatin receptor 2 (SSTR2). GM6 may also weakly antagonize cholinergic receptor muscarinic 4 (CHRM4), although this effect may be counterbalanced by up-regulation of CHRM4 following GM6 stimulation, which we have demonstrated in SH-SY5Y neuroblastoma cells. Using the same assays, we identified potential agonist activity with respect to adhesion G protein-coupled receptor B3 (ADGRB3/BAI3) and atypical chemokine receptor 3 (ACKR3/CXCR7).

The most post potent agonist activity of GM6 relates to insulin receptor alpha, for which GM6 acts as a positive allosteric modulator. The protein abundance of insulin receptor is also increased in GM6-stimulated cells, which we have confirmed through studies of multiple cell types, including human brain microvascular endothelial cells (HBMVECs), Ntera2 differentiated cells and GABAergic Neurons. We have identified additional receptors transcriptionally up-regulated by GM6 in SH-SY5Y neuroblastoma cells, including nerve growth factor receptor (NGFR) and fibroblast growth factor receptor like 1 (FGFRL1). GM6 further down-regulates the expression of key receptors mediating the inflammation response, such as TNF receptor superfamily member 19 (TNFRSF19), which may contribute to anti-inflammatory effects of the drug (discussed below). Finally, our gene expression profiling data have generated strong signals implicating the Notch and hedgehog signaling pathways, suggesting agonist activity with respect to Notch receptors (NOTCH1, 2, 3 and 4), patched 1 (PTCH1) and/or smoothened frizzled class receptor (SMO).

Collectively, these data have supported the hypothesis that, in contrast to many novel drug formulations, GM6 does not selectively interact with a single receptor target, but rather appears to interact with multiple receptors linked to a diverse set of pathways. These ligand-receptor interactions are further amplified by downstream transcriptional responses to GM6, which alter the abundance of additional receptors associated with neurogenesis and pro-survival responses.

(3) Signal Transduction

Signal transduction is the process by which extracellular signals are propagated intracellularly to activate signaling pathways that are in turn linked to downstream transcriptional responses. Our work has implicated a broad set of receptors mediating GM6 responses, and accordingly, we have identified a complex set of signaling pathways activated by GM6, which converge downstream to activate or inhibit core transcription factors.

Consistent with data demonstrating positive regulation of insulin receptor (IR), GM6-stimulated cells have increased phosphorylation of IR at tyrosine 972 in HBMVECs. This event likely indicates modulation of the insulin signaling cascade mediated by downstream proteins, such as Shc, PTB domain, IRS-1, PI3 kinase and SOCS. We have additionally observed decreased phosphorylation of the p85 regulatory subunit of phosphatidylinositide 3-kinase in Ntera2 differentiated cells. We expect that these signaling events would have downstream metabolic effects related to glucose uptake/release and the synthesis of carbohydrates, lipids and proteins, all of which may support neurogenesis and a broader pro-survival response. Other key proteins mediating GM6-directed signal transduction are likely to include Notch Intracellular domain (NICD), Deltex-1 (DTX1) and the mitogen-activated protein kinase (MAPK) cascade.

These findings have thus uncovered a diverse set of mediators linking extracellular signals to downstream cellular responses. These mediators transmit upstream signals and distribute effects of GM6 across a multi-pronged network of pathways, likely amplifying downstream responses and broadening the scope of biological processes involved in such responses. Our work has begun to characterize this chain of protein-protein interactions and phosphorylation events playing an essential role in cellular responses to GM6.

(4) Transcription Factors

Transcription factors are key convergence points for extracellular signaling pathways and play a critical role in coordinating a cell’s response to drugs or other stimuli. RNA-seq expression profiling in SH-SY5Y neuroblastoma cells has demonstrated that GM6 up-regulates genes are associated with C2H2 zinc finger transcription factors interacting with GC-rich motifs. In contrast, GM6 down-regulates genes associated with helix-turn-helix homeodomain transcription factors interacting with AT-rich motifs. Specific transcription factors up-regulated by GM6 include hes family bHLH transcription factor 7 (HES7), GLI family zinc finger 1 (GLI1), homeobox D11 (HOXD11), and signal transducer and activator of transcription 3 (STAT3). Up-regulation of HES7 and GLI1 expression by GM6 is consistent with Notch and hedgehog pathway activation, respectively. Moreover, DNA motifs known to interact with the HOXD11 and STAT3 transcription factors were enriched in sequence regions upstream of genes transcriptionally regulated by GM6. These findings have demonstrated that the gene expression response to GM6 is regulated by a core set of transcription factors. By applying genome-scale analyses such as RNA-seq we have begun to uncover the identify of transcription factors mediating responses to GM6, as well as the cis-regulatory codes linking these factors to specific DNA sequences.  

(5) Transcriptional response

We have identified 2867 protein-coding genes with expression significantly altered by GM6 (FDR < 0.10) in SH-SY5Y cells. A significant fraction of these genes are associated with neurogenesis and development, and we have further identified a subset of 77 GM6-regulated genes associated with ALS and linked to axon guidance and the intrinsic apoptosis pathway. Additional processes associated with ALS-associated genes up-regulated by GM6 include microtubule stability (TUBA4A, NEFL), synaptic transmission (STON1, CCL2, UNC13A, SLC1A1, VGF, ATXN1) and glutamate clearance (SLC1A1). Other genes down-regulated as part of the transcriptional response to GM6 have been associated with immunity, suggesting potential anti-inflammatory effects as well. Such expression responses include down-regulation of guanylate binding protein family member 6 (GBP6), CD4 molecule (CD4) and signal transducer and activator of transcription 4 (STAT4). To explore these anti-inflammatory responses further, we have extended our expression profiling analyses to include immune-associated cell types, such as jurkat T lymphocyte cells.

In jurkat T cells, genes down-regulated by GM6 were associated with acute-phase response proteins, immune response, inflammation and production of cytokines such as IL-1 and TNF. Proteins involved in the acute phase response are known to exhibit changes in serum concentration during inflammation. These proteins, such as C reactive protein, can be used to monitor systemic inflammation and liver is often a site of synthesis. Genes down-regulated by GM6 were associated with the acute-phase response (FN1, SERPINA1, FGA, AHSG, SERPINA3, SERPINF2, ORM1, F2). Notably, FGA encodes a component of fibrinogen, which is a coagulation factor whose abundance may correlate with erythrocyte sedimentation rate (ESR).

Genes down-regulated by GM6 in jurkat cells were additionally associated with immune system processes (FGA, APOA1, FGB, AMBP, FABP1, GPC3, APOA2, EGR1, DHRS2, ORM1, SP2, CYP27B1, NOTCH1, ZFAT), humoral immune response (FGA, FGB, NOTCH1) and regulation of inflammatory response (AHSG, A2M, C3, AGT). Decreased genes were also involved in triggering of immune responses and other downstream steps of immune reactions, such as response to lipopolysaccharide (APOB, NR1D1, CYP27B1, NOTCH1), response to bacterium (APOB, LYZ, FGA, FGB, PGC, NR1D1, CYP27B1, NOTCH1), regulation of immune effector process (RBP4, APOA1, FABP1, APOA2, PGC), positive regulation of lymphocyte proliferation (IGF2, JAK3, IRS2) and leukocyte migration (FN1, APOB, CD84, DBH, F2).

Cytokines are important mediators of immune and inflammatory reactions. Genes down-regulated by GM6 in the jurkat cell line were additionally linked to regulation of cytokine biosynthetic process (APOA2, TNFRSF8, THBS1), regulation of cytokine production (C3, AGT, SERPINF2, ORM1, CREBBP), response to interleukin-1 (FGB, EGR1, CREBBP), regulation of TNF production (TNFRSF8, ORM1, THBS1) and cellular response to TNF (APOB, APOA1, KRT8, KRT18). These findings demonstrate multiple gene expression responses in an immune-associated cell type (jurkat T lymphocytes) that may correspond to anti-inflammatory effects in vivo.

(6) Pharmacology

ALS is disease involving both upper and lower motor neurons, such that that a maximally effective treatment will likely need to penetrate the blood-brain barrier and also achieve good penetration into peripheral neurons. We have demonstrated that GM6 penetrates the blood brain barrier and is well absorbed by neuronal tissues. In our studies of C57BL/6 mice, endogenous GM6 levels were estimated to be 0.4 μM in the brain, but following GM6 injection (0.2 mg/kg), GM6 brain levels increased to 1.760 μM (4.4-fold increase). With more concentrated GM6 injection (2 mg/kg), brain levels increased to 12.92 μM (32.3-fold increase). These studies have confirmed the presence of an endogenous GM6 protein subunit within the central nervous system, and have additionally demonstrated that GM6 is able to cross the blood brain barrier to reach upper motor neurons within the central nervous system. Additional pharmacological studies demonstrated that GM6 has strong drug-like properties and good penetrance into tissues with estimated volume of distribution of 7.8 L/kg. GM6 additionally has good penetrance into cells with a cell partition of 5.2, indicating an intracellular GM6 concentration 5.2 times greater than its extracellular concentration. The estimated brain:plasma ratio is 1.65 consistent with good penetration into neuronal tissues. The tissue half-life was estimated to be 5.8 hours using microsomal clearance assays, and we have demonstrated that this half-life is not influenced by the presence of Riluzole.

(7) SOD1-G93A mouse model of ALS

The above findings demonstrate cellular mechanisms by which GM6 interacts with multiple extracellular receptors, leading to activation of signal transduction pathways, transcription factor mobilization, and modulation of gene expression activity in neuronal and immune cell types. To demonstrate in vivo efficacy of GM6 for ALS-like phenotypes, we utilized SOD1-G93A transgenic mice. In this model, GM6 was given intravenously starting at 80 days of age at concentrations of 1 mg/kg (n = 10) and 5 mg/kg (n = 10). A separate group of control mice received only vehicle treatment (n = 10). GM6 increased the age of disease onset by 12% at the lower dose (1 mg/kg) and by 27% at the higher dose (5 mg/kg) (P < 0.001). Likewise, GM6 increased the age at death by 16% at the lower dose (1 mg/kg) and by 30% at the higher dose (5 mg/kg) (P < 0.001). GM6 additionally led to dose-dependent improvements in disease progression, as evidenced by reduced decline of rota-rod performance and grip strength (14% increase in median performance at 1 mg/kg; 41% increase in median performance at 5 mg/kg; P < 0.001 in each case). We additionally observed improvements in clinical composite scores integrating several aspects of disease progression (weakness, tremors, reflex, and paralysis). Clinical composite scores are expected to increase in all SOD1-G93A transgenic mice, but the rate of increase was reduced in GM6-treated mice (23% increase in age at median score at 1 mg/kg, P = 0.001; 53% increase in age at median score at 5 mg/kg, P = 0.001).

These phenotypic improvements with GM6 treatment were correlated with improved spinal cord and CSF markers. We observed an increased percentage of ventral horn motor neurons (30% increase at 1 mg/kg; 40% increase at 5 mg/kg) and nerve growth factor (NGF) levels in CSF (60% increase at 1 mg/kg; 160% increase at 5 mg/kg). Interestingly, Cystatin C (CysC) levels were increased by 320% (1 mg/kg) and 670% (5 mg/kg) in GM6-treated SOD1-G93A mice. CysC is an endogenous cysteine protease inhibitor that inhibits cathepsin B, S and F and is thought to play a protective role in neurodegenerative disease. The abundance of CysC is decreased in CSF from ALS patients and increased CysC levels appears to correlate positively with ALS patient survival time. Consistent with increased CysC abundance, GM6 decreased abundance of cathepsin B (60% decrease at 1 mg/kg; 70% decrease at 5 mg/kg). We also observed decreased abundance of SOD1 protein (25% at 1 mg/kg; 50% at 5 mg/kg), CD68-positive microglia (50% at 1 mg/kg; 60% at 5 mg/kg), GFAP-positive astrocytes (70% at 1 mg/kg; 75% at 5 mg/kg) and TGF-beta (50% decrease at 1 mg/kg; 65% decrease at 5 mg/kg). Finally, GM6 led to strongly decreased abundance of key inflammatory markers such as TNF-alpha (50% decrease at 1 mg/kg; 60% decrease at 5 mg/kg) and IL-1beta (65% decrease at 1 mg/kg; 80% decrease at 5 mg/kg).

These findings have demonstrated efficacy for GM6 in the treatment of the ALS-like phenotype developing in a widely used and extensively validated ALS mouse model (SOD1-G93A transgenic mice). In this preclinical model, our results demonstrate effects of GM6 on disease biomarkers and phenotypic measures that are consistent with an effective ALS therapeutic, and have further provided proof-of-principle to support hypothesized mechanisms of action within an in vivo system.

(8) Phase 2A Clinical Trial

Given the promising preclinical data described above, we have performed a double-blind, randomized, placebo-controlled to evaluate effects of GM6 in human ALS patients (NCT01854294). The trial involved two main study sites at Columbia University Medical Center and Massachusetts General Hospital. The study enrolled 12 ALS patients meeting El Escorial criteria for ALS (n = 8 GM6-treated patients; n = 4 placebo-treated patient). Patients were given 6 intravenous doses of GM6 or placebo over a period of two weeks.

Consistent with prior studies demonstrating safety of GM6 in humans, adverse event rates were similar between GM6- and placebo-treated patients. The most common treatment-emergent adverse event was a mild rash (observed in 2 of the 8 GM6-treated patients). The study demonstrated promising trends with respect to key measures of functional status associated with ALS progression. Compared to a historical control cohort, the decline in ALS Functional Rating Scale (ALSFRS) was slowed in GM6-treated patients (P = 0.005). At one study site (Columbia), there was significantly slowed forced vital capacity (FVC) decline 10 weeks after GM6 treatment (P = 0.027). GM6 treatment also decreased plasma abundance of key ALS biomarkers, including TDP-43, Tau and SOD1 (P ≤ 0.037).

While these data do not yet demonstrate efficacy of GM6 as an ALS treatment, our findings have revealed several positive trends warranting further evaluation in a phase III clinical study. The findings support the hypothesis that GM6 favors neuron survival in ALS patients, slowing symptom progression and promoting a biomarker profile consistent with less severe disease.

(9) Conclusion

ALS is a debilitating disease that currently lacks highly effective pharmacological treatment options. Through decades of preclinical and clinical research, we developed the peptide drug GM6 as a potential treatment to improve neuron survival and inhibit symptom progression in ALS. Findings outlined in this summary demonstrate our progress in characterizing unique mechanisms of action for GM6 as a treatment for ALS. These mechanisms involve multiple extracellular receptors and the activation of intracellular signaling cascades, leading to altered transcription of hundreds of genes with broad hormone-like effects on cellular physiology. We have hypothesized that these effects can effectively promote neuron survival in the setting of ALS, resulting in the inhibition of symptom progression. In support of this, we have demonstrated efficacy of GM6 for treatment of the ALS-like phenotype in a widely used and validated mouse model (SOD1-G93A transgenic mice). We have extended these studies in a phase 2A clinical trial enrolling ALS patients, which confirmed safety of GM6 and a lack of drug-related adverse effects, while further demonstrating promising trends related to physical function measures and ALS disease biomarkers. These results together demonstrate unique mechanisms of action mediating the effects of GM6 on neuron survival, which involve multiple extracellular receptors, signal transduction pathways, transcription factors and transcriptionally regulated genes. Our findings further support the hypothesis that these mechanisms can effectively bolster neuron survival in ALS, as demonstrated by findings from a validated ALS mouse model and favorable trends from a double-blind, randomized, placebo-controlled phase 2A clinical trial.