Safety

Safety of GSNOR Inhibitors

Introduction: GSNOR Inhibition as a Mechanism Is Safe in Both Preclinical and Clinical Studies

GSNOR inhibitors inhibit only the enzyme S-nitrosoglutathione reductase (GSNOR). They do not bind themselves to the many targets they regulate; instead, they increase the concentration of S-nitrosoglutathione (GSNO), which trans-nitrosylates and thus regulates those targets. Despite GSNORis inhibiting only GSNOR, they have multiple mechanisms of action in that they are anti-inflammatory, antioxidant enzyme-inducing, sGC/cGMP-inducing, mitochondrial-sparing, and anti-fibrotic. Those mechanisms are a function of GSNO’s nitrosylating activity and not due to any of our GSNOR inhibitor’s interaction with the disease targets themselves. Many diseases share inflammation, oxidant damage, mitochondrial dysfunction, and fibrosis as mechanisms of pathology, so there is a great potential to treat many of them with GSNOR inhibition.

As discussed in more detail in the Science section, our data shows that GSNOR inhibition harnesses, through GSNO, the power of protein S-nitrosylation, one of the cell’s main signaling pathways that have been conserved over 2-3 billion years of evolution for multiple therapeutic benefits—and one much more amenable than other signaling pathways to pharmacologic control. There is no a priori reason why the nitrosylation pathway should have toxic consequences—otherwise, it would have been selected out of cell physiology by evolution. In fact, there is no evidence, as described below, that GSNOR inhibitors are toxic, including GSNO Therapeutics’ lead compound, GTI-850. Thus, we believe our drugs have the power to be truly transformative in medicine by being both safe and efficacious in a way that single target/single effect drugs are not. It could be a new paradigm in pharmacology that leads to the therapy of many diseases without toxicity.

Summary of GSNORis Clinical Studies

Nivalis Therapeutics performed multiple Phase I and two Phase II clinical trials of two GSNOR inhibitors (N6022 & N91115). While those drugs have different chemical structures and are different from GSNO Therapeutics’ GTI-850 and related compounds, they inhibit GSNOR, as do GSNO Therapeutics’ drugs.

They Found: No Preclinical Safety Issues for Either Drug, Including No Safety Pharmacology or Toxicology Issues; These Results Show That GSNOR Inhibition as a Mechanism of Action Has No Preclinical Safety Issues; As Discussed Above, GSNO Therapeutics Believes Its GSNORi GTI-850 Has No Off-Target Safety Issues in Addition to No On-Target, Mechanistic, Safety Issues.

No Evidence for SAEs in Phase I or II Human Clinical Studies With the Two Nivalis GSNOR Inhibitors and ~400 Pts.

That N6022 Was Active in Asthma by I.V. (It Is Not Orally Bioavailable) As Measured by Methacholine Challenge, With No SAEs in a Small Phase IIa Trial.

That N91115 Caused No SAEs in a Large Phase IIb and Was Active in Causing Weight Gain in CF Pts, but Failed to Statistically Increase FEV1. Lack of Weight Gain in CF Often Leads to Death, so Increasing Weight Gain is an Important Clinical Finding.

Perhaps the Reason N91115 Failed To Increase FEV1 Is Because the Preclinical Activity Against CF Fibroblasts Was Marginal, With a 2.3-Fold Increase in Cl- Transport vs. a 23-Fold Increase in Normal Subjects. After Its FEV1 Phase II Trial Failure, Nivalis Failed as a Viable Public Company and its IP was Sold.

Nivalis Showed That N91115 Is Not Carcinogenic in Preclinical Carcinogenicity Studies; This Result Shows That the GSNORi Mechanism Is Not Carcinogenic.

In summary, the Nivalis clinical studies establish proof of clinical safety and efficacy for GSNOR inhibition as a mechanism of therapeutic action.

Off-target toxicity occurs when a drug designed to bind to target also A binds to a protein or other cellular targets B, C, D, etc. Such off-target binding may not be a problem if the drug-bound targets do not produce toxicity or if the binding is weak. However, if any of the targets, once bound by the drug, produce side effects, the drug may be too toxic for use in patients and thus fail in clinical development. This problem is a drug by drug problem and must be assessed for each drug in preclinical and clinical development. Only those drugs with a sufficient ratio of the toxic dose/therapeutic dose are allowed by the FDA to be used in disease therapy. While there is no absolute limit, generally, that ratio should be at least 10-fold or higher unless the condition being treated is life-threatening and the side effects are considered acceptable to gain the therapeutic benefit. Aspirin in dogs, for example, has a ratio of around 4 and might not make it through safety assessment today if it were to come before the FDA for approval.

GSNO Therapeutics’ latest safety data shows that GTI-850, our lead compound, has a toxic dose to therapeutic dose ratio, or therapeutic index, of more than 100-fold, suggesting that the drug is quite safe.

GSNOR Inhibition Does Not Cause Nitrosative Stress

Initially, there was a concern about our technology causing nitrosative stress because our drugs increase the cellular concentration of GSNO, a nitrosylating agent. However, GSNO does not break down to NO in the cell under physiological conditions, and second, unlike NO in high concentrations, GSNO does not cause nitrosative stress. GSNOR inhibition does NOT increase the amount of free nitric oxide (NO), a radical which, in too high concentrations, can cause toxicity by nitrosative stress. In contrast, GSNOR inhibition reduces NOS2 and eNOS activity and the production of NO by feedback inhibition by NO and GSNO.

There are, however, other indications of the safety of our GSNOR inhibitors as a target. First, GSNO Thera expects to see little mechanistic toxicity from its GSNOR inhibitors because GSNOR knockout mice (-/-) with no GSNOR activity from conception until death develop, grow, reproduce, and behave normally but are less robust. So, the complete absence of GSNOR is neither lethal nor significantly disabling throughout a lifespan. Presumably, other GSNO denitrosylases, such as the Thioredoxin System, partially substitute for the absence of GSNOR in the KOs. However, GSNO Thera believes that the GSNOR knockdown mice (+/-), with only one of the two GSNOR alleles deleted, are a much closer analog to treatment with a GSNOR inhibitor that reduces, but doesn’t eliminate, the enzyme. The GSNOR knockdown (+/-) mice are completely normal, further supporting the idea that GSNOR inhibition does not cause mechanistic toxicity. Thus, the multiple mechanisms of therapeutic action by GSNOR inhibitors show no evidence of toxicity, even in the complete absence or half the normal amount of the enzyme from conception until death.

Off-target toxicity is a possibility for our novel inhibitors but is unlikely because of the unique shape of the GSNOR active site “pocket” into which we have designed our inhibitors to fit. Our inhibitors do not inhibit other members of the alcohol dehydrogenase family of enzymes, suggesting that it is unlikely that they will inhibit other proteins whose structures are even further away from GSNOR’s structure. Given the high therapeutic ratio (toxic dose/therapeutic dose) seen to date with GTI-334, GTI- 850, and GTI-850, that supposition appears to be substantiated. Furthermore, the lack of binding of GTI-334.1, GTI-891.1 and GTI-850 to 44 common toxicity targets suggests that they have little off-target toxicity.

GTI-891.1 and GTI-850: Summary of Metabolism, PK, and Stability Studies

Metabolic stability studies show that both drugs, which are structurally related, have sufficient stability in human, rat, mouse, and dog microsomes and hepatocytes to be good clinical candidates. Both drugs are highly bound to plasma proteins, which can increase their bioactivity. GTI-891.1 doesn’t inhibit the cytochrome P450 system enzymes, indicating that it won’t have any drug-drug interactions. Neither drug has Phase I metabolism (hydrolysis, oxidation, reduction, or cyclization) but is glucuronidated in Phase II metabolism, which is a probable mechanism of excretion. We also know that, in formal stability studies, GTI-891.1 is 100% stable for 30 days at room temperature and at 40 degrees C and for 2 years of bench stability at room temperature. It can be boiled in water and remains stable. Thus, it appears to have adequate stability for utility as a clinical agent.

Summary of GTI-891.1 Safety Studies

GTI-891.1: Acute I.V. Toxicity: Mice: piloerection at 400 mg/kg, I.V., but no deaths. Therapeutically active at 0.3-10.0 mg/kg = > 40-13,000-fold acute therapeutic index. Rats: no clinical or organ toxicity at 1000 mg/kg.
GTI-850: Sub-chronic 7-day QD toxicity study in mice at 300 mg/kg: no clinical signs, no gross organ toxicity, no hematological or clinical chemistry changes, and no histological toxicity.
GTI-891.1 and GTI-850: Eurofins’ Safety 44 off-target toxicity screen: no binding or enzyme inhibition. This shows no off-target toxicity to any of the 44 common targets of toxicity.
GTI-850: NASH study: No toxicity after 42 days of QD I.P. dosing at 10 mg/kg.
GTI-850: Diabetes: No toxicity after 56 days of QD oral dosing at 10 mg/kg.
GTI-891.1 and GTI-850: Ames test negative. The drugs are not mutagenic, which implies they are not carcinogenic.
GTI-891.1 and GTI-850: hERG channel negative. The drugs do not inhibit the cardiac potassium channel, which when inhibited, produce a potentially fatal long QT syndrome.

GSNO Therapeutics has a detailed preclinical development plan to finish the safety studies necessary for an IND. GSNO Therapeutics believes that all the evidence suggests that both GTI-891.1 and GTI-850 will complete those studies with no mechanistic or off-target toxicity that would prevent entrance into clinical trials. GTI-850 is our lead compound because it is more active in mouse efficacy studies, but GTI-891.1 serves as an active back-up.