Safety of GSNOR Inhibitors

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GSNOR inhibitors inhibit only the enzyme S-nitrosoglutathione reductase (GSNOR).  They do not themselves bind to the many targets they regulate, rather, they increase the concentration of S-nitrosoglutathione (GSNO) which transnitrosylates and thus regulates those targets.  Despite GSNORis inhibiting only GSNOR, they have multiple mechanisms of action, in that they are anti-inflammatory, anti-oxidant enzyme inducing, sGC/cGMP inducing, mitochondrial sparing, and anti-fibrotic.  Those mechanisms are a function of GSNO’s nitroylsting activity and not due to any of our GSNOR inhibitors 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 show that GSNOR inhibition harnesses, through GSNO, the power of nitrosylation, one of the cell's main signaling pathways, that has been conserved over more than 2-3 billion years of evolution, for multiple therapeutic benefits—and one much more amenable than other signaling pathways for 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 SAJE’s lead compound, SPL-850.  Thus, we believe our drugs have the power to be truly transformative in medicine 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.

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 themselves and also different than SAJE’s SPL-850 and related compounds, they inhibit GSNOR as do SAJE’s drugs. 

They found:

• No pre-clinical 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, SAJE believes its GSNORi SPL-850 has no off-target safety issues in addition to no on-target, mechanistic, safety issues.  In addition, N91115 was not carcinogenic in preclinical carcinogenicity studies. This result shows that the GSNORi mechanism is not carcinogenic.

• 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 increase FEV1. Lack of weight gain in CF occurs because primarily the pancreas, but also the liver, secretory ducts get clogged with mucus preventing digestive enzymes from reaching the gut to digest food. The fact that N91115 caused weight gain suggests that the drug was functioning in the pancreas and liver.  

• 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. Nivalis’ venture capitalist’s daughter has CF and he forced them to do a CF trial, against the judgement of the CMO and others. After its FEV1 Phase II trial failure, Nivalis also failed as a viable public company. SAJE is now the only company in the GSNOR field.

In summary: The Nivalis clinical studies establish proof of clinical safety and efficacy for GSNOR inhibition as a mechanism of therapeutic action

1. Mechanistic toxicity occurs when the inhibition or activation of the basic mechanism of drug action causes both a therapeutic effect and also a toxic effect. If the therapeutic effect occurs at lower concentrations than the toxic effect, then the drug might be used to treat a disease. However, if the therapeutic and toxic effects occur at concentrations that are too close to each other, then the drug may be too dangerous to use. In particular, some patients may metabolize the drug slower than the majority of patients and thus be exposed to a toxic concentration of drug and suffer unwanted side effects. Such drugs may be approved for use after small pivotal trials, but later be withdrawn from the market after use in larger populations. However, many mechanisms of drug action do not themselves cause toxicity. We believe that GSNOR inhibition is one of them. GSNOR regulates the nitrosylation pathways that have been in evolution for ~ 2-3 billion years. All eukaryotic organisms utilize nitrosylation for physiological control and GSNOR controls 90% of it.  If that pathway had toxic consequences, it would have been eliminated by evolution. So, while not proof, we believe that regulating the evolutionarily conserved nitrosylation pathways by inhibiting GSNOR, would be expected, by itself, to have no toxic side effects, which, to date, the preponderance of the evidence supports.

2. Off-target toxicity occurs when a drug designed to bind to target A also binds to 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, then 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 pre-clinical and clinical development. Only those drugs that have a sufficient ratio of the Toxic dose/Therapeutic dose are allowed by 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 around 4 and might not make it through safety assessment today, if it were to come before the FDA for approval. SAJE’s latest safety data shows that SPL-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.

Initially there was a concern about our technology causing nitrosative stress because our drugs increase the cellular concentration of GSNO, a nitrosylating agent. However, first, 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. The enzyme S-nitrosoglutathione reductase (GSNOR) in the presence of NADH reduces S-nitrosoglutathione (GSNO) to glutathione disulfide and hydroxyl amine. Inhibition of GSNOR activity causes an increase in the cellular pool of GSNO, which increases the level of cysteine nitrosylation on proteins that are in the tightly coupled evolutionarily selected nitrosylation pathways. 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, which prevents nitrosative stress by NO.

There are, however, other indications of the safety of our GSNOR inhibitiors as a target. First, SAJE 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, behave normally, and lead a normal lifespan. So, the complete absence of GSNOR is neither lethal nor significantly disabling throughout a lifespan. However, SAJE 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, by itself, 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 SPL-334, SPL 850, and SPL-891.1, that supposition appears to be substantiated. Furthermore, the lack of binding of both SPL-334.1, SPL-850, and SPL-891.1 to 44 common targets of toxicity, suggests that they have little off-target toxicity.

Metabolic stability studies show that both drugs have sufficient stability in human, rat, mouse, and dog microsomes and hepatocytes to be good clinical candidates for systemic administration. Both drugs are highly bound to plasma proteins which can increase their bioactivity. SPL-891.1 doesn’t inhibit the cytochrome P450 system enzymes, indicating that it won’t have any drug-drug interactions. As well 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 SPL-891.1 is 100% stable for 30 days at room temperature and at 40○ C, as well as for 2 years of bench stability at room temperature.  SPL-850 also has prolonged bench stability and can be heated to over 50C with no breakdown.  Thus, they both should have adequate stability for utility as a clinical agent.

1. SPL-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 IV.

2. SPL-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.

3. SPL-891.1 and SPL-850:  Eurofins’ Safety 44 off-target toxicity screen: no binding or enzyme inhibition. Shows no off-target toxicity to any of 44 common targets of toxicity.

4. SPL-891.1: NASH study: No toxicity after 42 days of qd I.P. dosing at 10 mg/kg.

5. SPL-891.1: Diabetes: No toxicity after 56 days of qd oral dosing at 10 mg/kg.

6. SPL-850 and SPL-891.1: Ames test negative. The drugs are not mutagenic which implies they are not carcinogenic.  

7. SPL-850 and SPL-891.1 hERG channel negative. The drugs do not inhibit the cardiac potassium channel which means they do not produce a fatal long QT syndrome.

8. SAJE has a detailed pre-clinical development plan to finish the safety studies necessary for an IND. SAJE believes that all the evidence suggests that both SPL-850 and SPL-891.1 will complete those studies with no mechanistic or off-target toxicity that would prevent entrance into clinical trials.  SPL-850 is our lead compound, but SPL-891.1 serves as a back-up.