Research and Development

Nitrosylation

In 1977 the gas, nitric oxide (NO), through its protein nitrosylation activity, was discovered by Ferid Murad, MD, PhD, one of our scientific advisors, to have important physiological signaling roles in many different cells and organs. That discovery resulted in the awarding of Nobel Prizes in 1998, including one to Dr. Murad. Since Dr. Murad’s discovery, many research efforts have tried to utilize NO for disease treatment by synthesizing novel chemical NO donors or by designing NO releasing devices. Most of those pharmacological efforts failed, in part because the gas has a short half-life of seconds and delivery of effective doses to target tissues has been insufficient for therapeutic effects. However, SAJE Pharma has licensed, invented, and developed a small molecule drug technology that increases the cellular concentration of a stable nitrosylating conjugate of NO, S-nitrosoglutathione, which captures, in a natural way, the therapeutic benefits of NO which can be used to treat many diseases with unmet medical needs. SAJE’s intellectual property portfolio, consisting of one issued US and EU patent and four composition of matter patent applications, protects the composition and use of its novel drug compounds.

GSNO and GSNOR Inhibition

SAJE’s small molecule drugs regulate nitrosylation by inhibiting S nitrosoglutathione reductase (GSNOR), the last and controlling step in the NO signaling pathway that has been honed by evolution in plants and animals for more than one billion years. GSNOR breaks down the stable, storage form of NO, which is S-nitrosoglutathione (GSNO), a conjugate of NO and glutathione. Many biologists believe that “Nitrosylation is the new Phosphorylation”, meaning that nitrosylation regulates cell pathways as directly as does phosphorylation. The big advantage for SAJE is that there is only one human GSNOR to inhibit as compared to many protein kinases and phosphatases, making it a much more “drug-able” target with less possibility for off-target toxicity. The goal of GSNOR inhibition therapy is not to eliminate the enzyme, but rather to reduce GSNOR activity enough to increase nitrosylation of those signaling pathways that produce therapeutic advantages in different diseases. GSNOR inhibition increases the GSNO storage form of nitric oxide which increases nitrosylation of accessible cysteines on key signaling proteins, all of which have been selected by evolution to play important physiological roles. The increase in nitrosylation leads to a change in protein structure, and thus function, which results in a cascade of biochemical responses and therapeutic benefits that include:

  1. Anti-inflammation: reduction in the number of eosinophils and lymphocytes that infiltrate inflamed tissue; inhibition of ICAM-1; inhibition of the cytokines: IFN-γ, TNF-α, TGF-β, IL-4, IL-5, IL-6, IL-12(p40), IL-12(p70), IL-13, Il-17, and IL-23 [References* 2, 3, 4 & SAJE unpublished].
  2. NFĸB: Inhibition of the activation of NFĸB by increasing the S-nitrosylation of Iĸĸβ, which inhibits its kinase activity and suppresses NFĸB activation [5] and, in turn, decreases the expression of inflammatory genes.
  3. Inhibition of the chemokines CCL-2 (MCP-1) and CCL11: CCL-2 recruits monocytes, memory T cells, and dendritic cells to the sites of inflammation produced by either tissue injury or infection. CCL-11 selectively recruits eosinophils by inducing their chemotaxis, and therefore, is implicated in allergic responses. Both are involved in induction of fibrosis in various tissues [2,3].
  4. Anti-Oxidant: induction of Nrf2/ARE system of anti-oxidant enzymes to inhibit the production of reactive oxygen species (ROS) [6], which are causative in the induction of fibrosis [ 7,8].
  5. Anti-fibrotic: SPL-334 not only prevents progression of fibrosis, but also reverses existing bleomycin induced fibrosis [3]--due to inhibition of ROS, CCL-2, CCL-11, and Connective tissue Growth Factor (CTGF). Reversal of existing fibrosis is almost unprecedented among clinical candidates. SPL-891.1 prevents NASH in a mouse model of the disease.
  6. EMT: Attenuation of epithelial-mesenchymal transition (EMT) as measured by decreased TGF-β induced collagen synthesis in human fibroblast cells, in vitro [3].
  7. Bronchodilation through opening of constricted bronchioles [2,4].
  8. Increased mucus clearance [2].
  9. Activation of Soluble Guanylyl Cyclase (sGC) which regulates the cyclic guanosine monophosphate (cGMP) system [5]. cGMP acts as a second messenger much like cyclic AMP. Its mechanism of action is activation of intracellular protein kinases in response to the binding of membrane impermeable peptide hormones to the external cell surface.The above benefits have been seen in the many different animal studies that SAJE and its collaborators have conducted. However, not every parameter has been measured in every experiment.
* see references above

Disease Applications

A unique advantage of SAJE’s program targeting GSNOR is the multitude and directionality of responses with a single small molecule. Such positive, pleiotropic, therapeutic effects, due to inhibiting one enzyme with small molecules, are rare in pharmacology. The fact that many important diseases are based on inflammation, oxidant damage, mucus accumulation, EMT, and fibrosis suggests that GSNOR inhibition should have wide application in many important disease areas, both orphan and major: i.e., oncologic, cardiovascular, CNS, metabolic, inflammatory, autoimmune, liver, kidney, respiratory, and multi-organ fibrotic diseases.

SAJE is primarily focused on fibrotic diseases with large unmet medical needs including Non-Alcholic Steatohepatitis (NASH), a disease associated with obesity, and idiopathic pulmonary fibrosis (IPF), an orphan disease. SAJE believes that the multiple therapeutic effects derived from inhibiting GSNOR will have great efficacy in treating both NASH and IPF, as is evidenced by the prevention and reversal of fibrosis in mouse models of the diseases [3, and SAJE, unpublished].

SAJE also has encouraging pre-clinical data in the ovalbumin and house dust mite models of asthma, some of which was published in Ferrini, et al., 2013 (Please see the list of publications above). SAJE is interested in out-licensing this application of its technology.

Using a Michael J. Fox grant, SAJE is testing its hypothesis that reducing the inflammation and oxidant damage that is causal in Parkinson’s Disease will have beneficial effects in an animal model of the disease. The challenge here is to invent a brain bioavailable GSNOR inhibitor, which SAJE is pursuing.

Other researchers have shown that GSNOR inhibition is efficacious in animal models of inflammatory bowel disease, endothelial vasodilatory function, hypertensive kidney damage, and chronic obstructive pulmonary disease (COPD).

In addition, SAJE has ongoing efficacy studies in in vitro and animal models of cancer progression and metastasis, immuno-oncology, rheumatoid arthritis, type II diabetes, glaucoma, cardiovascular disease, and aging.

Safety

GSNOR Inhibition is Safe in both Preclinical and Clinical Studies:

SAJE’s drugs inhibit only the enzyme S-nitrosoglutathione reductase (GSNOR), yet have multiple mechanisms of action, in that they are anti-inflammatory, anti-oxidant enzyme inducing, sGC/cGMP inducing, mitochondrial sparing, and anti-fibrotic. 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. Our data indicate that GSNOR inhibition harnesses the power of nitrosylation, one of the cell's main signaling pathways, that has been conserved over more than a 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. 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.

SPI-891.1 and SPL-850 Summary of Metabolism and Stability Studies:

Metabolic stability studies show that both drugs have sufficient stability in human, rat, mouse, and dog microsomes and hepatocytes to be good clinical candidates. Both drugs are highly bound to plamsa 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 and 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 ttemperature. Thus, it should have adequate stability for utility as a clinical agent.

Summary of SPL-891.1 Safety Studies:

  1. Acute I.V. Toxicity: Mice: piloerection at 400 mg/kg, I.V., but no deaths. Therapeutically active at 0.03-10.0 mg/kg = > 40-13,000-fold acute therapeutic index. Rats: no clinical or organ toxicity at 1000 mg/kg
  2. 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.
  3. NASH study: No toxicity after 42 days of qd I.P. dosing at 10 mg/kg.
  4. Diabetes: No toxicity after 56 days of qd oral dosing at 10 mg/kg.
  5. Ames test negative. The drug is not mutagenic which implies it is not carcinogenic.
  6. hERG channel negative. The drug does not inhibit the cardiac potassium channel which means it does not produce a fatal long QT syndrome.
  7. 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 SPL-891.1 will complete those studies with no mechanistic or off-target toxicity that would prevent entrance into clinical trials.

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 than SAJE’s SPL-891.1 and related compounds, they inhibit GSNOR as do SAJE’s drugs. They found:

Two Fundamental Types of Toxicity

  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. As discussed in this White Paper, we believe that GSNOR inhibition is one of them. GSNOR regulates the nitrosylation pathways that have been in evolution for ~ a billion years. All eukaryotic organisms utilize nitrosylation for physiological control and GSNOR is the main controlling enzyme. 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-891.1, one of our lead compounds, has a toxic dose to therapeutic dose ratio, or therapeutic index, of more than 13,333-fold, suggesting that the drug is quite safe.

GSNOR Inhibition Does Not Cause Nitrosavtive 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, 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 nitrosarive 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.

SAJE Pharma has shown that SPL-334 is active in the bleomycin model at a lowest dose of 0.3 mg/kg for some parameters and 1 mg/kg for other parameters in different therapeutic animal assays for up to three weeks of once a day dosing. No changes in body weight, behavior, or appearance were noted. The single dose MTD for oral SPL-334 is >192 mg/kg, so the therapeutic ratio is >192/0.3 or 192/1, which is between a 192 and a 640-fold therapeutic ratio for a single dose. In addition, no toxicity was seen at 3 mg/kg for 21 days of once a day dosing. Thus, there is a very large therapeutic window for SPL-334. Our second and third generation compounds are being tested now for single and multiple dose toxicity. SPL-891.1 has shown no toxicity after 1000 mg/kg i.v. and it is more potent a GSNOR inhibitor than SPL-334. Additionally, SPl-891.1 has no off-target toxicity nor Cyp inhibition.

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.

A scientist draws a molecule