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 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 500 million 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), and IL-13 [References above: 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. 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].
  4. Anti-fibrotic: SPL-334 not only prevents progression of fibrosis, but also reverses existing bleomycin induced fibrosis [3]--due to inhibition of ROS, CCL-11, and Connective tissue Growth Factor (CTGF). Reversal of existing fibrosis is almost unprecedented among clinical candidates.
  5. EMT: Attenuation of epithelial-mesenchymal transition (EMT) as measured by decreased TGF-β induced collagen synthesis in human fibroblast cells, in vitro [3].
  6. Bronchodilation through opening of constricted bronchioles [2,4].
  7. Increased mucus clearance [2].

The above benefits have been seen in the many different animal studies that SAJE and its collaborators have conducted.

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 usually rare in pharmacology. The fact that many important diseases are based on inflammation, oxidant damage, 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 including idiopathic pulmonary fibrosis (IPF), an orphan disease, with large unmet needs. SAJE believes that the multiple therapeutic effects derived from inhibiting GSNOR will have greater efficacy in treating IPF, as is evidenced by SPL-334’s reversal of existing fibrosis in the bleomycin mouse model of IPF [3]. Everything is in place to start the IND enabling studies required to begin clinical trials in IPF for our first lead compound, SPL-334.1 [an orally available salt of SPL-334].

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.

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

Safety

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 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 with no toxicity 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. Early results appear similarly promising.

There are, however, other indications of the safety of SPL -334 itself and of GSNOR inhibition as a target. First, SAJE expects to see little mechanistic toxicity from its GSNOR inhibitors because GSNOR knockout mice (-/-) with no GSNOR from conception until death, develop, grow, reproduce, behave normally, and lead a normal lifespan. So, the complete absence of GSNOR is neither lethal nor even mildly disabling. However, SAJE believes that the GSNOR knockdown mice (+/-) are a much closer analog to treatment with a GSNOR inhibitor that reduces, but doesn’t eliminate, the enzyme. The (+/-) mice are completely normal, further supporting the idea that GSNOR inhibition, by itself, does not cause mechanistic toxicity. Thus, the pleotropic mechanisms of therapeutic action by GSNOR inhibitors show no evidence of toxicity, even in the complete absence 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 (192-640-fold), that supposition appears to be substantiated.

One GSNOR inhibitor, N6022 from Nivalis Pharmaceuticals) has completed an i.v. Phase II clinical trial in asthma and was found to be efficacious and had no safety issues. That trial provides the first Clinical Proof of Concept for GSNOR as a target. Another GSNOR inhibitor is in a Phase II trial for cystic fibrosis (CF, see the Nivalis website) and was just granted orphan drug and fast track status by the FDA, which allowed an increased dose (400mg bid) and an expanded CF patient population. Together those trials provide an indication of the safety of the GSNOR target in patients and efficacy in the asthma trial with probable efficacy in the CF trial (or the FDA would not have allowed the changes in status and dose). Cpllectively, these studies provide “Clinical Proof of Principal: for the GSNOR target. While the chemical structures of Nivalis’ drugs and SAJE’s drugs are non-overlapping, and because we see no evidence of off-target toxicity with our drugs, SAJE predicts that its drugs will also be safe and effective in the clinic. All of these considerations greatly reduce the “risk profile” for SAJE’s drugs in later development.

A scientist draws a molecule