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

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. To date, SPL-891.1 has shown no toxicity after 100 mg/kg i.v. and it is more potent a GSNOR inhibitor than SPL-334. Early results appear similarly promising with SPL-875.1.

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 significantly 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 or half the normal amount of the enzyme from conception until death.

ff-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 and SPL-891.1, that supposition appears to be substantiated. Furthermore, the lack of binding of both SPL-334.1 and SPL-891.1 to common targets of toxicity, suggests that they have little off-target toxicity.

The Que group at Duke has completed a Phase I clinical trial of a GSNOR inhibitor, N6022. They showed that the compound was active by i.v. administration against a methacholine challenge in patients with mild asthma and that no safety issues were found. These results suggest that GSNOR inhibition, as a mechanism of action, has demonstrated “clinical proof of concept”.

A scientist draws a molecule