Development of SAJE's GSNOR Inhibitors

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As demonstrated in the Science section, GSNORi regulates at least 56 pathophysiological drivers of many of the most important diseases of today with the most unmet medical needs.  These diseases often have a complex set of drivers that makes single target/single effect drugs of limited efficacy against them.   The unique advantages of SAJE’s GSNOR target is the multitude and directionality of responses with a single small molecule. Such positive, multi-mechanism, therapeutic effects, due to inhibiting one enzyme with a single small molecule, are rare in pharmacology.  Many important diseases are based on inappropriate inflammation, oxidant/nitrosative damage, mitochondrial dysfunction, calcium dysregulation, endoplasmic reticulum stress, vascular dysfunction/hypoperfusion, hyperglycemia, misfolded proteins, MMP-9, BBB breakdown, and platelet aggregation.  GSNORi inhibits all of those pathological conditions for potent therapeutic benefits.  In addition, GSNORi activates: IL-4, IL-10, sGC/cGMP, Nrf-2 anti-oxidant system for ROS & RNS inhibition, and mitochondrial prohibitin for preventing/reversing mitochondrial dysfunction, and the neurotrophic factors BDNF, CNTF, Synaptophysin, and TrkB/pTrkB.  GSNORi also improves and reverses deficits in memory and learning, neurological functions, and fibrosis which suggests that GSNOR inhibition should have wide application in many important disease areas, both orphan and major: i.e., ophthalmic, neurodegenerative, cardiovascular, autoimmune, inflammatory, hepatic, renal, respiratory, and fibrotic diseases.  Obviously, not all of these diseases can be pursued in clinical trials by a small company such as SAJE.  

SAJE is currently focused on ophthalmic diseases as the fastest and most cost-effective approach to achieving clinical proof of concept for SPL-850.  The advantages of an initial ophthalmic clinical development program include that much less pre-clinical safety and PK studies are required and that the first clinical trial can be a Phase II trial in 120-150 patients in autoimmune allergic conjunctivitis.  Such trials take only 6 months from start to a data report.  A second Phase II/III trial in 120-150 patients, if successful, allows an NDA filing for that indication.  

Other ophthalmic indications for SPL-850 include many with large unmet medical needs:  dry eye, glaucoma, macular degeneration, UVeitis, diabetic retinopathy, and prevention of ocular fibrosis after surgery.  Current therapy to deliver drugs to the back of the eye for retinal degenerative diseases requires intravitreal injection.  We believe that SPL-850 could be an oral drug for retinal degenerative diseases.  The evidence for that possibility is that SPL-891 when delivered by IP injection prevented microglial activation in the mouse neural retina after LPS treatment.  We know that SPL-891 does not enter the brain, so not the retina either, but that GSNO enters the brain and, based on this result, presumably the retina.  This result is quite similar to the Hayashida “Circulation” paper (see SAJE publications list) where we showed that SPL-334, which also doesn’t enter the brain, caused increased nitrosylation on 226 brain proteins.  GSNO is a strong nitrosylating molecule, and brain bioavailable through the blood, so it is the only rational explanation for the results in both experiments.

Below is an outline of the rationales for GSNORis and SPL-850 in ophthalmic indications:

• GSNOR is present at high concentration in human ocular tissue 

• SPL 850,a GSNOR inhibitor, has been shown to be safe and is a small molecule non-steroid.

• Pre-clinical mouse model experiments conducted by ORA, a leader in ophthalmology drug development https://www.oraclinical.com/, yielded strong efficacy for SPL-850 in ORA’s autoimmune allergic conjunctivitis model which is highly correlated, approaching a 100% with results in human patients.  Few other animal models have such pre-clinical/clinical correlations.

• SPL850 was more active and safer than prednisolone, a steroid standard of care.

• ORA believes that the results in their allergic conjunctivitis model is also an excellent predictor of efficacy in dry eye. ORA claims SPL850’s results are so strong that an animal dry eye model, which they also perform, is unnecessary. 

• Based on the safety profile of SPL 850 and the preclinical mouse model results, IND enabling studies can be completed and an IND filed for Phase II studies targeted at treating first autoimmune allergic conjunctivitis and then dry eye.

• Improved drugs to treat dry eye disease are needed for this multibillion-dollar marketplace.

• SAJE has four  composition of matter patents currently pending on these structures, one of which has been filed worldwide.

• The US's annual cost of ophthalmic diseases is ~ $140 billion, which is more costly than three of the top seven major chronic illnesses, including heart disease, Alzheimer's, diabetes, and cancer.

While SAJE believes that its first clinical trial will show rapid and less expensive clinical proof of concept and a faster route to market, we are more than willing to pursue other disease indications to suit the needs of potential partners.  As demonstrated in the Science section, SPL-850 could provide potent efficacy and safety in other broad categories of indications:  autoimmune/inflammatory, neurodegenerative, cardiovascular, metabolic, respiratory, and fibrotic diseases.  Evidence exists for GSNOR inhibition having activity in all of those indications.

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.

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