A Phase 1 randomized study on the safety and pharmacokinetics of OCS-05, a neuroprotective disease modifying treatment for Acute Optic Neuritis and Multiple Sclerosis.

OCS-05 (aka BN201) is a peptidomimetic that binds to serum glucocorticoid kinase-2 (SGK2), displaying neuroprotective activity. The objective of this randomized, double-blind 2-part study was to test safety and pharmacokinetics of OCS-05 administered by intravenous (i.v.) infusion in healthy volunteers. Subjects (n = 48) were assigned to receive placebo (n = 12) or OCS-05 (n = 36). , Doses tested were 0.05, 0.2, 0.4, 0.8, 1.6, 2.4 and 3.2 mg/kg in the single ascending dose (SAD) part. In the multiple ascending dose (MAD) part, 2.4 and 3.0 mg/kg doses were administered with 2 h i.v. infusion for 5 consecutive days. Safety assessments included adverse events, blood tests, ECG, Holter monitoring, brain MRI and EEG. No serious adverse events were reported in the OCS-05 group (there was one serious adverse event in the placebo group). Adverse events reported in the MAD part were not clinically significant, and no changes on the ECG, EEG or brain MRI were observed. Single-dose (0.05-3.2 mg/kg) exposure (Cmax and AUC) increased in a dose-proportional manner. Steady state was reached by Day 4 and no accumulation was observed. Elimination half-life ranged from 3.35 to 8.23 h (SAD) and 8.63 to 12.2 h (MAD). Mean individual Cmax concentrations in the MAD part were well below the safety thresholds. OCS-05 administered as 2-h i.v. infusions of multiple doses up to 3.0 mg/Kg daily for up to 5 consecutive days was safe and well tolerated. Based on this safety profile, OCS-05 is currently being tested in a phase 2 trial in patient with acute optic neuritis (NCT04762017, date registration 21/02/2021).

Novel Inhaled Pan-JAK Inhibitor, LAS194046, Reduces Allergen-Induced Airway Inflammation, Late Asthmatic Response, and pSTAT Activation in Brown Norway Rats.

The Janus-activated kinase (JAK) family together with signal transducer and activator of transcription (STAT) signaling pathway has a key role in regulating the expression and function of many inflammatory cytokines. This has led to the discovery of JAK inhibitors for the treatment of inflammatory diseases, some of them already in the market. Considering the adverse effects associated with JAK inhibition by oral route, we wanted to explore whether JAK inhibition by inhaled route is enough to inhibit airway inflammation. The aim of this study was to characterize the enzymatic and cellular potency and the selectivity of LAS194046, a novel JAK inhibitor, compared with the reference compounds ruxolitinib and tofacitinib. The efficacy of this new JAK inhibitor is described in a model of ovalbumin (OVA)-induced airway inflammation in Brown Norway rats by inhaled administration. As potential markers of target engagement, we assessed the effect of LAS194046 on the STAT activation state. LAS194046 is a selective inhaled pan-JAK inhibitor that reduces allergen-induced airway inflammation, late asthmatic response, and phosphor-STAT activation in the rat OVA model. Our results show that topical inhibition of JAK in the lung, without relevant systemic exposure, is sufficient to reduce lung inflammation and improve lung function in a rat asthma model. In summary, JAK-STAT pathway inhibition by inhaled route constitutes a promising therapeutic option for lung inflammatory diseases.

Discovery of a Novel Inhaled PI3Kδ Inhibitor for the Treatment of Respiratory Diseases.

Oral PI3Kδ inhibitors such as Idelalisib and Duvelisib have shown efficacy as anticancer agents and Idelalisib has been approved for the treatment of three B-cell cancers. However, Idelalisib has a black box warning on its product label regarding the risks of fatal and serious toxicities including hepatic toxicity, severe diarrhea, colitis, pneumonitis, infections, and intestinal perforation. Some of these side effects are mechanism-related and could hinder the development of Idelalisib for less severe conditions. For respiratory diseases, compounds administered by inhalation are delivered directly to the site of action and may improve the therapeutic index of a drug, minimizing undesired side effects. This work describes the discovery and optimization of inhaled PI3Kδ inhibitors intended for the treatment of severe asthma and COPD. Once the potency was in the desired range, efforts were focused on identifying the particular physicochemical properties that could translate into better lung retention. This medicinal chemistry exercise led to the identification of LAS195319 as a candidate for clinical development.

Application of a phenotypic drug discovery strategy to identify biological and chemical starting points for inhibition of TSLP production in lung epithelial cells.

Thymic stromal lymphopoietin (TSLP) is a cytokine released by human lung epithelium in response to external insult. Considered as a master switch in T helper 2 lymphocyte (Th2) mediated responses, TSLP is believed to play a key role in allergic diseases including asthma. The aim of this study was to use a phenotypic approach to identify new biological and chemical starting points for inhibition of TSLP production in human bronchial epithelial cells (NHBE), with the objective of reducing Th2-mediated airway inflammation. To this end, a phenotypic screen was performed using poly I:C / IL-4 stimulated NHBE cells interrogated with a 44,974 compound library. As a result, 85 hits which downregulated TSLP protein and mRNA levels were identified and a representative subset of 7 hits was selected for further characterization. These molecules inhibited the activity of several members of the MAPK, PI3K and tyrosine kinase families and some of them have been reported as modulators of cellular phenotypic endpoints like cell-cell contacts, microtubule polymerization and caspase activation. Characterization of the biological profile of the hits suggested that mTOR could be a key activity involved in the regulation of TSLP production in NHBE cells. Among other targeted kinases, inhibition of p38 MAPK and JAK kinases showed different degrees of correlation with TSLP downregulation, while Syk kinase did not seem to be related. Overall, inhibition of TSLP production by the selected hits, rather than resulting from inhibition of single isolated targets, appeared to be due to a combination of activities with different levels of relevance. Finally, a hit expansion exercise yielded additional active compounds that could be amenable to further optimization, providing an opportunity to dissociate TSLP inhibition from other non-desired activities. This study illustrates the potential of phenotypic drug discovery to complement target based approaches by providing new chemistry and biology leads.

Discovery of a Potent, Selective, and Orally Available PI3Kδ Inhibitor for the Treatment of Inflammatory Diseases.

The delta isoform of the phosphatidylinositol 3-kinase (PI3Kδ) has been shown to have an essential role in specific immune cell functions and thus represents a potential therapeutic target for autoimmune and inflammatory diseases. Herein, the optimization of a series of pyrrolotriazinones as potent and selective PI3Kδ inhibitors is described. The main challenge of the optimization process was to identify an orally available compound with a good pharmacokinetic profile in preclinical species that predicted a suitable dosing regimen in humans. Structure-activity relationships and structure-property relationships are discussed. This medicinal chemistry exercise led to the identification of LAS191954 as a candidate for clinical development.

Mechanism of control of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase by threonine.

The regulatory domain of the bifunctional threonine-sensitive aspartate kinase homoserine dehydrogenase contains two homologous subdomains defined by a common loop-alpha helix-loop-beta strand-loop-beta strand motif. This motif is homologous with that found in the two subdomains of the biosynthetic threonine-deaminase regulatory domain. Comparisons of the primary and secondary structures of the two enzymes allowed us to predict the location and identity of the amino acid residues potentially involved in two threonine-binding sites of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase. These amino acids were then mutated and activity measurements were carried out to test this hypothesis. Steady-state kinetic experiments on the wild-type and mutant enzymes demonstrated that each regulatory domain of the monomers of aspartate kinase-homoserine dehydrogenase possesses two nonequivalent threonine-binding sites constituted in part by Gln(443) and Gln(524). Our results also demonstrated that threonine interaction with Gln(443) leads to inhibition of aspartate kinase activity and facilitates the binding of a second threonine on Gln(524). Interaction of this second threonine with Gln(524) leads to inhibition of homoserine dehydrogenase activity.

Overproduction, purification, and characterization of recombinant aspartate semialdehyde dehydrogenase from Arabidopsis thaliana.

In plant and microorganisms, aspartate semialdehyde dehydrogenase (ASDH) produces the branch point intermediate between the lysine and threonine/methionine pathways. In this study, we report the first cDNA cloning, purification, and characterization of a plant ASDH. The Arabidopsis thaliana ASDH is an homodimeric enzyme composed of subunits of 36 kDa. The plant enzyme exhibited a specific activity of 26 micromol NADPH oxidized min(-1) mg(-1) of protein with a K(M) value for NADPH of 92 microM. ASDH showed cooperative behavior for aspartyl phosphate with a K(0.5) value of 37 microM.

Overproduction, purification, and characterization of recombinant bifunctional threonine-sensitive aspartate kinase-homoserine dehydrogenase from Arabidopsis thaliana.

In plant, the first and the third steps of the synthesis of methionine and threonine are catalyzed by a bifunctional enzyme, aspartate kinase-homoserine dehydrogenase (AK-HSDH). In this study, we report the first purification and characterization of a highly active threonine-sensitive AK-HSDH from plants (Arabidopsis thaliana). The specific activities corresponding to the forward reaction of AK and reverse reaction of HSDH of AK-HSDH were 5.4 micromol of aspartyl phosphate produced min(-1) mg(-1) of protein and 18.8 micromol of NADPH formed min(-1) mg(-1) of protein, respectively. These values are 200-fold higher than those reported previously for partially purified plant enzymes. AK-HSDH exhibited hyperbolic kinetics for aspartate, ATP, homoserine, and NADP with K(M) values of 11.6 mM, 5.5 mM, 5.2 mM, and 166 microM, respectively. Threonine was found to inhibit both AK and HSDH activities by decreasing the affinity of the enzyme for its substrates and cofactors. In the absence of threonine, AK-HSDH behaved as an oligomer of 470 kDa. Addition of the effector converted the enzyme into a tetrameric form of 320 kDa.