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Coronaviruses (CoV) are a large family of enveloped, RNA viruses that circulate in mammals and birds but have crossed the species barrier to infect humans seven times. Of these, three pathogenic strains have caused zoonotic infections in humans that result in severe respiratory syndromes including the Middle East Respiratory Syndrome (MERS-CoV), severe acute respiratory syndrome (SARS-CoV), and now SARS-CoV-2 coronaviruses, the latter of which is the cause of the ongoing pandemic of coronavirus disease 2019 (COVID-19). Here, we describe a panel of synthetic monoclonal antibodies, built on a human framework, that bind SARS-CoV-2 spike protein, compete for binding with ACE2, and potently inhibit infection by SARS-CoV-2. These antibodies were found to have a range of neutralization potencies against live virus infection in Vero E6 cells, potently inhibiting authentic SARS-CoV-2 virus at sub-nanomolar concentrations. These antibodies represent strong immunotherapeutic candidates for treatment of COVID-19.

GBO-006 was shown to be a highly specific and selective PLK2 inhibitor that promoted mitotic arrest in various cancer cell lines, subsequently resulting in their apoptotic death. Intraperitoneal alternate day dosing of GBO-006 using 100 % DMSO as formulation showed significant tumor regression in xenograft models, demonstrating proof of concept of PLK2 inhibition in vivo. These studies necessitated the development of a suitable and GRAS (generally considered as safe) preformulation for pharmacokinetic and efficacy studies. GBO-006 possesses challenging physicochemical and biopharmaceutical properties like poor solubility in aqueous media, low permeability and a crystalline nature. Different methods like cosolvency, complexation and micellar solubilization were employed to improve the solubility of GBO-006. A strategy of co-solvency is used to solubilize the GBO-006 up to 10 mg/mL. A formulation with 20 % DMSO, 40 % PEG 400, 30 % of 100 mM citrate buffer (pH 3.0) and 10 % solutol displayed clear solution without any visual precipitation of the drug even after 2 weeks of storage. GBO-006 showed moderate clearance in rat and high systemic clearance in mouse and dog. It showed poor oral bioavailability across all species. Intraperitoneal dosing of GBO-006 demonstrated the linear exposure. GBO-006 showed significant inhibition of tumor progression.

Platelet-activating factor (PAF) is released in numerous clinical situations. PAF primes or directly activates polymorphonuclear (PMN) leukocytes, which results in release of oxyradicals (O 2 − , H2O2, .OH) and hypochlorous acid (HOCl). The authors investigated the effects of PAF (1 µg/kg IV) in the absence and in the presence of antioxidants (superoxide dismutase [SOD], catalase [CAT], dimethylthiourea [DMTU]) and methionine, a quencher of HOCl, on cardiac function and contractility; blood lactate, gases, and pH levels; serum creatine kinase activity (CK); chemiluminescent activity of PMN leukocytes; and cardiac tissue malondialdehyde (MDA) in anesthetized dogs. Hemodynamic measurements and collection of blood samples for various biochemical measurements were made before and at various intervals up to two hours after PAF administration in the presence and absence of various antioxidants. PAF produced a decrease in indices of cardiac function and contractility and an increase in systemic and pulmonary vascular resistance. There were decreases in the blood pH and PMN leukocyte chemiluminescence and increases in blood lactate, serum CK activity, and tissue MDA content. SOD plus catalase or DMTU plus methionine reduced the effects of PAF on cardiac function and contractility, blood lactate and pH, serum CK, and cardiac tissue MDA. The antioxidants only partially antagonized the deleterious effects of PAF. The combination of SOD + CAT was superior to that of DMTU + methionine in reducing the deleterious effects of PAF. These results suggest that PAF-induced depression of cardiac function and contractility, and the increase in systemic and pulmonary vascular resistance, may be partly mediated by the release of oxyradicals and HOCl from PMN leukocytes. Antioxidants may be beneficial in reducing the deleterious effects of PAF on the cardiovascular system.

We investigated the effects of chronic volume overload in the absence or presence of vitamin E supplements on the cardiac function and contractility, cardiac malondialdehyde (MDA) —a lipid peroxidation product —cardiac antioxidant enzyme activity and antioxidant reserve in canine model. The dogs were divided into three groups of seven dogs each: group I, control; group II, mitral regurgitation (MR) of 4 months duration; and group III, MR of 4 months duration receiving vitamin E (40 U /kg /daily) orally. MR was created by detaching two or more chordae tendinae to raise left atrial pressure to 2.5 to three times normal. MR produced a decrease in the index of myocardial contractility with little change in myocardial function. Decrease in myocardial (left and right ventricles) contractility was associated with an increase in cardiac MDA, and a decrease in cardiac antioxidant reserve and antioxidant enzyme activity. Prevention of volume overload-induced decrease in myocardial contractility by vitamin E was associated with a decrease in cardiac MDA and an increase in cardiac antioxidant reserve and glutathione peroxidase activity towards control levels. Superoxide dismutase and catalase activity remained depressed in vitamin E-treated group. The results indicate that chronic volume overload decreases the contractility of both right and left ventricles and is associated with oxidative stress in both ventricles. These results support the hypothesis that oxygen free radicals are involved in the chronic volume overload-induced cardiac depression.

PTEN somatic mutations occur in a large percentage of human cancers, from breast to prostate. Deletions of PTEN e.g. in prostate cancers are associated with tumor aggression and poor outcome, and up to 70 percent of prostate cancer patients have lost one copy of the PTEN gene at the time of diagnosis. Recent pharmacologic and genetic studies have identified the β isoform of PI3K to be required for growth of PTEN-deficient tumors in vivo and highly potent compounds are currently being explored in the clinic. These clinical candidates, however, are either limited by poor bioavailability or moderate selectivity against the delta isoform, thus warranting more selective compounds with better exposure. We have undertaken a comprehensive knowledge-based discovery of selective inhibitors of PI3Kβ with admissible pharmacokinetic and drug like properties. We have successfully identified a chemical series with potency ranging as low as 2.5 nM for PI3Kβ with >100 - fold selectivity for PI3Kδ. The α and γ isoform selectivity was around 1000 and 500 fold respectively. Compounds did not decrease insulin sensitivity and T cell proliferation thus mitigating the PI3Kα and PI3Kδ mediated side effects of nonselective inhibitors. In vitro biochemical potency translated into cell survival assay and pAKT target engagement in recombinant and wild type mammalian cell lines. In vivo tumor regression was observed in xenograft models of prostate cancer. The series did not show any observable hERG or CYP liabilities. The considerable potency, high selectivity and good bioavailability of this novel class of compounds not only makes them attractive for prostate cancer, but also for rare disease indications like hamartoma and other drug resistant cancers that are dependent on PTEN deletions. Citation Format: Arnab R. Chowdhury, Jeyaraj D. Athisayamani, Sayan Mitra, Srinivas Maddi, Ram Sudheer Adluri, Sridhar R. Bethi, Hemant Joshi, Vijaya G. Tirunagaru, Jang B. Gupta. Discovery of selective PI3Kb inhibitors for PTEN-deficient tumors and other indications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 373.

Kinase gene fusions have been implicated as oncogenic drivers promoting cell survival and proliferation. TrkA gene fusions have been reported in a number of cancer types including lung, colorectal, papillary thyroid, sarcoma, cholangiocarcinoma and other tumors. We have identified novel, potent, selective and orally bioavailable TrkA inhibitors for the treatment of TrkA-driven cancers. One of the inhibitors, GVK-TrkI showed potent activity with an IC50 of 0.8 nM and 1.5 nM in the cell based assays for WT TrkA (AD293 cells expressing TrkA) and TPM3-NTRK1 fusion (KM12 cells) respectively. Potent activity of 12.5 nM IC50 was observed in TrkA Z-lyte kinase assay. Significant change in TrkA Z-lyte kinase IC50 was not observed at multiple ATP concentrations ranging from 100 uM to 1 mM suggesting non-ATP competitive mode of inhibition. Excellent selectivity (>1000x) was observed towards TrkB and TrkC in the cell based assays. GVK-TrkI inhibited TrkA autophosphorylation in a concentration dependent manner up to 24 h in KM12 cells in vitro. GVK-TrkI inhibited cell proliferation in KM12 cells with a GI50 of 34 nM while exhibiting no activity in cell lines without TrkA fusion. Comparable activity was observed in Ba/F3 cell line expressing MPRIP-NTRK1 fusion, demonstrating inhibition of TrkA driven cell proliferation irrespective of the N-terminal fusion with a different gene, MPRIP. In the KM12 xenograft model, treatment with GVK-TrkI at various doses and dosing regimens of 20 mpk bid, 50 mpk bid and 100 mpk qd resulted in dose dependent inhibition with almost 100% tumor growth inhibition (TGI) at 100 mpk qd with 3 out of 8 mice showing complete tumor regression. Besides, at 50 mpk in various dosing regimens of qd, bid and bid alternate day showed good correlation of TGI with compound exposure. GVK-TrkI was well-tolerated, causing no weight loss or death compared with vehicle control. Based on the evidence of ATP binding site mutations contributing to clinical resistance with kinase inhibitor drugs, GVK-TrkI is predicted to be effective as a second generation TrkA inhibitor with a non-ATP binding site mechanism compared to pan-Trk inhibitors currently being pursued in clinic. These results suggest that GVK-TrkI is an effective therapeutic option in TrkA driven cancers with TrkA gene fusions, activating mutations and cancers with NGF/TrkA overexpression resulting in constitutive activation of TrkA pathway. Citation Format: Vijaya G. Tirunagaru, Kumaragurubaran Nagaswamy, Sayan Mitra, Srinivas Maddi, Ram Sudheer Adluri, Hemant Joshi, Jang B. Gupta. Discovery and characterization of a selective TrkA inhibitor for the treatment of TrkA-driven cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2643.

The effects of platelet-activating factor (PAF) on cardiac function and con tractility and its mechanism of action are not fully understood. The authors investigated the effects of PAF in the absence or presence of a potent PAF antag onist CV-6209 on the cardiac function and contractility; lipid peroxidation prod uct malondialdehyde (MDA), an indirect measure of oxygen free radicals; serum creatine kinase (CK); blood lactate; and pH in anesthetized dogs. CV- 6209 (1 mg/kg, IV) did not produce significant changes in the various parame ters studied. PAF (1 μg/kg, IV) produced decreases in the cardiac function (cardiac index, left ventricular work index) and indices of cardiac contractility [(+) and (-) dp/dt, dp/dt at CPIP, (dp/dt)/IP, dp/dt at CPIP/IP, Vmax] and in creases in the systemic and pulmonary vascular resistance. It also produced increases in cardiac MDA, serum CK, blood lactate, and H+ and decreases in blood pH and HCO3- . CV-6209 completely prevented the PAF-induced changes in the hemodynamic and biochemical parameters. These results suggest that PAF-induced cardiac depression may be due to PAF-induced release of oxyradicals from neutrophils and that PAF antagonist may be useful in counteracting the deleterious effects of PAF on the cardiovas cular system.

The effects of .OH on the isolated tracheal smooth muscles (TSM), from control, polyethylene glycol (PEG)-glucose oxidase (GO)-, and GO+PEG catalase-treated rabbits were investigated. GO or GO+catalase were given intravenously each week for 4 mo. .OH produced relaxation of basal and ACh-precontracted tension in TSM of control rabbits. The relaxant effect was attenuated by removal of epithelium, whereas it was converted to contraction in the indomethacin-pretreated muscle. .OH produced contraction of TSM and ACh-precontracted muscle in GO-treated rabbits. The contractile response was abolished in preparations denuded of epithelium or pretreated with indomethacin. .OH produced relaxation in basal tension, but a small contraction in ACh-precontracted muscle of GO+catalase-treated rabbits. The contractile response to .OH was unaffected by indomethacin pretreatment; however, it was converted to relaxation in the preparations denuded of epithelium. Contractile response of TSM to ACh was augmented in deepithelialized, indomethacin-, or GO-treated preparations. H2O2 damaged the tracheal epithelium. These results suggest that 1) .OH-induced relaxation/contraction of TSM is partly epithelium dependent and is mediated by bronchodilator/bronchoconstrictor arachidonic acid metabolites, 2) the airway smooth muscle with healthy epithelium responds to .OH differently from those with dysfunctional or damaged epithelium, and 3) hyperresponsiveness of the airways to ACh may be related to the epithelial dysfunction.

We investigated the effects of H2O2 generated by glucose (G) and glucose oxidase (GO) on the isolated rabbit tracheal smooth muscle suspended in Krebs-Ringer solution. H2O2 generated by G+GO was measured with luminol-dependent chemiluminescence. G+GO in the concentrations of 1x (1.80 microM G, 0.075 U/ml GO) and 2, 4, and 8x generated 1.35, 3.2, 6.10, and 6.00 microM of H2O2, respectively. H2O2 produced relaxation of rabbit tracheal smooth muscle, relaxed acetylcholine (ACh)-precontracted muscle, and reduced muscle responsiveness to ACh. These effects were concentration dependent. H2O2, however, produced contraction of guinea pig tracheal smooth muscle. Catalase completely inhibited the H2O2-induced relaxation of ACh-precontracted tracheal smooth muscle. H2O2-induced relaxation was greater in preparations with intact epithelium (65%) than in those denuded of epithelium (40%). The relaxant effects of H2O2 in the presence of an inhibitor of nitric oxide synthesis (NG-monomethyl-L-arginine), an inhibitor of guanylate cyclase (methylene blue), an inhibitor of cyclooxygenase (indomethacin), and an ATP-sensitive K+ channel blocker (glipizide) were 44, 44, 39, and 48%, respectively. H2O2-induced relaxation in the presence of indomethacin in preparations with denuded epithelium was 29%. These results suggest that H2O2-induced relaxation of tracheal smooth muscle is partly epithelium dependent and is mediated by inhibitory arachidonic acid metabolites, epithelium-derived relaxing factor (nitric oxide), ATP-sensitive K+ channels, and the synthesis and release of prostaglandins from epithelium and the underlying smooth muscle.

Introduction: The highly selective, ATP competitive PLK2 inhibitor GBO-006 was previously shown by us to arrest growth of triple negative breast cancer (TNBC) at 30mg/kg dose (mpk) in MDAMB-231 xenograft model. However, the maximum formulation strength was 20mg/ml and was not adequate for toxicology studies. GBO-006 was found to be crystalline and poorly soluble ( Experimental procedures: Nanosizing of GBO-006 by ‘bottom-up’ and ‘combination of bottom-up and top-down’ technologies did not yield particles in the desirable nanosize range. Nanosizing GBO-006 to less than 400 nm (d0.9) particle sizes was feasible by top-down technology using bead milling and a high shear microfluidics processor. During initial trials, lower strength formulations (5 to 25 mg/mL) were nanosized and stabilized using bead milling with non-ionic surfactant(s), Tween 80 & poloxamer 188, in addition to polymers such as PVP K12. Microfluidization was not pursued further due to clogging of the interaction chamber at higher concentrations (50 mg/mL). Results: A crystalline, lipid nanoparticle of GBO-006 was feasible by bead milling and further assessed for pharmacokinetic evaluation and efficacy studies. Intraperitoneal dose escalation studies in mice showed a dose-dependent linear increase in plasma exposure of GBO-006. Fifty percent reduction in MDAMB-231 xenograft tumor volume was observed with 1.5 mpk of GBO-006 after qd dosing. Significant accumulation of GBO-006 was observed in spleen and liver upon chronic dosing (21 days). We hypothesized that accumulation was likely due to reticulo-endothelial system (RES) mediated uptake, which was further proven by in vitro experiments with differentiated macrophages. Conclusion: We have successfully developed a nanosuspension formulation for GBO-006. Notably, this nanosuspension showed similar efficacy to previous formulations at much lower doses (1.5 mpk), however particle size of 260 nm accentuated RES uptake. Ongoing studies are focused on decreasing particle size below 150 nm and incorporating a negative zeta potential to bypass RES uptake and minimize tissue distribution. Citation Format: Vijaya G. Tirunagaru, Arnab Roy Chowdhury, Jeyaraj Athisayamani Duraiswamy, Srinivasa Rao Maddi, Sayan Mitra, Chandra Deb, Ram Sudheer Adluri, Jang B. Gupta. Development of lipid-based nanosuspension formulation of first-in-class PLK2 inhibitor GBO-006 to treat triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1661. doi:10.1158/1538-7445.AM2015-1661

We investigated the effect of exogenously generated superoxide anions (O(2)(-)), hydrogen peroxide (H(2)O(2)) and hydroxyl radicals (.OH) on isolated rabbit tracheal smooth muscle suspended in Krebs-Ringer solution. The ability of oxygen free radicals (OFRs) to affect acetyicholine (Ach)-induced contraction in these muscles was also investigated. OFRs, in general, produced a concentration-dependent relaxation of the tracheal smooth muscle in the doses used. However, in large concentrations, O(2)(-) and H(2)O(2) produced effects which were smaller than those obtained with lower concentrations. The relaxant effects of these oxyradicals were progressive and lasted throughout the 20min observation period. At all concentrations used, the OFRs tended to abolish or reduce Ach-induced contraction in a concentration-dependent manner. O(2)(-) was more potent than H(2)O(2) or DHF in relaxing the Ach-precontracted muscle and in inhibiting the response of the muscle to Ach. OFR-induced relaxation of the Ach-contracted muscle was not due to inactivation of the Ach by OFRs. Relaxation produced by OFRs was greater in preparations with intact epithelium than in those denuded of epithelium. The relaxant effects were blocked by indomethacin, a cyclooxygenase inhibitor. OFRs in the presence of indomethacin produced contraction only in the preparations with intact epithelium, suggesting a release of contractile factor(s) from epithelium. These results suggest that OFRs relax rabbit tracheal smooth muscle. The relaxation appears to be mediated through the synthesis and release of prostaglandins from the epithelium and smooth muscles.

Exogenous oxygen free radicals (OFRs) have been reported to depress cardiac function and contractility. The decrease in cardiac function and contractility in chronic volume overload could be due to increased levels of OFRs. We therefore investigated the effects of volume overload of 9 months duration in the absence and presence of catalase treatment on cardiac function and contractility; left ventricular malondialdehyde (MDA), a lipid peroxidation product; and left ventricular antioxidant reserve, measured as left ventricular chemiluminescence (LV-CL) in dogs. Dogs were divided into three groups: group I, control; group II, mitral regurgitation (MR) of 9 months duration; and group III, same as group II but supplemented with polyethylene glycol (PEG)-catalase (5000IU/kg/week, intravenously).

Various methods have been used in the past to assess the implication of oxygen free radicals (OFR) in ischemia-reperfusion-induced cardiac injury. Luminol-enhanced tert-butyl-initiated chemiluminescence in cardiac tissue reflects oxidative stress and is a very sensitive method. It was used to elucidate the role of OFR in cardiac injury due to ischemia and reperfusion. Studies were conducted on perfused isolated rabbit hearts in three groups (n = 8 in each): I, control; II, submitted to global ischemia for 30 min; III, submitted to ischemia for 30 min followed by reperfusion for 60 min. The heart tissue was then assayed for chemiluminescence (CL); content of malondialdehyde (MDA), an indicator of OFR-induced cardiac injury; and activity of tissue levels of antioxidants [superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px)]. The control values for left and right ventricular CL and malondialdehyde were 81.1 +/- 15.4 (S.E.) and 182.4 +/- 50.3 (S.E.), mv.min.mg protein-1; and 0.024 +/- 0.006 (S.E.) and 0.324 +/- 0.005 (S.E.) nmoles.mg protein-1 respectively. Ischemia produced an increase in the cardiac CL (3.3 to 4.4 fold) and MDA content (2 to 2.6 fold). Reperfusion following ischemia also produced similar changes in CL and MDA content. The control values for activity of left ventricular SOD, catalase, and GSH-Px were 45.77 +/- 1.73 (S.E.) U.mg protein-1, 5.35 +/- 0.51 (S.E.) K.10(-3).sec-1.mg protein-1, and 77.50 +/- 7.70 (S.E.) nmoles NADPH.min-1.mg protein-1 respectively. Activities of SOD and catalase decreased during ischemia but were similar to control values in ischemic-reperfused hearts. The GSH-Px activity of left ventricle was unaffected by ischemia, and ischemia-reperfusion. GSH-Px activity of the right ventricle increased with ischemia, and ischemic-reperfusion. These results indicate that cardiac tissue chemiluminescence would be a useful and sensitive tool for the detection of oxygen free radical-induced cardiac injury.

It has been suggested that oxygen free radicals (OFR) depress the excitation-contraction coupling in cardiac muscle. It is possible that a decrease in the cardiac contractility in the failing heart may be due to an increased OFR producing activity of polymorphonuclear (PMN) leukocytes. We studied the OFR producing activity (chemiluminescence) of PMN leukocytes from blood in dogs with heart failure due to chronic volume overload. The animals were divided into two groups: I) normal, (n = 10): II) dogs with mitral insufficiency (MI) of 6 to 9 months duration, (n = 10). Hemodynamic studies were done to establish the presence of heart failure. Blood samples were collected to measure PMN leukocyte chemiluminescence. There was a decrease in the cardiac index and index of myocardial contractility (dp/dt/IIP) and an increase in the left ventricular end-diastolic pressure in dogs with MI indicating left ventricular failure. The peak chemiluminescent activity of the PMN leukocytes in blood of dogs with failure was about four folds greater than that in the blood from normal dogs. These results suggest that there may be an increased OFR generation in dogs with volume overload heart failure. The decrease in the myocardial contractility in the failing heart might be due to an increase in the OFR produced by the PMN leukocytes.

Mongrel dogs of either sex with ligation of their left anterior descending coronary artery were employed for the present study to investigate the biochemical basis of the cardioprotective action of dilazep. Dilazep (0.2 mg/kg intravenous) was administered 20 minutes after coronary arterial occlusion. Ligation of the artery for 40 minutes increased the tissue lactate concentration and decreased the adenosine triphosphate molecules within the occluded bed. Dilazep decreased the lactate concentration and improved the adenosine triphosphate content of this bed significantly. Dilazep also lowered the tissue cyclic adenosine monophosphate concentration and free fatty acid extraction of the ischemic myocardium as studied 40 minutes after coronary arterial ligation. It can be inferred that less free fatty acid extraction and calcium antagonistic action of dilazep helps in restoring mitochondrial function. Furthermore, decreased tissue lactate concentration resulted after better perfusion of the occluded bed and helped in greater generation of adenosine triphosphate molecules. These favorable biochemical and metabolic changes contribute to the cardioprotective action of dilazep.