Carter, Rebecca W., McKenzie Begaye, and Nancy L. Kanagy. Acute and chronic NOS inhibition enhances ␣2-adrenoreceptor-stimulated RhoA and Rho kinase in rat aorta.

The availability of effective, reliably accessible, and affordable treatments for snakebite envenoming is a critical and long unmet medical need. Recently, small, synthetic toxin-specific inhibitors with oral bioavailability used in conjunction with antivenom have been identified as having the potential to greatly improve outcomes after snakebite. Varespladib, a small, synthetic molecule that broadly and potently inhibits secreted phospholipase A2 (sPLA2s) venom toxins has renewed interest in this class of inhibitors due to its potential utility in the treatment of snakebite envenoming. The development of varespladib and its oral dosage form, varespladib-methyl, has been accelerated by previous clinical development campaigns to treat non-envenoming conditions related to ulcerative colitis, rheumatoid arthritis, asthma, sepsis, and acute coronary syndrome. To date, twenty-nine clinical studies evaluating the safety, pharmacokinetics (PK), and efficacy of varespladib for non-snakebite envenoming conditions have been completed in more than 4600 human subjects, and the drugs were generally well-tolerated and considered safe for use in humans. Since 2016, more than 30 publications describing the structure, function, and efficacy of varespladib have directly addressed its potential for the treatment of snakebite. This review summarizes preclinical findings and outlines the scientific support, the potential limitations, and the next steps in the development of varespladib’s use as a snakebite treatment, which is now in Phase 2 human clinical trials in the United States and India.

Introduction: Snakebite is an urgent, unmet global medical need causing significant morbidity and mortality worldwide. Varespladib is a potent inhibitor of venom secretory phospholipase A2 (sPLA2) that can be administered orally via its prodrug, varespladib-methyl. Extensive preclinical data support clinical evaluation of varespladib as a treatment for snakebite envenoming (SBE). The protocol reported here was designed to evaluate varespladib-methyl for SBE from any snake species in multiple geographies. Methods and Analysis: BRAVO (Broad-spectrum Rapid Antidote: Varespladib Oral for snakebite) is a multicenter, randomized, double-blind, placebo-controlled, phase 2 study to evaluate the safety, tolerability, and efficacy of oral varespladib-methyl plus standard of care (SoC) vs. SoC plus placebo in patients presenting with acute SBE by any venomous snake species. Male and female patients 5 years of age and older who meet eligibility criteria will be randomly assigned 1:1 to varespladib-methyl or placebo. The primary outcome is the Snakebite Severity Score (SSS) that has been modified for international use. This composite outcome is based on the sum of the pulmonary, cardiovascular, nervous, hematologic, and renal systems components of the updated SSS. Ethics and Dissemination: This protocol was submitted to regulatory authorities in India and the US. A Clinical Trial No Objection Certificate from the India Central Drugs Standard Control Organisation, Drug Controller General-India, and a Notice to Proceed from the US Food and Drug Administration have been obtained. The study protocol was approved by properly constituted, valid institutional review boards or ethics committees at each study site. This study is being conducted in compliance with the April 1996 ICH Guidance for Industry GCP E6, the Integrated Addendum to ICH E6 (R2) of November 2016, and the applicable regulations of the country in which the study is conducted. The trial is registered on Clinical trials.gov, NCT#04996264 and Clinical Trials Registry-India, 2021/07/045079 000062.

PKC augments calcium sensitivity in spontaneously hypertensive rats and contributes to alpha(2)-adrenergic receptor (AR) contraction in rabbit saphenous vein. We showed previously that denuded aortic rings from N(omega)-nitro-l-arginine-treated hypertensive rats (LHR) contract more to CaCl(2) and to the alpha(2)-AR agonist UK-14304 than do rings from normotensive rats (NR). We hypothesized that enhanced PKC activity or a change in PKC isoform contributes to augmented calcium sensitivity and enhanced alpha(2)-AR contraction in LHR aorta. Current studies demonstrate that non-isoform-specific PKC inhibitors reduced UK-14304 contraction in both NR and LHR aorta. However, the calcium-dependent PKC inhibitor Gö-6976 only attenuated contraction in LHR aorta. Additionally, UK-14304 translocated PKC-delta to the membrane in NR aorta, whereas PKC-alpha was translocated to the membrane in LHR aorta. Finally, in ionomycin-permeabilized aorta Gö-6976 eliminated enhanced basal and augmented alpha(2)-AR-stimulated calcium sensitivity in LHR aorta but did not affect NR contraction. Together, these data suggest that PKC-alpha contributes to augmented calcium sensitivity and alpha(2)-AR reactivity after chronic nitric oxide synthase inhibition hypertension.

We have demonstrated enhanced contractile sensitivity to the alpha(2)-adrenoreceptor (alpha(2)-AR) agonist UK-14304 in arteries from rats made hypertensive with chronic nitric oxide synthase (NOS) inhibition (LHR) compared with arteries from normotensive rats (NR); additionally, this contraction requires Ca(2+) entry. We hypothesized that tyrosine kinases augment alpha(2)-AR contraction in LHR arteries by increasing Ca(2+). The tyrosine kinase inhibitor tyrphostin 23 significantly attenuated UK-14304 contraction of denuded thoracic aortic rings from NR and LHR. However, tyrphostin 23 did not alter UK-14304 contraction in ionomycin-permeabilized aorta, which indicates that tyrosine kinases regulate intracellular Ca(2+) concentration. The Src family inhibitor PP1 and the epidermal growth factor receptor kinase inhibitor AG-1478 did not alter alpha(2)-AR contraction, whereas the mitogen-activated protein kinase extracellular signal-regulated kinase kinase inhibitor PD-98059 attenuated the contraction. Contraction to CaCl(2) in ionomycin-permeabilized LHR rings was greater than in NR rings. UK-14304 augmented CaCl(2) contraction in ionomycin-permeabilized rings from both groups but to a greater extent in LHR aorta. Together, these data suggest that alpha(2)-AR stimulates contraction via two pathways. One, which is enhanced with NOS inhibition hypertension, activates Ca(2+) sensitivity and is independent of tyrosine kinases. The other is tyrosine kinase dependent and regulates intracellular Ca(2+) concentration.

This laboratory exercise uses a simple preparation and a straightforward protocol to illustrate many of the basic principles of vascular biology covered in an introductory physiology course. The design of this laboratory allows students to actively participate in an exercise demonstrating the regulation of arterial tone by endothelial and extrinsic factors. In addition, this hands-on laboratory allows students to gather data using well-known basic biomedical research techniques. Specifically, students are introduced to an isolated organ-chamber technique that is widely used to study cellular mechanisms of many tissues including vascular smooth muscle contraction and dilation. On the basis of student evaluations, participation in the experiments and interpreting data reinforce lecture materials on smooth muscle and endothelial cell function and illustrate mechanisms regulating vascular tone. Students come away with a greater understanding of vascular biology, a deeper appreciation of integrative physiology, and an understanding of the process of conducting tissue-chamber experiments.

The African viperid snake genera Atheris, Cerastes, and Proatheris are closely related, similar in size, but occupy extremely divergent ecological niches (arboreal in tropical rainforests, fossorial in deserts, and swamp-dwelling, respectively). Their venoms have not previously been subjected to comparative analyses for their action upon the coagulation of blood, most notably with significant data deficiencies from Atheris and Proatheris. In contrast, the closely related genus Echis is well-documented as capable of producing potent procoagulant effects. In light of this, we set out to compare the coagulotoxic actions of Atheris ceratophora, A. chlorechis, A. desaixi, A. nitschei, A. squamigera, C. cerastes, C. cerastes gasperettii, C. vipera, and Proatheris superciliaris and explore potential pharmacological interventions to reestablish normal blood coagulation. All venoms displayed extremely potent procoagulant effects, over twice as fast as the most potent Echis reported to date. Although Cerastes is used in the immunising mixture of two different regionally available antivenoms (Inoserp-MENA with C. cerastes, C. cerastes gasperettii, C. vipera and Saudi Arabian polyvalent with C. cerastes), none of the other species in this study are included in the immunising mixture of any antivenom. Notably, all the Cerastes species were only neutralised by the Inoserp-MENA antivenom. C. cerastes venom was not neutralised well by the Saudi Arabian antivenom, with the low levels of recognition for any of the Cerastes venoms suggesting a strong regional variation in the venom of this species, as the C. cerastes venom tested was of African (Tunisian) origin versus Saudi locality used in that antivenom’s production. The other antivenoms (Micropharm EchiTAbG, ICP EchiTAb-Plus-ICP, Inosan Inoserp Pan-Africa, Premium Serums PANAF Sub-Sahara Africa, South African Vaccine Producers Echis, South African Vaccine Producers Polyvalent) all displayed trivial-to-no ability to neutralise the procoagulant toxicity of any of the Atheris, Cerastes, or Proatheris venoms. Comparative testing of the enzyme inhibitors DMPS, marimastat, and prinomastat, revealed a very potent neutralising capacity of marimastat, with prinomastat showing lower but still significant potency at the same molar concentration, while a 5× molar concentration of DMPS had no apparent effect on procoagulant venom effects normalized by the other inhibitors. These results and methods contribute to the body of knowledge of potential clinical effects and data necessary for evidence-based advancement of clinical management strategies.