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Endothelin B (ETB) receptors present in abundance the central nervous system (CNS) have been shown to have significant implications in its development and neurogenesis. We have targeted ETB receptors stimulation using a highly specific agonist, IRL-1620, to treat CNS disorders. In a rat model of cerebral ischemia intravenous administration IRL-1620 significantly reduced infarct volume and improved neurological and motor functions compared to control. This improvement, in part, is due to an increase in neuroregeneration. We also investigated the role of IRL-1620 in animal models of Alzheimer's disease (AD). IRL-1620 improved learning and memory, reduced oxidative stress and increased VEGF and NGF in Aβ treated rats. IRL-1620 also improved learning and memory in an aged APP/PS1 transgenic mouse model of AD. These promising findings prompted us to initiate human studies. Successful chemistry, manufacturing and control along with mice, rat and dog toxicological studies led to completion of a human Phase I study in healthy volunteers. We found that a dose of 0.6 μg/kg of IRL-1620 can be safely administered, three times every four hours, without any adverse effect. A Phase II clinical study with IRL-1620 has been initiated in patients with cerebral ischemia and mild to moderate AD. [ABSTRACT FROM AUTHOR]

Background: Centhaquine (CQ) (Lyfaquin ® ) is in late stage clinical development as a safe and effective first-in-class resuscitative agent for hemorrhagic shock patients (NCT02408731, NCT04056065, and NCT04045327). Acute kidney injury (AKI) is known to be associated with hemorrhagic shock. Hence, effect of CQ on protection of kidneys from damage due to hemorrhagic shock was investigated. Methods: To assess effect of CQ on AKI in shock, we created a rat model with hemorrhagic shock and AKI. Renal arteries were clamped and de-clamped to induce AKI like ischemia/reperfusion model and hemorrhage was carried out by withdrawing blood for 30 min. Rats were resuscitated with CQ (0.02 mg/kg) for 10 min. MAP, heart rate (HR), and renal blood flow (RBF) were monitored for 120 min. Results: CQ produced a significant improvement in RBF compared to vehicle ( p < 0.003) even though MAP and HR was similar in CQ and vehicle groups. Blood lactate level was lower ( p = 0.0064) in CQ than vehicle at 120 min post-resuscitation. Histopathological analysis of tissues indicated greater renal damage in vehicle than CQ. Western blots showed higher HIF-1α ( p = 0.0152) and lower NGAL ( p = 0.01626) levels in CQ vs vehicle. Immunofluorescence in the kidney cortex and medulla showed significantly higher ( p < 0.045) expression of HIF-1α and lower expression of Bax ( p < 0.044) in CQ. Expression of PHD 3 ( p < 0.0001) was higher, while the expression of Cytochrome C ( p = 0.01429) was lower in the cortex of CQ than vehicle. Conclusion: Results show CQ (Lyfaquin ® ) increased renal blood flow, augmented hypoxia response, decreased tissue damage and apoptosis following hemorrhagic shock induced AKI, and may be explored to prevent/treat AKI. Translational Statement: Centhaquine (CQ) is safe for human use and currently in late stage clinical development as a first-in-class resuscitative agent to treat hemorrhagic shock. In the current study, we have explored a novel role of CQ in protection from hemorrhagic shock induced AKI, indicating its potential to treat/prevent AKI., Competing Interests: AG is an employee of Pharmazz, Inc., and has issued and pending patents related to this study. Midwestern University is the patent assignee with AG as an inventor of this technology, while Pharmazz Inc. holds its exclusive worldwide license and is engaged in the clinical development and commercialization of centhaquine (CQ) for human use. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ranjan, Zhang, Briyal and Gulati.)

Background: Preterm infants experience rapid brain growth during early post-natal life making them vulnerable to drugs acting on central nervous system. Morphine is administered to premature neonates for pain control and caffeine for apnea of prematurity. Simultaneous use of morphine and caffeine is common in the neonatal intensive care unit. Prior studies have shown acute neurotoxicity with this combination, however, little information is available on the mechanisms mediating the neurotoxic effects. The objective of this study was to determine the effects of morphine and caffeine, independently and in combination on mitochondrial dysfunction (Drp1 and Mfn2), neural apoptosis (Bcl-2, Bax, and cell damage) and endothelin (ET) receptors (ET A and ET B ) in neonatal rat brain. Methods: Male and female rat pups were grouped separately and were divided into four different subgroups on the basis of treatments-saline (Control), morphine (MOR), caffeine (CAFF), and morphine + caffeine (M+C) treatment. Pups in MOR group were injected with 2 mg/kg morphine, CAFF group received 100 mg/kg caffeine, and M+C group received both morphine (2 mg/kg) and caffeine (100 mg/kg), subcutaneously on postnatal days (PND) 3-6. Pups were euthanized at PND 7, 14, or 28. Brains were isolated and analyzed for mitochondrial dysfunction, apoptosis markers, cell damage, and ET receptor expression via immunofluorescence and western blot analyses. Results: M+C showed a significantly higher expression of Bax compared to CAFF or MOR alone at PND 7, 14, 28 in female pups ( p < 0.05) and at PND 7, 14 in male pups ( p < 0.05). Significantly ( p < 0.05) increased expression of Drp1, Bax, and suppressed expression of Mfn2, Bcl-2 at PND 7, 14, 28 in all the treatment groups compared to the control was observed in both genders. No significant difference in the expression of ET A and ET B receptors in male or female pups was seen at PND 7, 14, and 28. Conclusion: Concurrent use of morphine and caffeine during the first week of life increases apoptosis and cell damage in the developing brain compared to individual use of caffeine and morphine., (Copyright © 2020 Kasala, Briyal, Prazad, Ranjan, Stefanov, Donovan and Gulati.)

The development of effective drugs for stroke is urgently required as it is the 2nd largest killer in the world and its incidence is likely to increase in the future. We have demonstrated cerebral endothelin B receptors (ETBR) as a potential target to treat acute cerebral ischemic stroke. However, the mechanism of ETBR mediated neural regeneration and repair remains elusive. In this study, a permanent middle cerebral artery occluded (MCAO) rat model was used. Sovateltide (an ETBR agonist) injected intravenously showed better survival and neurological and motor function improvement than control. Higher neuronal progenitor cells (NPCs) differentiation along with better mitochondrial morphology and biogenesis in the brain of sovateltide rats were noted. Exposure of cultured NPCs to hypoxia and sovateltide also showed higher NPC differentiation and maturation. This study shows a novel role of ETBR in NPCs and mitochondrial fate determination in cerebral ischemia, and in improving neurological deficit after stroke.

Oxidative stress results from excessive reactive oxygen species formation and/or inadequate antioxidant defense. Premature and critically ill infants are especially susceptible due to an immature intrinsic antioxidant system that cannot fully compensate for a free radical load. Oxidative stress is also associated with endothelial dysfunction and alterations in Endothelin-1 (ET-1) signaling pathways. However, the effects of the complex interaction between oxidative stress and ET-1 in newborns are not well-understood. The objective of this pilot study was to determine the relationship between levels of common oxidative stress biomarkers [glutathione (GSH), malondialdehyde (MDA)] and ET-1 in newborns of different gestational ages. In a level IV NICU, 63 neonates were prospectively enrolled and divided into groups based on gestational age at birth: Early Preterm (24 0/7-30 6/7 weeks), Late Preterm (31 0/7-36 6/7 weeks), and Term (37 0/7-42 weeks). Umbilical cord (1.5 mL) and 24(±4) h of life (24 h) (1 mL) blood samples were collected for GSH, MDA, and ET-1 analyses. GSH, MDA, and ET-1 were determined using established methodology. Mean cord MDA levels for all age groups, Early Preterm (2.93 ± 0.08 pg/ml), Late Preterm (2.73 ± 0.15 pg/ml), and Term (2.92 ± 0.13 pg/ml), were significantly higher than those at 24 h of life ( p < 0.001). Mean cord ET-1 levels were significantly higher than 24 h samples in both Early and Late Preterm groups ( p < 0.05). Cord and 24 h ET-1 levels did not correlate with MDA and GSH levels at birth (r 2 = 0.03, p > 0.05 and r 2 = 0.001, p > 0.05, respectively) or 24 h of life (r 2 = 0.001, p > 0.05 and r 2 = 0.03, p > 0.05, respectively). Preterm neonates exposed to prenatal corticosteroids (1.87 ± 0.31 pg/ml) had lower cord MDA levels than non-exposed neonates (2.85 ± 0.12 pg/ml) ( p < 0.05). Both cord and 24 h OS markers were significantly higher in neonates treated with oxygen therapy ( p < 0.005 and p < 0.05, respectively) than those who did not receive supplemental oxygen. Oxidative stress markers (MDA and GSH) and ET-1 levels act independently. MDA is higher in cord blood than at 24 h of life regardless of gestational age. In preterm neonates, ET-1 levels are higher in umbilical cord blood compared to 24 h of life., (Copyright © 2020 Stefanov, Briyal, Pais, Puppala and Gulati.)

Introduction: Neonatal HIE is associated with high morbidity and mortality. Current research, is focused on developing alternative treatments to therapeutic hypothermia for treatment of HIE. The endocannabinoid system is known to be influential in neuronal protection. Activation of brain CB2 receptors, has been shown to reduce inflammatory markers and decrease infarct volume in adult cerebral ischemic models. Methods: Rat pups were divided into six groups: 1-Placebo; 2-JWH133; 3-HIE + Placebo; 4-HIE + JWH133; 5-HIE + Hypothermia + Placebo; and 6-HIE + Hypothermia + JWH133. HIE was induced in in groups 3-6 by right carotid ligation on postnatal day 7 followed by placement in a hypoxic chamber. Pups in groups 5 and 6 were treated with hypothermia. Western blot analysis was used to analyze brain tissue for acute inflammatory markers (IL-6, TNFα, MIP1α, and RANTES), immunoregulatory cytokines (TGFβ and IL-10), and CB2 receptor expression. DNA fragmentation in the brains of pups was determined via TUNEL staining post HIE. Results: The combination of JWH133 and hypothermia significantly reduced tumor necrosis factor α (TNFα) (-57.7%, P = 0.0072) and macrophage inflammatory protein 1α (MIP1α) (-50.0%, P = 0.0211) as compared to placebo. DNA fragmentation was also significantly reduced, with 6.9 ± 1.4% TUNEL+ cells in HIE+JWH133 and 12.9 ± 2.2% in HIE+Hypothermia + JWH133 vs. 16.6 ± 1.9% in HIE alone. No significant difference was noted between groups for the expression of interleukins 6 and 10, RANTES, or TGFβ. After 8 h, CB2 receptor expression increased nearly 2-fold in the HIE and HIE + JWH133 groups (+214%, P = 0.0102 and +198%, P = 0.0209, respectively) over placebo with no significant change in the hypothermia groups. By 24 h post HIE, CB2 receptor expression was elevated over five times that of placebo in the HIE ( P < 0.0001) and HIE + JWH133 ( P = 0.0002) groups, whereas hypothermia treatment maintained expression similar to that of placebo animals. Conclusion: These results indicate that the combination of CB2 agonist and hypothermia may be neuroprotective in treating HIE, opening the door for further studies to examine alternative or adjuvant therapies to hypothermia., (Copyright © 2020 Gupta, Hornick, Briyal, Donovan, Prazad and Gulati.)

Endothelin-B receptor agonist, IRL-1620, provides significant neuroprotection following cerebral ischemia in rats. Whether this neuroprotection is due to inhibition of apoptosis is unknown. IRL-1620-treated rats following permanent middle cerebral artery occlusion (MCAO) showed significant improvement in neurological and motor functions along with a decrease in infarct volume at 24 h (-81.3%) and day 7 (-73.0%) compared to vehicle group. Cerebral blood flow (CBF) significantly improved in IRL-1620-treated animals compared to vehicle by day 7 post MCAO. IRL-1620-treated rats showed an increase in phospho-Akt and decrease in Bad level 7 h post-occlusion compared to vehicle, while Akt and Bad expression was similar in cerebral hemispheres at 24 h post-MCAO. The phospho-Bad level was lower in vehicle- but not in IRL-1620-treated rats at 24 h. Anti-apoptotic Bcl-2 expression decreased, while pro-apoptotic Bax expression increased in vehicle-treated MCAO rats, these changes were attenuated (P < 0.01) by IRL-1620. Mitochondrial membrane-bound Bax intensity significantly decreased in IRL-1620 compared to vehicle-treated MCAO rats. IRL-1620 treatment reduced (P < 0.001) the number of TUNEL-positive cells compared to vehicle at 24 h and day 7 post MCAO. The results demonstrate that IRL-1620 is neuroprotective and attenuates neural damage following cerebral ischemia in rats by increasing CBF and reducing apoptosis.

Vancomycin has been associated with acute kidney injury in preclinical and clinical settings; however, the precise exposure profiles associated with vancomycin-induced acute kidney injury have not been defined. We sought to determine pharmacokinetic/pharmacodynamics indices associated with the development of acute kidney injury using sensitive urinary biomarkers. Male Sprague-Dawley rats received clinical-grade vancomycin or normal saline as an intraperitoneal injection. Total daily doses between 0 and 400 mg/kg of body weight were administered as a single dose or 2 divided doses over a 24-h period. At least five rats were utilized for each dosing protocol. A maximum of 8 plasma samples per rat were obtained, and urine was collected over the 24-h period. Kidney injury molecule-1 (KIM-1), clusterin, osteopontin, cystatin C, and neutrophil gelatinase-associated lipocalin levels were determined using Milliplex multianalyte profiling rat kidney panels. Vancomycin plasma concentrations were determined via a validated high-performance liquid chromatography methodology. Pharmacokinetic analyses were conducted using the Pmetrics package for R. Bayesian maximal a posteriori concentrations were generated and utilized to calculate the 24-h area under the concentration-time curve (AUC), the maximum concentration ( C ), and the minimum concentration. Spearman's rank correlation coefficient ( max ), and the minimum concentration. Spearman's rank correlation coefficient ( r s ) was used to assess the correlations between exposure parameters, biomarkers, and histopathological damage. Forty-seven rats contributed pharmacokinetic and toxicodynamic data. KIM-1 was the only urinary biomarker that correlated with both composite histopathological damage ( r s = 0.348, P = 0.017) and proximal tubule damage ( r s = 0.342, P = 0.019). The vancomycin AUC and C max were most predictive of increases in KIM-1 levels ( r s = 0.438 and P = 0.002 for AUC and r s = 0.451 and P = 0.002 for C max ). Novel urinary biomarkers demonstrate that kidney injury can occur within 24 h of vancomycin exposure as a function of either AUC or C max ., (Copyright © 2017 American Society for Microbiology.)

Aim: ET A receptor antagonists reverse opioid tolerance but the involvement of ET B receptors is unknown. In morphine or oxycodone tolerant mice we investigated (1) the effect of ET B receptor agonist, IRL-1620, on analgesic tolerance; (2) changes in expression of the brain ET A and ET B receptors; and (3) alterations in the brain VEGF, NGF, PI3K and notch-1 expression., Main Methods: Body weight, body temperature, and tail-flick latency were assessed before and after a challenge dose of morphine or oxycodone in vehicle or IRL-1620 treated mice. Expression studies were carried out using Western blots., Key Findings: Tail flick latency to a challenge dose of opioid was significantly increased by IRL-1620 from 39% to 100% in morphine tolerant and from 8% to 83% in oxycodone tolerant mice. Morphine or oxycodone did not alter ET A or ET B receptor expression. IRL-1620 had no effect on ET A however it increased (61%) expression of ET B receptors. IRL-1620-induced increase in ET B receptor expression was attenuated by morphine (39.8%) and oxycodone (51.8%). VEGF expression was not affected by morphine or oxycodone and was unaltered by IRL-1620. However, NGF and PI3K expression was decreased (P < 0.001) by morphine and oxycodone and was unaffected by IRL-1620. Notch-1 expression was not altered by morphine, oxycodone or IRL-1620., Significance: ET B receptor agonist, IRL-1620, restored analgesic tolerance to morphine and oxycodone, but it did not affect morphine and oxycodone induced decrease in NGF/PI3K expression. It is concluded that IRL-1620 attenuates opioid tolerance without the involvement of NGF/PI3K pathway.