Mechanistic Insights of Neuroprotective Efficacy of Verapamil-Loaded Carbon Quantum Dots against LPS-Induced Neurotoxicity in Rats.

Alzheimer's disease (AD) is a neurodegenerative disease that badly impacts patients and their caregivers. AD is characterized by deposition of amyloid beta (Aß) and phosphorylated tau protein (pTau) in the brain with underlying neuroinflammation. We aimed to develop a neuroprotective paradigm by loading verapamil (VRH) into hyaluronic acid-modified carbon quantum dots (CQDs) and comparing its effectiveness with the free form in an AD-like model in rats induced by lipopolysaccharide (LPS). The experimental rats were divided into seven groups: control, LPS, CQDs, early free VRH (FVRH), late FVRH, early verapamil carbon quantum dots (VCQDs), and late VCQDs. Characterizations of VCQDs, the behavioral performance of the rats, histopathological and immunohistochemical changes, some AD hallmarks, oxidative stress biomarkers, neuro-affecting genes, and DNA fragmentation were determined. VRH was successfully loaded into CQDs, which was confirmed by the measured parameters. VRH showed enhancement in cognitive functions, disruption to the architecture of the brain, decreased Aß and pTau, increased antioxidant capacity, modifiable expression of genes, and a decline in DNA fragmentation. The loaded therapy was superior to the free drug. Moreover, the early intervention was better than the late, confirming the implication of the detected molecular targets in the development of AD. VRH showed multifaceted mechanisms in combating LPS-induced neurotoxicity through its anti-inflammatory and antioxidant properties, thereby mitigating the hallmarks of AD. Additionally, the synthesized nanosystem approach exhibited superior neuroprotection owing to the advantages offered by CQDs. However, finding new actionable biomarkers and molecular targets is of decisive importance to improve the outcomes for patients with AD.

Intracoronary adenosine compared with adrenaline and verapamil in the treatment of no-reflow phenomenon following primary PCI in STEMI patients.

BACKGROUND: no-reflow can complicate up to 25% of pPCI and is associated with significant morbidity and mortality. We aimed to compare the outcomes of intracoronary epinephrine and verapamil with intracoronary adenosine in the treatment of no-reflow after primary percutaneous coronary intervention (pPCI). METHODS: 108 STEMI patients had no-reflow during pPCI were assigned into four groups. Group 1, in which epinephrine and verapamil were injected through a well-cannulated guiding catheter. Group 2, in which same drugs were injected in the distal coronary bed through a microcatheter or perfusion catheter. Group 3, in which adenosine was injected through a guiding catheter. Group 4, in which adenosine was injected in distal coronary bed. Primary end point was the achievement of TIMI III flow and MBG II or III. Secondary end point was major adverse cardiovascular and cerebrovascular events (MACCEs) during hospital stay. RESULTS: The study groups did not differ in their baseline characteristics. Primary end point was achieved in 15 (27.8%) patients in the guide-delivery arm compared with 34 (63%) patients in the local-delivery arm, p < 0.01. However, the primary end point did not differ between the epinephrine/verapamil group and the adenosine group (27 (50%) vs 22 (40.7%), p = 0.334). The secondary end points were similar between the study groups. CONCLUSION: Local delivery of epinephrine, verapamil and adenosine in the distal coronary bed is more effective in achieving TIMI III flow with MBG II or III compared with their guide-delivery in patients who suffered no-reflow during pPCI. There was no difference between epinephrine/verapamil Vs. adenosine.

Machine learning predicts cerebral vasospasm in patients with subarachnoid haemorrhage.

BACKGROUND: Cerebral vasospasm (CV) is a feared complication which occurs after 20-40% of subarachnoid haemorrhage (SAH). It is standard practice to admit patients with SAH to intensive care for an extended period of resource-intensive monitoring. We used machine learning to predict CV requiring verapamil (CVRV) in the largest and only multi-center study to date. METHODS: Patients with SAH admitted to UCLA from 2013 to 2022 and a validation cohort from VUMC from 2018 to 2023 were included. For each patient, 172 unique intensive care unit (ICU) variables were extracted through the primary endpoint, namely first verapamil administration or no verapamil. At each institution, a light gradient boosting machine (LightGBM) was trained using five-fold cross validation to predict the primary endpoint at various hospitalization timepoints. FINDINGS: A total of 1750 patients were included from UCLA, 125 receiving verapamil. LightGBM achieved an area under the ROC (AUC) of 0.88 > 1 week in advance and ruled out 8% of non-verapamil patients with zero false negatives. Our models predicted "no CVRV" vs "CVRV within three days" vs "CVRV after three days" with AUCs = 0.88, 0.83, and 0.88, respectively. From VUMC, 1654 patients were included, 75 receiving verapamil. VUMC predictions averaged within 0.01 AUC points of UCLA predictions. INTERPRETATION: We present an accurate and early predictor of CVRV using machine learning with multi-center validation. This represents a significant step towards optimized clinical management and resource allocation in patients with SAH. FUNDING: Robert E. Freundlich is supported by National Center for Advancing Translational Sciences federal grant UL1TR002243 and National Heart, Lung, and Blood Institute federal grant K23HL148640; these funders did not play any role in this study. The National Institutes of Health supports Vanderbilt University Medical Center which indirectly supported these research efforts. Neither this study nor any other authors personally received financial support for the research presented in this manuscript. No support from pharmaceutical companies was received.

Unraveling Verapamil's Multidimensional Role in Diabetes Therapy: From ß-Cell Regeneration to Cholecystokinin Induction in Zebrafish and MIN6 Cell-Line Models.

This study unveils verapamil's compelling cytoprotective and proliferative effects on pancreatic ß-cells amidst diabetic stressors, spotlighting its unforeseen role in augmenting cholecystokinin (CCK) expression. Through rigorous investigations employing MIN6 ß-cells and zebrafish models under type 1 and type 2 diabetic conditions, we demonstrate verapamil's capacity to significantly boost ß-cell proliferation, enhance glucose-stimulated insulin secretion, and fortify cellular resilience. A pivotal revelation of our research is verapamil's induction of CCK, a peptide hormone known for its role in nutrient digestion and insulin secretion, which signifies a novel pathway through which verapamil exerts its therapeutic effects. Furthermore, our mechanistic insights reveal that verapamil orchestrates a broad spectrum of gene and protein expressions pivotal for ß-cell survival and adaptation to immune-metabolic challenges. In vivo validation in a zebrafish larvae model confirms verapamil's efficacy in fostering ß-cell recovery post-metronidazole infliction. Collectively, our findings advocate for verapamil's reevaluation as a multifaceted agent in diabetes therapy, highlighting its novel function in CCK upregulation alongside enhancing ß-cell proliferation, glucose sensing, and oxidative respiration. This research enriches the therapeutic landscape, proposing verapamil not only as a cytoprotector but also as a promoter of ß-cell regeneration, thereby offering fresh avenues for diabetes management strategies aimed at preserving and augmenting ß-cell functionality.

Verapamil inhibits respiratory syncytial virus infection by regulating Ca2+ influx.

AIMS: The study evaluated the antiviral effect of Verapamil against respiratory syncytial virus (RSV) and investigated its underlying mechanism. MATERIALS AND METHODS: RSV-infected BALB/c mice were treated with Verapamil. Body weight, survival rates, viral load, lung damage, inflammatory factors, and the expression of RSV fusion (F) protein were analyzed. In cellular studies, intracellular Ca2+ and viral titers were measured in the presence of Verapamil, Calcium Chloride, and EGTA. A time-of-addition assay assessed the antiviral effect of Verapamil. KEY FINDINGS: Mice infected with RSV and treated with Verapamil exhibited a significant decrease in weight loss, an increase in survival rates, and reductions in viral titers, RSV F protein expression, inflammatory responses, and lung tissue injury. Verapamil reduced intracellular calcium levels, which correlated with reduced viral titers. The addition of calcium chloride reversed the anti-viral effects mediated by Verapamil, while EGTA potentiated them. The antiviral activity of Verapamil was observed during the early phase of RSV infection, likely by blocking Ca2+ channels and inhibiting virus replication. SIGNIFICANCE: Verapamil effectively inhibits RSV infection by blocking calcium channels and reducing intracellular calcium levels, thereby impeding viral replication. Thus, Verapamil shows promise as a treatment for RSV.

Intranasal etripamil for rapid treatment of paroxysmal supraventricular tachycardia.

Paroxysmal supraventricular tachycardia (PSVT) is a common arrhythmia that, although usually benign, can occur unpredictably, cause disabling symptoms and significantly impair quality of life. If spontaneous resolution does not occur, the only current self-treatment is for the patient to attempt vagal maneuvers, however, these are frequently unsuccessful. Hospital attendance is then required for intravenous therapy. Etripamil, an intranasal calcium channel blocker similar to verapamil, may be able to fill this therapeutic gap, allowing rapid self-treatment of PSVT at home. This narrative review discusses the latest evidence for etripamil and its potential role in future clinical practice.

Paroxysmal supraventricular tachycardia (PSVT) is an abnormal heart rhythm, causing the heart to beat rapidly. There are several ways to treat PSVT. This article discusses a new therapy, etripamil. One treatment involves breathing techniques called 'vagal maneuvers'. These avoid medication and sometimes stop the abnormal rhythm, however, in many cases, this does not work. An alternative is a tablet taken when symptoms occur. Unfortunately, tablets take time to absorb, meaning symptoms may continue until the medication takes effect, and this approach does not work for everyone. If these approaches fail, patients suffering from PSVT may need to seek treatment at a hospital. This may involve intravenous therapy, with certain drugs causing unpleasant sensations of chest discomfort. Some patients may also be kept in the hospital for monitoring. Although PSVT can often be cured via a catheter ablation procedure, this is invasive (involving wires inserted via veins in the groin), so not everyone wishes to pursue this, and in some cases, it cannot be performed safely. There is a need for a rapid, safe, and effective treatment that patients can administer at home when PSVT occurs. Etripamil shows promise. Because it is a nasal spray, etripamil allows rapid absorption into the body ­ much faster than a tablet. Etripamil is not yet available on the market; however, several studies have demonstrated its effectiveness and safety, so it may be available in the near future. Promising evidence for etripamil in certain groups, such as elderly patients, is still lacking.

Inhibitory mechanisms of docosahexaenoic acid on carbachol-, angiotensin II-, and bradykinin-induced contractions in guinea pig gastric fundus smooth muscle.

We studied the inhibitory actions of docosahexaenoic acid (DHA) on the contractions induced by carbachol (CCh), angiotensin II (Ang II), and bradykinin (BK) in guinea pig (GP) gastric fundus smooth muscle (GFSM), particularly focusing on the possible inhibition of store-operated Ca2+ channels (SOCCs). DHA significantly suppressed the contractions induced by CCh, Ang II, and BK; the inhibition of BK-induced contractions was the strongest. Although all contractions were greatly dependent on external Ca2+, more than 80% of BK-induced contractions remained even in the presence of verapamil, a voltage-dependent Ca2+ channel inhibitor. BK-induced contractions in the presence of verapamil were not suppressed by LOE-908 (a receptor-operated Ca2+ channel (ROCC) inhibitor) but were suppressed by SKF-96365 (an SOCC and ROCC inhibitor). BK-induced contractions in the presence of verapamil plus LOE-908 were strongly inhibited by DHA. Furthermore, DHA inhibited GFSM contractions induced by cyclopiazonic acid (CPA) in the presence of verapamil plus LOE-908 and inhibited the intracellular Ca2+ increase due to Ca2+ addition in CPA-treated 293T cells. These findings indicate that Ca2+ influx through SOCCs plays a crucial role in BK-induced contraction in GP GFSM and that this inhibition by DHA is a new mechanism by which this fatty acid inhibits GFSM contractions.

Verapamil-Loaded Cubosomes for Enhancing Intranasal Drug Delivery: Development, Characterization, Ex Vivo Permeation, and Brain Biodistribution Studies.

Verapamil hydrochloride (VRP), an antihypertensive calcium channel blocker drug has limited bioavailability and short half-life when taken orally. The present study was aimed at developing cubosomes containing VRP for enhancing its bioavailability and targeting to brain for cluster headache (CH) treatment as an off-label use. Factorial design was conducted to analyze the impact of different components on entrapment efficiency (EE%), particle size (PS), zeta potential (ZP), and percent drug release. Various in-vitro characterizations were performed followed by pharmacokinetic and brain targeting studies. The results revealed the significant impact of glyceryl monooleate (GMO) on increasing EE%, PS, and ZP of cubosomes with a negative influence on VRP release. The remarkable effect of Poloxamer 407 (P407) on decreasing EE%, PS, and ZP of cubosomes was observed besides its influence on accelerating VRP release%. The DSC thermograms indicated the successful entrapment of the amorphous state of VRP inside the cubosomes. The design suggested an optimized formulation containing GMO (50% w/w) and P407 (5.5% w/w). Such formulation showed a significant increase in drug permeation through nasal mucosa with high Er value (2.26) when compared to VRP solution. Also, the histopathological study revealed the safety of the utilized components used in the cubosomes preparation. There was a significant enhancement in the VRP bioavailability when loaded in cubosomes owing to its sustained release favored by its direct transport to brain. The I.N optimized formulation had greater BTE% and DTP% at 183.53% and 90.19%, respectively in comparison of 41.80% and 59% for the I.N VRP solution.

Use of Intravenous Lipid Emulsion Therapy and Insulin in a Case of Tarka Intoxication.

INTRODUCTION: Tarka (trandolapril/verapamil hydrohloride extended-release) is a fixed-dose combination antihypertensive drug formed from verapamil hydrochloride and trandolapril. Toxicologic manifestations of Tarka overdose are altered mental status, bradycardia, hypotension, atrioventricular block (first-degree), hyperglycemia, metabolic acidosis, and shock. CASE PRESENTATION: We report a case of Tarka toxicity in a 2-year-old girl who presented with altered mental status, cardiogenic shock, hypotension, bradycardia, severe metabolic acidosis, hyperglycemia, and first-degree atrioventricular block. We started fluid resuscitation, epinephrine, norepinephrine, and insulin. Because of the patient's hyperlactatemia and hypotension despite standard therapies, we initiated intravenous lipid emulsion (ILE) therapy, after which her condition improved promptly. DISCUSSION: Tarka overdose may be life-threatening as it can cause cardiogenic shock. In our patient, the regression of lactate elevation in a short time with ILE therapy and the improvement of her general condition highlight the importance of ILE. CONCLUSIONS: ILE is an alternative treatment method for acute lipophilic drug intoxications, such as Tarka.

Pharmacogenomic study-A pilot study of the effect of pharmacogenomic phenotypes on the adequate dosing of verapamil for migraine prevention.

OBJECTIVE: To investigate factors affecting the efficacy and tolerability of verapamil for migraine prevention using individual pharmacogenomic phenotypes. BACKGROUND: Verapamil has a wide range of dosing in headache disorders without reliable tools to predict the optimal doses for an individual. METHODS: This is a retrospective chart review examining adults with existing pharmacogenomic reports at Mayo Clinic who had used verapamil for migraine. Effects of six cytochrome P450 phenotypes on the doses of verapamil for migraine prevention were assessed. RESULTS: Our final analysis included 33 migraine patients (82% with aura). The mean minimum effective and maximum tolerable doses of verapamil were 178.2(20-320) mg and 227.9(20-480) mg. A variety of CYP2C9, CYP2D6, and CYP3A5 phenotypes were found, without significant association with the verapamil doses after adjusting for age, sex, body mass index, and smoking status. CONCLUSIONS: We demonstrated a wide range of effective and tolerable verapamil doses used for migraine in a cohort with various pharmacogenomic phenotypes.

Verapamil and tangeretin enhances the M1 macrophages to M2 type in lipopolysaccharide-treated mice and inhibits the P-glycoprotein expression by downregulating STAT1/STAT3 and upregulating SOCS3.

LPS induced sepsis is a complex process involving various immune cells and signaling molecules. Dysregulation of macrophage polarization and ROS production contributed to the pathogenesis of sepsis. PGP is a transmembrane transporter responsible for the efflux of a number of drugs and also expressed in murine macrophages. Natural products have been shown to decrease inflammation and expression of efflux transporters. However, no treatment is currently available to treat LPS induced sepsis. Verapamil and Tangeretin also reported to attenuate lipopolysaccharide-induced inflammation. However, the effects of verapamil or tangeretin on lipopolysaccharide (LPS)-induced sepsis and its detailed anti-inflammatory mechanism have not been reported. Here, we have determined that verapamil and tangeretin protects against LPS-induced sepsis by suppressing M1 macrophages populations and also through the inhibition of P-glycoprotein expression via downregulating STAT1/STAT3 and upregulating SOCS3 expression in macrophages. An hour before LPS (10 mg/kg) was administered; mice were given intraperitoneal injections of either verapamil (5 mg/kg) or tangeretin (5 mg/kg). The peritoneal macrophages from different experimental groups of mice were isolated. Hepatic, pulmonary and splenic morphometric analyses revealed that verapamil and tangeretin decreased the infiltration of neutrophils into the tissues. Verapamil and tangeritin also enhanced the activity of SOD, CAT, GRX and GSH level in all the tissues tested. verapamil or tangeretin pre-treated mice shifted M1 macrophages to M2 type possibly through the inhibition of P-glycoprotein expression via downregulating STAT1/STAT3 and upregulating SOCS3 expression. Hence, both these drugs have shown protective effects in sepsis via suppressing iNOS, COX-2, oxidative stress and NF-κB signaling in macrophages. Therefore, in our study we can summarize that mice were treated with either Vera or Tan before LPS administration cause an elevated IL-10 by the macrophages which enhances the SOCS3 expression, and thereby able to limits STAT1/STAT3 inter-conversion in the macrophages. As a result, NF-κB activity is also getting down regulated and ultimately mitigating the adverse effect of inflammation caused by LPS in resident macrophages. Whether verapamil or tangeretin offers such protection possibly through the inhibition of P-glycoprotein expression in macrophages needs clarification with the bio availability of these drugs under PGP inhibited conditions is a limitation of this study.

The importance of heteroresistance and efflux pumps in bedaquiline-resistant Mycobacterium tuberculosis isolates from Iran.

BACKGROUND: Tuberculosis (TB) continues to pose a threat to communities worldwide and remains a significant public health issue in several countries. We assessed the role of heteroresistance and efflux pumps in bedaquiline (BDQ)-resistant Mycobacterium tuberculosis isolates. METHODS: Nineteen clinical isolates were included in the study, of which fifteen isolates were classified as MDR or XDR, while four isolates were fully susceptible. To evaluate BDQ heteroresistance, the Microplate Alamar Blue Assay (MABA) method was employed. For screening mixed infections, MIRU-VNTR was performed on clinical isolates. Mutations in the atpE and Rv0678 genes were determined based on next-generation sequencing data. Additionally, real-time PCR was applied to assess the expression of efflux pump genes in the absence and presence of verapamil (VP). RESULTS: All 15 drug-resistant isolates displayed resistance to BDQ. Among the 19 total isolates, 21.05% (4/19) exhibited a heteroresistance pattern to BDQ. None of the isolates carried a mutation of the atpE and Rv0678 genes associated with BDQ resistance. Regarding the MIRU-VNTR analysis, most isolates (94.73%) showed the Beijing genotype. Fifteen (78.9%) isolates showed a significant reduction in BDQ MIC after VP treatment. The efflux pump genes of Rv0676c, Rv1258c, Rv1410c, Rv1634, Rv1819, Rv2459, Rv2846, and Rv3065 were overexpressed in the presence of BDQ. CONCLUSIONS: Our results clearly demonstrated the crucial role of heteroresistance and efflux pumps in BDQ resistance. Additionally, we established a direct link between the Rv0676c gene and BDQ resistance. The inclusion of VP significantly reduced the MIC of BDQ in both drug-susceptible and drug-resistant clinical isolates.

Efflux pump effects on levofloxacin resistance in Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) inherently displays resistance to most antibiotics, with the underlying drug resistance mechanisms remaining largely unexplored. Efflux pump is believed to play an important role in mediating drug resistance. The current study examined the potential of efflux pump inhibitors to reverse levofloxacin (LFX) resistance in M. abscessus. The reference strain of M. abscessus (ATCC19977) and 60 clinical isolates, including 41 M. abscessus subsp. abscessus and 19 M. abscessus subsp. massilense, were investigated. The drug sensitivity of M. abscessus against LFX alone or in conjunction with efflux pump inhibitors, including verapamil (VP), reserpine (RSP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or dicyclohexylcarbodiimide (DCC), were determined by AlarmarBlue microplate assay. Drug-resistant regions of the gyrA and gyrB genes from the drug-resistant strains were sequenced. The transcription level of the efflux pump genes was monitored using qRT-PCR. All the tested strains were resistant to LFX. The drug-resistant regions from the gyrA and gyrB genes showed no mutation associated with LFX resistance. CCCP, DCC, VP, and RSP increased the susceptibility of 93.3% (56/60), 91.7% (55/60), 85% (51/60), and 83.3% (50/60) isolates to LFX by 2 to 32-fold, respectively. Elevated transcription of seven efflux pump genes was observed in isolates with a high reduction in LFX MIC values in the presence of efflux pump inhibitors. Efflux pump inhibitors can improve the antibacterial activity of LFX against M. abscessus in vitro. The overexpression of efflux-related genes in LFX-resistant isolates suggests that efflux pumps are associated with the development of LFX resistance in M. abscessus.

Calcium channel inhibitor and extracellular calcium improve aminoglycoside-induced hair cell loss in zebrafish.

Aminoglycosides are commonly used antibiotics for treatment of gram-negative bacterial infections, however, they might act on inner ear, leading to hair-cell death and hearing loss. Currently, there is no targeted therapy for aminoglycoside ototoxicity, since the underlying mechanisms of aminoglycoside-induced hearing impairments are not fully defined. This study aimed to investigate whether the calcium channel blocker verapamil and changes in intracellular & extracellular calcium could ameliorate aminoglycoside-induced ototoxicity in zebrafish. The present findings showed that a significant decreased number of neuromasts in the lateral lines of zebrafish larvae at 5 days' post fertilization after neomycin (20 µM) and gentamicin (20 mg/mL) exposure, which was prevented by verapamil. Moreover, verapamil (10-100 µM) attenuated aminoglycoside-induced toxic response in different external calcium concentrations (33-3300 µM). The increasing extracellular calcium reduced hair cell loss from aminoglycoside exposure, while lower calcium facilitated hair cell death. In contrast, calcium channel activator Bay K8644 (20 µM) enhanced aminoglycoside-induced ototoxicity and reversed the protective action of higher external calcium on hair cell loss. However, neomycin-elicited hair cell death was not altered by caffeine, ryanodine receptor (RyR) agonist, and RyR antagonists, including thapsigargin, ryanodine, and ruthenium red. The uptake of neomycin into hair cells was attenuated by verapamil and under high external calcium concentration. Consistently, the production of reactive oxygen species (ROS) in neuromasts exposed to neomycin was also reduced by verapamil and high external calcium. Significantly, zebrafish larvae when exposed to neomycin exhibited decreased swimming distances in reaction to droplet stimulus when compared to the control group. Verapamil and elevated external calcium effectively protected the impaired swimming ability of zebrafish larvae induced by neomycin. These data imply that prevention of hair cell damage correlated with swimming behavior against aminoglycoside ototoxicity by verapamil and higher external calcium might be associated with inhibition of excessive ROS production and aminoglycoside uptake through cation channels. These findings indicate that calcium channel blocker and higher external calcium could be applied to protect aminoglycoside-induced listening impairments.

Molecular, immunological, and physiological evidences of a sphingosine-activated plasma membrane Ca2+-channel in Trypanosoma equiperdum.

The hemoparasite Trypanosoma equiperdum belongs to the Trypanozoon subgenus and includes several species that are pathogenic to animals and humans in tropical and subtropical areas across the world. As with all eukaryotic organisms, Ca2+ is essential for these parasites to perform cellular processes thus ensuring their survival across their life cycle. Despite the established paradigm to study proteins related to Ca2+ homeostasis as potential drug targets, so far little is known about Ca2+ entry into trypanosomes. Therefore, in the present study, the presence of a plasma membrane Ca2+-channel in T. equiperdum (TeCC), activated by sphingosine and inhibited by verapamil, is described. The TeCC was cloned and analyzed using bioinformatic resources, which confirmed the presence of several domains, motifs, and a topology similar to the Ca2+ channels found in higher eukaryotes. Biochemical and confocal microscopy assays using antibodies raised against an internal region of human L-type Ca2+ channels indicate the presence of a protein with similar predicted molar mass to the sequence analyzed, located at the plasma membrane of T. equiperdum. Physiological assays based on Fura-2 signals and Mn2+ quenching performed on whole parasites showed a unidirectional Ca2+ entry, which is activated by sphingosine and blocked by verapamil, with the distinctive feature of insensitivity to nifedipine and Bay K 8644. This suggests a second Ca2+ entry for T. equiperdum, different from the store-operated Ca2+ entry (SOCE) previously described. Moreover, the evidence presented here for the TeCC indicates molecular and pharmacological differences with their mammal counterparts, which deserve further studies to evaluate the potential of this channel as a drug target.

Efflux Pump Inhibitor Potentiates the Antimicrobial Photodynamic Inactivation of Multidrug-Resistant Acinetobacter baumannii.

Background: Acinetobacter baumannii, a nosocomial pathogen, poses a major public health problem due to generating resistance to several antimicrobial agents. Antimicrobial photodynamic inactivation (APDI) employs a nontoxic dye as a photosensitizer (PS) and light to produce reactive oxygen species that destroy bacterial cells. The intracellular concentration of PS could be affected by factors such as the function of efflux pumps to emit PS from the cytosol. Objective: To evaluate the augmentation effect of an efflux pump inhibitor, verapamil, three multidrug-resistant A. baumannii were subjected to APDI by erythrosine B (EB). Methods and results: The combination of EB and verapamil along with irradiation at 530 nm induced a lethal effect and more than 3 log colony-forming unit reduction to all A. baumannii strains in planktonic state. In contrast, EB and irradiation alone could produce only a sublethal effect on two of the strains. Conclusions: These data suggest that verapamil increases the intracellular concentration of EB, which potentiates the lethal efficacy of APDI. Verapamil could be applied with EB and green light to improve their antimicrobial efficacy against A. baumannii-localized infections.

Methylglyoxal induces cardiac dysfunction through mechanisms involving altered intracellular calcium handling in the rat heart.

Methylglyoxal (MGO) is an endogenous, highly reactive dicarbonyl metabolite generated under hyperglycaemic conditions. MGO plays a role in developing pathophysiological conditions, including diabetic cardiomyopathy. However, the mechanisms involved and the molecular targets of MGO in the heart have not been elucidated. In this work, we studied the exposure-related effects of MGO on cardiac function in an isolated perfused rat heart ex vivo model. The effect of MGO on calcium homeostasis in cardiomyocytes was studied in vitro by the fluorescence indicator of intracellular calcium Fluo-4. We demonstrated that MGO induced cardiac dysfunction, both in contractility and diastolic function. In rat heart, the effects of MGO treatment were significantly limited by aminoguanidine, a scavenger of MGO, ruthenium red, a general cation channel blocker, and verapamil, an L-type voltage-dependent calcium channel blocker, demonstrating that this dysfunction involved alteration of calcium regulation. MGO induced a significant concentration-dependent increase of intracellular calcium in neonatal rat cardiomyocytes, which was limited by aminoguanidine and verapamil. These results suggest that the functionality of various calcium channels is altered by MGO, particularly the L-type calcium channel, thus explaining its cardiac toxicity. Therefore, MGO could participate in the development of diabetic cardiomyopathy through its impact on calcium homeostasis in cardiac cells.

Mechanism of Induction of P-gp Activity During MET Induced by DEX in Lung Cancer Cell Line.

Lung cancer metastasis often leads to a poor prognosis for patients. Mesenchymal-epithelial transition (MET) is one key process associated with metastasis. MET has also been linked to multidrug drug resistance (MDR). MDR arises from the overactivity of drug efflux transporters such as P-glycoprotein (P-gp) which operate at the cell plasma membrane, under the regulatory control of the scaffold proteins ezrin (Ezr), radixin (Rdx), and moesin (Msn), collectively known as ERM proteins. The current study was intended to clarify the functional changing of P-gp and the underlying mechanisms in the context of dexamethasone (DEX)-induced MET in lung cancer cells. We found that the mRNA and membrane protein expression of Ezr and P-gp was increased in response to DEX treatment. Moreover, the DEX-treated group exhibited an increase in Rho123 efflux, and it was reversed by treatment with the P-gp inhibitor verapamil or Ezr siRNA. The decrease in cell viability with paclitaxel (PTX) treatment was mitigated by pretreatment with DEX. The increased expression and activation of P-gp during the progression of lung cancer MET was regulated by Ezr. The regulatory mechanism of P-gp expression and activity may differ depending on the cell status.

Virtual clinical QT exposure-response studies - A translational computational approach.

BACKGROUND AND PURPOSE: A recent paradigm shift in proarrhythmic risk assessment suggests that the integration of clinical, non-clinical, and computational evidence can be used to reach a comprehensive understanding of the proarrhythmic potential of drug candidates. While current computational methodologies focus on predicting the incidence of proarrhythmic events after drug administration, the objective of this study is to predict concentration-response relationships of QTc as a clinical endpoint. EXPERIMENTAL APPROACH: Full heart computational models reproducing human cardiac populations were created to predict the concentration-response relationship of changes in the QT interval as recommended for clinical trials. The concentration-response relationship of the QT-interval prolongation obtained from the computational cardiac population was compared against the relationship from clinical trial data for a set of well-characterized compounds: moxifloxacin, dofetilide, verapamil, and ondansetron. KEY RESULTS: Computationally derived concentration-response relationships of QT interval changes for three of the four drugs had slopes within the confidence interval of clinical trials (dofetilide, moxifloxacin and verapamil) when compared to placebo-corrected concentration-ΔQT and concentration-ΔQT regressions. Moxifloxacin showed a higher intercept, outside the confidence interval of the clinical data, demonstrating that in this example, the standard linear regression does not appropriately capture the concentration-response results at very low concentrations. The concentrations corresponding to a mean QTc prolongation of 10 ms were consistently lower in the computational model than in clinical data. The critical concentration varied within an approximate ratio of 0.5 (moxifloxacin and ondansetron) and 1 times (dofetilide, verapamil) the critical concentration observed in human clinical trials. Notably, no other in silico methodology can approximate the human critical concentration values for a QT interval prolongation of 10 ms. CONCLUSION AND IMPLICATIONS: Computational concentration-response modelling of a virtual population of high-resolution, 3-dimensional cardiac models can provide comparable information to clinical data and could be used to complement pre-clinical and clinical safety packages. It provides access to an unlimited exposure range to support trial design and can improve the understanding of pre-clinical-clinical translation.

Fluid-solid coupling numerical simulation of the effects of different doses of verapamil on cancellous bone in type 2 diabetic rats.

BACKGROUND: The purpose of this study was to investigate the effects of four different doses of verapamil on the mechanical behaviors of solid and the characteristics of fluid flow in cancellous bone of distal femur of type 2 diabetes rats under dynamic external load. METHODS: Based on the micro-CT images, the finite element models of cancellous bones and fluids at distal femurs of rats in control group, diabetes group, treatment groups VER 4, VER 12, VER 24, and VER 48 (verapamil doses of 4, 12, 24, and 48 mg/kg/day, respectively) were constructed. A sinusoidal time-varying displacement load with an amplitude of 0.8 µm and a period of 1s was applied to the upper surface of the solid region. Then, fluid-solid coupling numerical simulation method was used to analyze the magnitudes and distributions of von Mises stress, flow velocity, and fluid shear stress of cancellous bone models in each group. RESULTS: The results for mean values of von Mises stress, flow velocity and FSS (t = 0.25s) were as follows: their values in control group were lower than those in diabetes group; the three parameters varied with the dose of verapamil; in the four treatment groups, the values of VER 48 group were the lowest, they were the closest to control group, and they were smaller than diabetes group. Among the four treatment groups, VER 48 group had the highest proportion of the nodes with FSS = 1-3 Pa on the surface of cancellous bone, and more areas in VER 48 group were subjected to fluid shear stress of 1-3 Pa for more than half of the time. CONCLUSION: It could be seen that among the four treatment groups, osteoblasts on the cancellous bone surface in the highest dose group (VER 48 group) were more easily activated by mechanical loading, and the treatment effect was the best. This study might help in understanding the mechanism of verapamil's effect on the bone of type 2 diabetes mellitus, and provide theoretical guidance for the selection of verapamil dose in the clinical treatment of type 2 diabetes mellitus.