Intensive Care Interventions Among Children With Toxicologic Exposures to Cardiovascular Medications.

OBJECTIVES: Interventions requiring a PICU are rare in toxicologic exposures, but cardiovascular medications are high-risk exposures due to their hemodynamic effects. This study aimed to describe prevalence of and risk factors for PICU interventions among children exposed to cardiovascular medications. DESIGN: Secondary analysis of Toxicology Investigators Consortium Core Registry from January 2010 to March 2022. SETTING: International multicenter research network of 40 sites. PATIENTS: Patients 18 years old or younger with acute or acute-on-chronic toxicologic exposure to cardiovascular medications. Patients were excluded if exposed to noncardiovascular medications or if symptoms were documented as unlikely related to exposure. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of 1,091 patients in the final analysis, 195 (17.9%) received PICU intervention. One hundred fifty-seven (14.4%) received intensive hemodynamic interventions and 602 (55.2%) received intervention in general. Children less than 2 years old were less likely to receive PICU intervention (odds ratio [OR], 0.42; 95% CI, 0.20-0.86). Exposures to alpha-2 agonists (OR, 2.0; 95% CI, 1.11-3.72) and antiarrhythmics (OR, 4.26; 95% CI, 1.41-12.90) were associated with PICU intervention. In the sensitivity analysis removing atropine from the composite outcome PICU intervention, only exposures to calcium channel antagonists (OR, 2.12; 95% CI, 1.09-4.11) and antiarrhythmics (OR, 4.82; 95% CI, 1.57-14.81) were independently associated with PICU intervention. No independent association was identified between PICU intervention and gender, polypharmacy, intentionality or acuity of exposure, or the other medication classes studied. CONCLUSIONS: PICU interventions were uncommon but were associated with exposure to antiarrhythmic medications, calcium channel antagonists, and alpha-2 agonists. As demonstrated via sensitivity analysis, exact associations may depend on institutional definitions of PICU intervention. Children less than 2 years old are less likely to require PICU interventions. In equivocal cases, age and exposure to certain cardiovascular medication classes may be useful to guide appropriate disposition.

Central role of TRAP1 in the ameliorative effect of oleanolic acid on the mitochondrial-mediated and endoplasmic reticulum stress-excitated apoptosis induced by ochratoxin A.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin that is widely distributed in foodstuffs and feeds, meanwhile oleanolic acid (OA) is ubiquitous in various fruit skins, food materials, and medicinal herbs. Due to that OA has a nephroprotective effect, it has the poteintial to counteract OTA-induced nephrotoxicity by nutritional intervention of OA. Furthermore, tumor necrosis factor receptor-associated protein 1 (TRAP1) acts as the core of endoplasmic reticulum (ER)-mitochondria crosstalk, becoming our focus in the mechanism investigation. In this study, the cell viability, apoptosis rate, and protein expressions of human proximal tubule epithelial-originated kidney-2 (HK-2) cells in response to OTA and/or OA were determined. Results indicated that a 24 h-treatment of 1-5 µM OTA could notably induce mitochondrial-mediated and ER stress (ERS)-excitated apoptosis via inhibiting TRAP1, thereby activating CypD, Bax, Cyt-C, Cleaved Caspase-9, Cleaved Caspase-3, GRP78, p-PERK, p-eIF2α, ATF4, and CHOP and inhibiting Bcl-2 (P < 0.05). Results of the RNA interference of TRAP1 further ascertained its anti-apoptotic function via inhibiting CypD, Bax, GRP78, and CHOP and enhancing Bcl-2 (P < 0.05). The pre-treatment of 2 µM OA for 2 h could remarkably relieve OTA-induced suppression of TRAP1 (P < 0.05). In conclusion, TRAP1 played a central role in the ameliorative effect of OA on the mitochondrial-mediated and ERS-excitated apoptosis induced by OTA.

Astaxanthin Alleviates Ochratoxin A-Induced Cecum Injury and Inflammation in Mice by Regulating the Diversity of Cecal Microbiota and TLR4/MyD88/NF-κB Signaling Pathway.

Ochratoxin A (OTA) is a common environmental pollutant found in a variety of foods and grains, and excessive OTA consumption causes serious global health effects on animals and humans. Astaxanthin (AST) is a natural carotenoid that has anti-inflammatory, antiapoptotic, immunomodulatory, antitumor, antidiabetes, and other biological activities. The present study is aimed at investigating the effects of AST on OTA-induced cecum injury and its mechanism of action. Eighty C57 mice were randomly divided into four groups, including the control group, OTA group (5 mg/kg body weight), AST group (100 mg/kg body weight), and AST intervention group (100 mg/kg body weight AST+5 mg/kg body weight OTA). It was found that AST decreased the endotoxin content, effectively prevented the shortening of mouse cecum villi, and increased the expression levels of tight junction (TJ) proteins, consisting of occludin, claudin-1, and zonula occludens-1 (ZO-1). AST increased the number of goblet cells, the contents of mucin-2 (MUC2), and defensins (Defa5 and ß-pD2) significantly, while the expression of mucin-1 (MUC1) decreased significantly. The 16S rRNA sequencing showed that AST affected the richness and diversity of cecum flora, decreased the proportion of lactobacillus, and also decreased the contents of short-chain fatty acids (SCFAs) (acetate and butyrate). In addition, AST significantly decreased the expression of TLR4, MyD88, and p-p65, while increasing the expression of p65. Meanwhile, the expression of inflammatory factors including TNF-α and INF-γ decreased, while the expression of IL-10 increased. In conclusion, AST reduced OTA-induced cecum injury by regulating the cecum barrier function and TLR4/MyD88/NF-κB signaling pathway.

N-acetylcysteine prevents verapamil-induced cardiotoxicity with no effect on the noradrenergic arch-associated neurons in zebrafish.

There is a strong association between calcium channel blockers (CCBs) and heart failure. CCB toxicity is very common due to overdose and underlying medical conditions. CCBs also have been shown to affect the nervous system. Recently, we demonstrated that the antioxidant N-acetylcysteine (NAC) prevented ketamine-induced cardiotoxicity, developmental toxicity and neurotoxicity. Functionally, we attributed NAC's beneficial effect to its ability to increase cellular calcium. Here, we hypothesized that if there was an involvement of calcium in NAC's preventative effects on ketamine toxicity, NAC might also ameliorate toxicities induced by verapamil, an L-type CCB used to treat hypertension. Using zebrafish embryos, we show that in the absence of NAC, verapamil (up to 100 µM) dose-dependently reduced heart rate and those effects were prevented by NAC co-treatment. Furthermore, a 2-h treatment with NAC rescued reduction of heart rate induced by pre-treatment of 50 and 100 µM of verapamil for 18 h. Verapamil up to 100 µM and NAC up to 1.5 mM did not have any adverse effects on the expression of tyrosine hydroxylase in the noradrenergic neurons of the arch-associated cluster (AAC) located near the heart. NAC did not change cysteine levels in the embryos suggesting that the beneficial effect of NAC on verapamil toxicity may not involve its antioxidant property. In our search for compounds that can prevent CCB toxicity, this study, for the first time, demonstrates protective effects of NAC against verapamil's adverse effects on the heart.

Chemical and Pharmacological Screening of Rhinella icterica (Spix 1824) Toad Parotoid Secretion in Avian Preparations.

The biological activity of Rhinella icterica parotoid secretion (RIPS) and some of its chromatographic fractions (RI18, RI19, RI23, and RI24) was evaluated in the current study. Mass spectrometry of these fractions indicated the presence of sarmentogenin, argentinogenin, (5ß,12ß)-12,14-dihydroxy-11-oxobufa-3,20,22-trienolide, marinobufagin, bufogenin B, 11α,19-dihydroxy-telocinobufagin, bufotalin, monohydroxylbufotalin, 19-oxo-cinobufagin, 3α,12ß,25,26-tetrahydroxy-7-oxo-5ß-cholestane-26-O-sulfate, and cinobufagin-3-hemisuberate that were identified as alkaloid and steroid compounds, in addition to marinoic acid and N-methyl-5-hydroxy-tryptamine. In chick brain slices, all fractions caused a slight decrease in cell viability, as also seen with the highest concentration of RIPS tested. In chick biventer cervicis neuromuscular preparations, RIPS and all four fractions significantly inhibited junctional acetylcholinesterase (AChE) activity. In this preparation, only fraction RI23 completely mimicked the pharmacological profile of RIPS, which included a transient facilitation in the amplitude of muscle twitches followed by progressive and complete neuromuscular blockade. Mass spectrometric analysis showed that RI23 consisted predominantly of bufogenins, a class of steroidal compounds known for their cardiotonic activity mediated by a digoxin- or ouabain-like action and the blockade of voltage-dependent L-type calcium channels. These findings indicate that the pharmacological activities of RI23 (and RIPS) are probably mediated by: (1) inhibition of AChE activity that increases the junctional content of Ach; (2) inhibition of neuronal Na+/K+-ATPase, leading to facilitation followed by neuromuscular blockade; and (3) blockade of voltage-dependent Ca2+ channels, leading to stabilization of the motor endplate membrane.

Traditional Chinese medicine practices used in COVID-19 (Sars-cov 2/Coronavirus-19) treatment in clinic and their effects on the cardiovascular system.

OBJECTIVE: The aim of this study was to evaluate the effectiveness of plants used in the formulations of traditional Chinese medicine (TCM), which were also used in clinical trials to treat patients with the novel coronavirus COVID-19, and to assess their effects on the cardiovascular system. METHODS: A literature review of PubMed, ResearchGate, ScienceDirect, the Cochrane Library, and TCM monographs was conducted and the effects of the plants on the cardiovascular system and the mechanisms of action in COVID-19 treatment were evaluated. RESULTS: The mechanism of action, cardiovascular effects, and possible toxicity of 10 plants frequently found in TCM formulations that were used in the clinical treatment of COVID-19 were examined. CONCLUSION: TCM formulations that had been originally developed for earlier viral diseases have been used in COVID-19 treatment. Despite the effectiveness seen in laboratory and animal studies with the most commonly used plants in these formulations, the clinical studies are currently insufficient according to standard operating procedures. More clinical studies are needed to understand the safe clinical use of traditional plants.

Comparative effectiveness of methylene blue versus intravenous lipid emulsion in a rodent model of amlodipine toxicity.

Context: Calcium channel blocker (CCB) poisonings are the leading cause of death from cardiovascular medication-related overdoses. Current treatments (calcium salts, vasopressors, inotropes) are often insufficient. Intravenous lipid emulsion (ILE) and methylene blue (MB) show promise in treating CCB overdoses unresponsive to conventional therapy. Objective: To compare the effectiveness of MB versus ILE in a rodent model of amlodipine (AML) poisoning with survival as the primary outcome and hemodynamic parameters as secondary outcomes. Materials and methods: Sixty-four adult male albino rats were anesthetized and cannulated for non-invasive hemodynamic measurement. Rats received amlodipine intraperitoneally (42 mg/kg). We then divided the rats into four groups: AML only without antidote, AML followed by ILE (24.8 mL/kg over 10 min), AML followed by normal saline (an equivalent volume of ILE), and AML followed by IV MB (2 mg/kg over 5 min). They received study treatments at 5, 30, and 60 minutes from the start of the protocol and with observation for 2 hours. Results: Survival time in ILE group was greater than in the control and NS groups. Differences between ILE and MB and between MB and NS were not significant. Hemodynamic parameters significantly increased in ILE group compared to the MB group at the 30, 60 and 120 min assessments but not after induction of AML poisoning and at 5 min assessment. Conclusions: Survival was greatest in rats treated with ILE. Both MB and NS had little effect on survival when compared to control animals. Both ILE and MB improved hemodynamics.

Substituent effect of N-benzylated gramine derivatives that prevent the PP2A inhibition and dissipate the neuronal Ca2+ overload, as a multitarget strategy for the treatment of Alzheimer's disease.

Following the premises of the multitarget-directed ligands approach for the drug R&D against neurodegenerative diseases, where Alzheimer's disease (AD) outstands, we have synthesized and evaluated analogues of the gramine derivative ITH12657 (1-benzyl-5-methyl-3-(piperidin-1-ylmethyl-1H-indole, 2), which had shown important neuroprotective properties, such as blocking effect of voltage-gated Ca2+ channels (VGCC), and prevention of phosphoprotein phosphatase 2A (PP2A) inhibition. The new analogues present different substitutions at the pending phenyl ring, what slightly modified their pharmacological characteristics. The VGCC blockade was enhanced in derivatives possessing nitro groups, while the pro-PP2A feature was ameliorated by the presence of fluorine. Chlorine atoms supplied good activities over the two biological targets aimed; nevertheless that substitution provoked loss of viability at 100-fold higher concentrations (10 µM), what discards them for a deeper pharmacological study. Overall, the para-fluorine derivative of ITH12657 was the most promising candidate for further preclinical assays.

Assessment of hepatic metabolism-dependent nephrotoxicity on an organs-on-a-chip microdevice.

Drug-induced nephrotoxicity is one of the most frequent adverse events in pharmacotherapy. It has resulted in numerous clinical trial failures and high drug development costs. The predictive capabilities of existing in vitro models are limited by their inability to recapitulate the complex process of drug metabolism at the multi-organ level in vivo. We present a novel integrated liver-kidney chip that allows the evaluation of drug-induced nephrotoxicity following liver metabolism in vitro. The liver-kidney chip consists of two polydimethylsiloxane layers with compartmentalized micro-channels separated by a porous membrane. Hepatic and renal cells were co-cultured in separate micro-chambers on a single chip. Ifosfamide and verapamil were used as model drugs, and their metabolites produced by hepatic metabolism were identified using mass spectrometry, respectively. The metabolites triggered significantly distinct nephrotoxic effects as assessed by cell viability, lactate dehydrogenase leakage and permeability of renal cells. This in vitro liver-kidney model facilitates the characterization of drug metabolism in the liver as well as the assessment of subsequent nephrotoxicity in a single assay. Obviously, this multi-organ platform is simple and scalable, and maybe widely applicable to the evaluation of drug metabolism and safety during the early phases of drug development.

Synthesis and biological evaluation of fluoro-substituted 3,4-dihydroquinazoline derivatives for cytotoxic and analgesic effects.

As a bioisosteric strategy to overcome the poor metabolic stability of lead compound KYS05090S, a series of new fluoro-substituted 3,4-dihydroquinazoline derivatives was prepared and evaluated for T-type calcium channel (Cav3.2) block, cytotoxic effects and liver microsomal stability. Among them, compound 8h (KCP10068F) containing 4-fluorobenzyl amide and 4-cyclohexylphenyl ring potently blocked Cav3.2 currents (>90% inhibition) at 10µM concentration and exhibited cytotoxic effect (IC50=5.9µM) in A549 non-small cell lung cancer cells that was comparable to KYS05090S. Furthermore, 8h showed approximately a 2-fold increase in liver metabolic stability in rat and human species compared to KYS05090S. Based on these overall results, 8h (KCP10068F) may therefore represent a good backup compound for KYS05090S for further biological investigations as novel cytotoxic agent. In addition, compound 8g (KCP10067F) was found to partially protect from inflammatory pain via a blockade of Cav3.2 channels.

Efficacy of osthole for Echinococcus granulosus in vitro and Echinococcus multilocularis in vivo.

Echinococcosis is a zoonotic infection caused by cestode species of the genus Echinococcus; in addition, this zoonosis has long been neglected as a parasitic disease and has limited treatment options. Clinical drugs such as benzimidazole derivatives have limited treatment efficacy. The current study evaluated a novel drug, osthole, with low toxicity and high activity against Echinococcus in vitro and in vivo. The results in vitro indicated that the viability of Echinococcus granulosus protoscoleces in the group treated with osthole (120µM) decreased by 100% within 3days. In vivo experiments were conducted using parasite-infected mice. For this purpose, three groups of infected mice were treated daily for 6 weeks with albendazole (ABZ, 100mg/kg, positive control group), osthole (100mg/kg, experimental group), or honey/PBS (100mg/kg, negative control group), respectively. The osthole- and ABZ-treated groups presented a significant reduction in wet weight of metacestodes, increase in the level of interleukin (IL)-4 and the percentage of eosinophils compared with the control group. Osthole exhibited a high activity against echinococcosis in vivo. In addition, the toxicity of osthole was evaluated via an in vitro 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, as well as via morphological observation and calculation of liver and kidney function indexes in vivo. No obvious toxic effects of osthole were observed in our study. Therefore, this novel drug may be a promising alternative to benzimidazole in anti-echinococcosis chemotherapy.

Molecular basis of toxicity of N-type calcium channel inhibitor MVIIA.

MVIIA (ziconotide) is a specific inhibitor of N-type calcium channel, Cav2.2. It is derived from Cone snail and currently used for the treatment of severe chronic pains in patients unresponsive to opioid therapy. However, MVIIA produces severe side-effects, including dizziness, nystagmus, somnolence, abnormal gait, and ataxia, that limit its wider application. We previously identified a novel inhibitor of Cav2.2, ω-conopeptide SO-3, which possesses similar structure and analgesic activity to MVIIA's. To investigate the key residues for MVIIA toxicity, MVIIA/SO-3 hybrids and MVIIA variants carrying mutations in its loop 2 were synthesized. The substitution of MVIIA's loop 1 with the loop 1 of SO-3 resulted in significantly reduced Cav2.2 binding activity in vitro; the replacement of MVIIA loop 2 by the loop 2 of SO-3 not only enhanced the peptide/Cav2.2 binding but also decreased its toxicity on goldfish, attenuated mouse tremor symptom, spontaneous locomotor activity, and coordinated locomotion function. Further mutation analysis and molecular calculation revealed that the toxicity of MVIIA mainly arose from Met(12) in the loop 2, and this residue inserts into a hydrophobic hole (Ile(300), Phe(302) and Leu(305)) located between repeats II and III of Cav2.2. The combinative mutations of the loop 2 of MVIIA or other ω-conopeptides may be used for future development of more effective Cav2.2 inhibitors with lower side effects.

ω-Tbo-IT1-New Inhibitor of Insect Calcium Channels Isolated from Spider Venom.

Novel disulfide-containing polypeptide toxin was discovered in the venom of the Tibellus oblongus spider. We report on isolation, spatial structure determination and electrophysiological characterization of this 41-residue toxin, called ω-Tbo-IT1. It has an insect-toxic effect with LD50 19 µg/g in experiments on house fly Musca domestica larvae and with LD50 20 µg/g on juvenile Gromphadorhina portentosa cockroaches. Electrophysiological experiments revealed a reversible inhibition of evoked excitatory postsynaptic currents in blow fly Calliphora vicina neuromuscular junctions, while parameters of spontaneous ones were not affected. The inhibition was concentration dependent, with IC50 value 40 ± 10 nM and Hill coefficient 3.4 ± 0.3. The toxin did not affect frog neuromuscular junctions or glutamatergic and GABAergic transmission in rat brains. Ca(2+) currents in Calliphora vicina muscle were not inhibited, whereas in Periplaneta americana cockroach neurons at least one type of voltage gated Ca(2+) current was inhibited by ω-Tbo-IT1. Thus, the toxin apparently acts as an inhibitor of presynaptic insect Ca(2+) channels. Spatial structure analysis of the recombinant ω-Tbo-IT1 by NMR spectroscopy in aqueous solution revealed that the toxin comprises the conventional ICK fold containing an extended ß-hairpin loop and short ß-hairpin loop which are capable of making "scissors-like mutual motions".

Inhibition of Voltage-Gated Calcium Channels After Subchronic and Repeated Exposure of PC12 Cells to Different Classes of Insecticides.

We previously demonstrated that acute inhibition of voltage-gated calcium channels (VGCCs) is a common mode of action for (sub)micromolar concentrations of chemicals, including insecticides. However, because human exposure to chemicals is usually chronic and repeated, we investigated if selected insecticides from different chemical classes (organochlorines, organophosphates, pyrethroids, carbamates, and neonicotinoids) also disturb calcium homeostasis after subchronic (24 h) exposure and after a subsequent (repeated) acute exposure. Effects on calcium homeostasis were investigated with single-cell fluorescence (Fura-2) imaging of PC12 cells. Cells were depolarized with high-K(+) saline to study effects of subchronic or repeated exposure on VGCC-mediated Ca(2+) influx. The results demonstrate that except for carbaryl and imidacloprid, all selected insecticides inhibited depolarization (K(+))-evoked Ca(2+) influx after subchronic exposure (IC50's: approximately 1-10 µM) in PC12 cells. These inhibitory effects were not or only slowly reversible. Moreover, repeated exposure augmented the inhibition of the K(+)-evoked increase in intracellular calcium concentration induced by subchronic exposure to cypermethrin, chlorpyrifos, chlorpyrifos-oxon, and endosulfan (IC50's: approximately 0.1-4 µM). In rat primary cortical cultures, acute and repeated chlorpyrifos exposure also augmented inhibition of VGCCs compared with subchronic exposure. In conclusion, compared with subchronic exposure, repeated exposure increases the potency of insecticides to inhibit VGCCs. However, the potency of insecticides to inhibit VGCCs upon repeated exposure was comparable with the inhibition previously observed following acute exposure, with the exception of chlorpyrifos. The data suggest that an acute exposure paradigm is sufficient for screening chemicals for effects on VGCCs and that PC12 cells are a sensitive model for detection of effects on VGCCs.

Thieno[2,3-b]pyridines--a new class of multidrug resistance (MDR) modulators.

To identify new potent multidrug resistance modulators, we have synthesized a series of novel thieno[2,3-b]pyridines and furo[2,3-b]pyridines, and examined their structure-activity relationships. All synthesized compounds were tested to determine BCRP1, P-gp, and MRP1 inhibitor activity, and most potent MDR modulators were also screened for their toxicity, cytotoxicity and Ca(2+) channel antagonist activity. Among these compounds, thieno[2,3-b]pyridine (6r) was found to exhibit a potent P-gp inhibitory action with EC50 = 0.3 ± 0.2 µM, MRP1 inhibitory action with EC50 = 1.1 ± 0.1 µM and BCRP1 inhibitory action with EC50 = 0.2 ± 0.05 µM and may represent suitable candidate for further pharmacological studies.

Inhibition of voltage-gated calcium channels as common mode of action for (mixtures of) distinct classes of insecticides.

Humans are exposed to distinct structural classes of insecticides with different neurotoxic modes of action. Because calcium homeostasis is essential for proper neuronal function and development, we investigated the effects of insecticides from different classes (pyrethroid: (α-)cypermethrin; organophosphate: chlorpyrifos; organochlorine: endosulfan; neonicotinoid: imidacloprid) and mixtures thereof on the intracellular calcium concentration ([Ca(2+)]i). Effects of acute (20 min) exposure to (mixtures of) insecticides on basal and depolarization-evoked [Ca(2+)]i were studied in vitro with Fura-2-loaded PC12 cells and high resolution single-cell fluorescence microscopy. The data demonstrate that cypermethrin, α-cypermethrin, endosulfan, and chlorpyrifos concentration-dependently decreased depolarization-evoked [Ca(2+)]i, with 50% (IC50) at 78nM, 239nM, 250nM, and 899nM, respectively. Additionally, acute exposure to chlorpyrifos or endosulfan (10µM) induced a modest increase in basal [Ca(2+)]i, amounting to 68 ± 8nM and 53 ± 8nM, respectively. Imidacloprid did not disturb basal or depolarization-evoked [Ca(2+)]i at 10µM. Following exposure to binary mixtures, effects on depolarization-evoked [Ca(2+)]i were within the expected effect additivity range, whereas the effect of the tertiary mixture was less than this expected additivity effect range. These results demonstrate that different types of insecticides inhibit depolarization-evoked [Ca(2+)]i in PC12 cells by inhibiting voltage-gated calcium channels (VGCCs) in vitro at concentrations comparable with human occupational exposure levels. Moreover, the effective concentrations in this study are below those for earlier described modes of action. Because inhibition of VGCCs appears to be a common and potentially additive mode of action of several classes of insecticides, this target should be considered in neurotoxicity risk assessment studies.

The venom of the spider Selenocosmia jiafu contains various neurotoxins acting on voltage-gated ion channels in rat dorsal root ganglion neurons.

Selenocosmia jiafu is a medium-sized theraphosid spider and an attractive source of venom, because it can be bred in captivity and it produces large amounts of venom. We performed reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses and showed that S. jiafu venom contains hundreds of peptides with a predominant mass of 3000-4500 Da. Patch clamp analyses indicated that the venom could inhibit voltage-gated Na+, K+ and Ca2+ channels in rat dorsal root ganglion (DRG) neurons. The venom exhibited inhibitory effects on tetrodotoxin-resistant (TTX-R) Na+ currents and T-type Ca2+ currents, suggesting the presence of antagonists to both channel types and providing a valuable tool for the investigation of these channels and for drug development. Intra-abdominal injection of the venom had severe toxic effects on cockroaches and caused death at higher concentrations. The LD50 was 84.24 µg/g of body weight in the cockroach. However, no visible symptoms or behavioral changes were detected after intraperitoneal injection of the venom into mice even at doses up to 10 mg/kg body weight. Our results provide a basis for further case-by-case investigations of peptide toxins from this venom.

Successful reduction of off-target hERG toxicity by structural modification of a T-type calcium channel blocker.

To obtain an optimized T-type calcium channel blocker with reduced off-target hERG toxicity, we modified the structure of the original compound by introducing a zwitterion and reducing the basicity of the nitrogen. Among the structurally modified compounds we designed, compounds 5 and 6, which incorporate amides in place of the original compound's amines, most appreciably alleviated hERG toxicity while maintaining T-type calcium channel blocking activity. Notably, the benzimidazole amide 5 selectively blocked T-type calcium channels without inhibiting hERG (hERG/T-type⩾220) and L-type channels (L-type/T-type=96), and exhibited an excellent pharmacokinetic profile in rats.

In vitro cytotoxicity on human ovarian cancer cells by T-type calcium channel blockers.

The growth inhibition of human cancer cells via T-type Ca(2+) channel blockade has been well known. Herein, a series of new 3,4-dihydroquinazoline derivatives were synthesized via a brief SAR study on KYS05090 template and evaluated for both T-type Ca(2+) channel (Cav3.1) blockade and cytotoxicity on three human ovarian cancer cells (SK-OV-3, A2780 and A2780-T). Most of compounds except 6i generally exhibited more potent cytotoxicity on SK-OV-3 than mibefradil as a positive control regardless of the degree of T-type channel blockade. In particular, eight compounds (KYS05090, 6a and 6c-6h) showing strong channel blockade exhibited almost equal and more potent cytotoxicity on A2780 when compared to mibefradil. On A2780-T paclitaxel-resistant human ovarian carcinoma, two compounds (KYS05090 and 6d) were 20-fold more active than mibefradil. With respect to cell cycle arrest effect on A2780 and A2780-T cells, KYS05090 induced large proportion of sub-G1 phase in the cell cycle progression of A2780 and A2780-T, meaning the induction of cancer cell death instead of cell cycle arrest via blocking T-type Ca(2+) channel. Among new analogues, compounds 6g and 6h induced cell cycle arrest at G1 phase of A2780 and A2780-T cells in dose-dependent manner and exhibited strong anti-proliferation effects of ovarian cancer cells by blocking T-type Ca(2+) channel. Furthermore, 6g and 6h possessing strong cytotoxic effects could induce apoptosis of A2780 cells, which was detected by confocal micrographs using DAPI staining.

Spinal actions of ω-conotoxins, CVID, MVIIA and related peptides in a rat neuropathic pain model.

BACKGROUND AND PURPOSE: Antagonists of the N-type voltage gated calcium channel (VGCC), Cav 2.2, have a potentially important role in the treatment of chronic neuropathic pain. ω-conotoxins, such MVIIA and CVID are effective in neuropathic pain models. CVID is reported to have a greater therapeutic index than MVIIA in neuropathic pain models, and it has been suggested that this is due to faster reversibility of binding, but it is not known whether this can be improved further. EXPERIMENTAL APPROACH: We examined the potency of CVID, MVIIA and two intermediate hybrids ([K10R]CVID and [R10K]MVIIA) to reverse signs of neuropathic pain in a rat nerve ligation model in parallel with production of side effects. We also examined the potency and reversibility to inhibit primary afferent synaptic neurotransmission in rat spinal cord slices. KEY RESULTS: All ω-conotoxins produced dose-dependent reduction in mechanical allodynia. They also produced side effects on the rotarod test and in a visual side-effect score. CVID displayed a marginally better therapeutic index than MVIIA. The hybrids had a lesser effect in the rotarod test than either of their parent peptides. Finally, the conotoxins all presynaptically inhibited excitatory synaptic neurotransmission into the dorsal horn and displayed recovery that was largely dependent upon the magnitude of inhibition and not the conotoxin type. CONCLUSIONS AND IMPLICATIONS: These findings indicate that CVID provides only a marginal improvement over MVIIA in a preclinical model of neuropathic pain, which appears to be unrelated to reversibility from binding. Hybrids of these conotoxins might provide viable alternative treatments.