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Background: The widespread misuse of synthetic cannabinoids (SCs) has led to a notable increase in reported adverse effects, raising significant health concerns. SCs use has been particularly associated with acute kidney injury (AKI). However, the pathogenesis of SCs-induced AKI is not well-understood., Methods: We investigated the nephrotoxic effect of acute administration of N-[(1S)- 1-(aminocarbonyl)-2-methylpropyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (AB-FUBINKA) (3 mg/kg for 5 days) in mice. Various parameters of oxidative stress, inflammation, and apoptosis have been quantified. The expressions of mitochondrial complexes (I-V) in renal tissues were also assessed., Results: Our findings showed that AB-FUBINACA induced substantial impairment in the renal function that is accompanied by elevated expression of renal tubular damage markers; KIM-1 and NGAL. Administration of AB-FUBINACA was found to be associated with a significant increase in the expression of oxidative stress markers (iNOS, NOX4, NOX2, NOS3) and the level of lipid peroxidation in the kidney. The expression of pro-inflammatory markers (IL-6, TNF-alpha, NF-kB) was also enhanced following exposure to AB-FUBINACA. These findings were also correlated with increased expression of major apoptosis regulatory markers (Bax, caspase-9, caspase-3) and reduced expression of mitochondrial complexes I, III, and IV., Conclusion: These results indicate that AB-FUBINACA can trigger oxidative stress and inflammation, and activate caspase-dependent apoptosis in the kidney, with these processes being possibly linked to disruption of mitochondrial complexes and could be an underlying mechanism of SCs-induced nephrotoxicity., Competing Interests: Declarations. Conflict of interest: The authors declare that there is no conflict of interest. Ethics approval: This animal experiments of this study were conducted in accordance the guidelines of the ethics committee for use of animals in research and approved by the Institutional Review Board at the Yarmouk University (No. IACUC/2021/14). Consent for publication: Not applicable., (© 2024. The Author(s).)

Purpose: There is very little information about the toxicological and pathological effects of synthetic cannabinoids, which have cannabis-like properties. This study was carried out to histopathologically, hematologically, and biochemically determine the toxic effects of acute and subacute exposure to a novel synthetic cannabinoid 1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)indazole-3-carboxamide in internal organs of adult male rats., Methods: The cannabinoid was injected intraperitoneally at three doses (0.5, 1.0, and 2.0 mg/kg, body weight). The cannabinoid was administered to acute groups for 2 days and to subacute groups for 14 days. Observations were made for 14 days and various changes such as mortality, injury, and illness were recorded daily. Hematological and biochemical changes were evaluated and histopathological analyses in lung, liver, and kidney tissues were also performed., Results: No mortality was observed. It was observed that there were fluctuations in hematological and serum biochemical parameters. Among the oxidative stress parameters, significant decreases in superoxide dismutase, catalase levels and significant increases in lipid peroxidation levels were determined. Serious pathological changes such as necrosis, vacuolation, congestion, and fibrosis were observed in the internal organs in a dose-dependent and time-dependent manner. It was also found that the synthetic cannabinoid triggered apoptosis in the organs. The results demonstrated that the most affected organ by the cannabinoid was the kidney., Conclusion: This study showed for the first time that CUMYL-4CN-BINACA adversely affects healthy male albino rats. It can be estimated that the abuse of the cannabinoid may harm human health in the same way., (© 2023. The Author(s), under exclusive licence to Japanese Association of Forensic Toxicology.)

Sepsis is a life-threatening condition caused by a dysregulated host response to infection. The development of sepsis is associated with excessive nitric oxide (NO) production, which plays an important role in controlling vascular homeostasis. 7-nitroindazole (7-NI) is a selective inhibitor of neuronal nitric oxide synthase (NOS-1) with potential application for treating NO imbalance conditions. However, 7-NI exhibits a low aqueous solubility and a short plasma half-life. To circumvent these biopharmaceutical limitations, pegylated (NEPEG 7NI ) and non-pegylated nanoemulsions (NENPEG 7 NI ) containing 7-NI were developed. This study evaluates the pharmacokinetic profiles and toxicological properties of 7-NI loaded into the nanoemulsions. After a single intravenous administration of the free drug and the nanoemulsions at a dose of 10 mg.kg -1 in Wistar rats, 7-NI was widely distributed in the organs. The pharmacokinetic parameters of C max , t 1/2 , and AUC 0-t were significantly increased after administration of the NEPEG 7NI , compared to both free 7-NI and NENPEG 7NI (p < 0.05). No observable adverse effects were observed after administering the free 7-NI, NEPEG 7NI , or NENPEG 7NI in the animals after a single dose of up to 3.0 mg.kg -1 . The results indicated that 7-NI-loaded nanoemulsions are safe, constituting a promising approach to treating sepsis., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Drug-induced liver injury (DILI) is the leading cause of acute liver failure and a major concern in drug development. Altered bile acid homeostasis via inhibition of the bile salt export pump (BSEP) is one mechanism of DILI. Dasatinib, pazopanib, and sorafenib are tyrosine kinase inhibitors (TKIs) that competitively inhibit BSEP and increase serum biomarkers for hepatotoxicity in ∼25-50% of patients. However, the mechanism(s) of hepatotoxicity beyond competitive inhibition of BSEP are poorly understood. This study examined mechanisms of TKI-mediated hepatotoxicity associated with altered bile acid homeostasis. Dasatinib, pazopanib, and sorafenib showed bile acid-dependent toxicity at clinically relevant concentrations, based on the C-DILI assay using sandwich-cultured human hepatocytes (SCHH). Among several bile acid-relevant genes, cytochrome P450 (CYP) 7A1 mRNA was specifically upregulated by 6.2- to 7.8-fold (dasatinib) and 5.7- to 9.3-fold (pazopanib), compared with control, within 8 hours. This was consistent with increased total bile acid concentrations in culture medium up to 2.3-fold, and in SCHH up to 1.4-fold, compared with control, within 24 hours. Additionally, protein abundance of sodium taurocholate co-transporting polypeptide (NTCP) was increased up to 2.0-fold by these three TKIs. The increase in NTCP protein abundance correlated with increased function; dasatinib and pazopanib increased hepatocyte uptake clearance (CL uptake ) of taurocholic acid, a probe bile acid substrate, up to 1.4-fold. In conclusion, upregulation of CYP7A1 and NTCP in SCHH constitute novel mechanisms of TKI-associated hepatotoxicity. SIGNIFICANCE STATEMENT: Understanding the mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors (TKIs) is fundamental to development of effective and safe intervention therapies for various cancers. Data generated in sandwich-cultured human hepatocytes, an in vitro model of drug-induced hepatotoxicity, revealed that TKIs upregulate bile acid synthesis and alter bile acid uptake and excretion. These findings provide novel insights into additional mechanisms of bile acid-mediated drug-induced liver injury, an adverse effect that limits the use and effectiveness of TKI treatment in some cancer patients., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Pazopanib is a tyrosine kinase inhibitor that is generally used for the treatment of metastatic renal cell cancer and advanced soft tissue sarcoma. It can cause various degrees of hepatotoxicity. Our study aimed to investigate the effect of taxifolin on pazopanib-induced liver toxicity. A total of 18 rats were divided into three groups: the pazopanib (PP), pazopanib plus taxifolin (TPP), and control (C) group. Taxifolin was administered to the TPP (n=6) group with a dose of 50 mg/kg. Distilled water was orally admnistered to the C (n=6) and PP (n=6) groups as a solvent. Subsequently, pazopanib 200 mg/kg was administered to the TPP and PP groups via the stomach. This procedure was repeated once a day for four weeks. Then, all rats were sacrificed, and their livers were removed. Malondialdehyde (MDA), total glutathione (tGSH), total oxidant status (TOS), and total antioxidant status (TAS) levels were evaluated. MDA and TOS levels were higher in the PP group compared with the levels of the other parameters (P<0.001). tGSH and TAS levels were lower in the PP group than in the TPP and C groups (P<0.001), and the aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels were higher. Furthermore, liver tissue damage, including hemorrhage, hydropic degeneration, and necrosis was observed in the PP group. Administration of taxifolin before pazopanib significantly improved degenerative changes. Our study demonstrated that the administration of taxifolin is significantly effective in preventing pazopanib-induced hepatotoxicity in rats.

Introduction: Synthetic cannabinoids are abused substances with strong psychoactive effects. Little is known about the effects on neurotransmission and the toxicity of the second-generation cannabinoid 5F-APINAC. The objective was to assess the influence of short- and long-term exposures of 5F-APINAC on metabolites associated with neurotransmission on zebrafish., Methods: Short-term ("acute", 4 h) and long-term ("chronic", 96 h) exposures to 5F-APINAC were performed at 0.001, 0.01, 0.1, 1.0 and 10 μM. Intervention groups were compared with a vehicle control. Each group n = 20 zebrafish eggs/larvae. Metabolites related to neurotransmission were determined., Results: In chronic exposure, larvae exposed to 10 μM 5F-APINAC presented morphological and developmental alterations. GABA had the lowest concentrations at higher exposure in acute (p < 0.01) and chronic (p < 0.001) experiments. Glutamine showed a descending trend in the acute experiment, but an ascending trend in the chronic exposure (p < 0.05). In chronic exposure, tryptophan presented an overall descending trend, but with a neat increase at 10 μM 5F-APINAC (p < 0.001). Tryptamine in acute exposure presented lower (p < 0.05) concentrations at higher doses. Dopamine and acetylcholine presented highest (p < 0.05) concentrations in the acute and chronic exposures, but with a drop at the highest doses in the chronic experiments. In chronic exposure, xanthurenic acid decreased, except for the highest dose. Picolinic acid was increased at the highest doses in the chronic experiment (p < 0.001)., Conclusions: Short- and long-term exposures induced metabolomic alterations associated with the gamma-aminobutyric acid/glutamic acid, dopaminergic/adrenergic, cholinergic neurotransmitter systems, and the kynurenine pathway. Chronic exposure at 10 μM 5F-APINAC was associated with embryotoxicity confirmed by teratogenesis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Marijuana or synthetic cannabinoids and alcohol are often used together, with these combinations causing motor impairments that can subsequently lead to motor vehicle accidents. This study investigated the combined use of both synthetic cannabinoids and ethanol and their effect on motor coordination in mice in addition to examining the neurochemical changes in the cerebellum. Ethanol (2 g/kg, i.p.) significantly induced motor impairment in the accelerating rotarod test in mice. Furthermore, ethanol-induced motor impairments were further accentuated when combined with the synthetic cannabinoid, JWH-018 or AB-CHMINACA. The enhancement effects of the synthetic cannabinoids were completely antagonized by pretreatment with the selective CB 1 receptor antagonist AM251, but not by the selective CB 2 receptor antagonist AM630. Neurochemical study results showed that ethanol caused a reduction in the extracellular glutamate levels in the cerebellum during periods of ethanol-induced motor impairment. In addition to the enhanced motor impairment seen when ethanol was combined with JWH-018, these combinations also enhanced the reduction of the extracellular glutamate levels in the cerebellum. We additionally used microelectrode array recordings to examine the effects of ethanol and/or JWH-018 on the spontaneous network activity in primary cultures from mouse cerebellum. Results showed that ethanol combined with JWH-018 significantly reduced spontaneous neuronal network activity in the primary cerebellar culture. Our findings demonstrate that ethanol-induced motor impairments are enhanced by synthetic cannabinoids, with these effects potentially mediated by CB 1 receptors. An accentuated reduction of neurotransmissions in the cerebellum may play an important role in motor impairments caused by ethanol combined with synthetic cannabinoids., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Benign prostatic hyperplasia (BPH) is a progressive proliferative disease, the incidence of which is constantly increasing due to aging of population. In this research, a hexokinase-II enzyme inhibiting agent, lonidamine - the use of which is limited in BPH treatment due to high hepatic toxicity observed after three months of treatment - was selected as an active agent, based on its mechanism of action in treating BPH. The aim of this study was to evaluate in vivo therapeutic efficacy and hepatic toxicity of lipid-polymer hybrid nanoparticles of lonidamine in a rat BPH model created in rat prostates. After local injections of hybrid nanoparticles of lonidamine were administered to the rat prostates, hyperplasic structures of prostates were evaluated in terms of prostatic index values, immunohistochemical evaluations, and histopathological findings. Liver blood enzyme values were also determined to specify hepatic toxicity. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction and histopathological methods to determine intravital degenerative destruction in liver. Through this study, lonidamine-loaded hybrid nanoparticles were found to reduce the hepatic toxicity and increase therapeutic efficiency of lonidamine. Therefore, lonidamine-entrapped hybrid nanoparticles may provide a promising, and very safe, drug delivery strategy in the treatment of BPH., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In previous studies, we have identified several families of 5-nitroindazole derivatives as promising antichagasic prototypes. Among them, 1-(2-aminoethyl)-2-benzyl-5-nitro-1,2-dihydro-3H-indazol-3-one, (hydrochloride) and 1-(2-acetoxyethyl)-2-benzyl-5-nitro-1,2-dihydro-3H-indazol-3-one (compounds 16 and 24, respectively) have recently shown outstanding activity in vitro over the drug-sensitive Trypanosoma cruzi CL strain (DTU TcVI). Here, we explored the activity of these derivatives against the moderately drug-resistant Y strain (DTU TcII), in vitro and in vivo. The outcomes confirmed their activity over replicative forms, showing IC50 values of 0.49 (16) and 5.75 μm (24) towards epimastigotes, 0.41 (16) and 1.17 μm (24) against intracellular amastigotes. These results, supported by the lack of toxicity on cardiac cells, led to better selectivities than benznidazole (BZ). Otherwise, they were not as active as BZ in vitro against the non-replicative form of the parasite, i.e. bloodstream trypomastigotes. In vivo, acute toxicity assays revealed the absence of toxic events when administered to mice. Moreover, different therapeutic schemes pointed to their capability for decreasing the parasitaemia of T. cruzi Y acute infected mice, reaching up to 60% of reduction at the peak day as monotherapy (16), 79.24 and 91.11% when 16 and 24 were co-administered with BZ. These combined therapies had also a positive impact over the mortality, yielding survivals of 83.33 and 66.67%, respectively, while untreated animals reached a cumulative mortality of 100%. These findings confirm the 5-nitroindazole scaffold as a putative prototype for developing novel drugs potentially applicable to the treatment of Chagas disease and introduce their suitability to act in combination with the reference drug.

AKB48 and its fluorinated derivative 5F-AKB48 are synthetic cannabinoids (SCs) which have caused hospitalizations and deaths in human users. Abuse of SCs is dangerous because users may mistake them for natural cannabis, which is generally considered to be unlikely to elicit adverse effects. The present studies were designed to investigate the in vitro oxidative metabolism of 5F-AKB48 by human microsomal fractions from different organs and sexes as well as recombinant human cytochrome P450s (P450s). Mass spectrometry data tentatively provides evidence for the existence of mono-, di-, and trihydroxylated metabolites in a successive metabolism. Experiments utilizing P450s revealed that the most active enzymes (CYP2D6, CYP2J2, CYP3A4, and CYP3A5) effectively produced mono- and dihydroxylated metabolites, while CYP3A4/5 also produced significant amounts of the trihydroxylated metabolite. Moreover, although the affinity and potency of Phase I metabolite 4OH-5F-AKB48 is reduced when compared to that of the parent drug, this metabolite nevertheless retains similar high affinity for CB1 receptors, and greater efficacy for G protein activation, when compared to THC. Finally, 5F-AKB48 produced time- and dose-dependent cannabimimetic effects in mice which were more potent, but shorter acting, than those of Δ 9 -THC, and were attenuated by prior treatment with the CB1 antagonist rimonabant. Based on our data, we hypothesize that while many cases of toxicity result from genetic mutations, which can lead to a decrease or even absence of activity for Phase I drug-metabolizing enzymes, other P450s could potentially increase their role in the metabolism of these SCs. Because many metabolites of SCs remain biologically active, they could contribute to the deleterious effects of these substances., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Structure-based drug design enabled the discovery of 8 , HTL22562, a calcitonin gene-related peptide (CGRP) receptor antagonist. The structure of 8 complexed with the CGRP receptor was determined at a 1.6 Å resolution. Compound 8 is a highly potent, selective, metabolically stable, and soluble compound suitable for a range of administration routes that have the potential to provide rapid systemic exposures with resultant high levels of receptor coverage (e.g., subcutaneous). The low lipophilicity coupled with a low anticipated clinically efficacious plasma exposure for migraine also suggests a reduced potential for hepatotoxicity. These properties have led to 8 being selected as a clinical candidate for acute treatment of migraine.

The increased diffusion of the so-called novel psychoactive substances (NPS) and their continuous change in structure andconceivably activity has led to the need of a rapid screening method to detect their biological effects as early as possible after their appearance in the market. This problem is very felt in forensic pathology and toxicology, so the preclinical study is fundamental in the approach to clinical and autopsy cases of difficult interpretation intoxication. Zebrafish is a high-throughput suitable model to rapidly hypothesize potential aversive or beneficial effects of novel molecules. In the present study, we measured and compared the behavioral responses to two novel neuroactive drugs, namely APINAC, a new cannabimimetic drug, and methiopropamine (MPA), a methamphetamine-like compound, on zebrafish larvae (ZL) and adult mice. By using an innovative statistical approach (general additive models), it was found that the spontaneous locomotor activity was impaired by the two drugs in both species: the disruption extent varied in a dose-dependent and time-dependent manner. Sensorimotor function was also altered: i) the visual object response was reduced in mice treated with APINAC, whereas it was not after exposure to MPA; ii) the visual placing responses were reduced after treatment with both NPS in mice. Furthermore, the visual motor response detected in ZL showed a reduction after treatment with APINAC during light-dark and dark-light transition. The same pattern was found in the MPA exposed groups only at the dark-light transition, while at the transition from light to dark, the individuals showed an increased response. In conclusion, the present study highlighted the impairment of spontaneous motor and sensorimotor behavior induced by MPA and APINAC administration in both species, thus confirming the usefulness of ZL as a model for a rapid behavioural-based drug screening., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Synthetic cannabinoids are an emerging clinical and public health concern. The current study aimed to determine: (1) The characteristics and circumstances of death of all recorded cases of synthetic cannabinoid-related sudden or unnatural death in Australia, (2) The toxicology of cases and (3) Their major organ pathology. Methods: Retrospective study of all cases in Australia in which synthetic cannabinoid use was a mechanism contributory to death ( n   =  55) retrieved from the National Coronial Information System (2000-2017). Information was collected on cause of death, demographics, drug use history, circumstances of death, toxicology and major organ pathology. Results: The mean age was 37.2 years and 91.1% were male. Causes of death comprised of accidental toxicity (38.2%), accidental toxicity/cardiovascular disease (9.1%), natural disease (20.0%), suicide (10.9%) and traumatic accident (10.9%). The most common clinical presentation proximal to death was sudden collapse (25.5%). Cardiovascular disease was prominent: severe atherosclerosis (20.0%), myocardial replacement fibrosis (18.0%), cardiomegaly (12.0%). The most frequent synthetic cannabinoids were the indazolecarboxemides (61.8%), most commonly AB-CHMINACA (38.2%). The most frequent other substances were alcohol (34.5%) and Δ 9 -THC (23.6%). Conclusions: AB-CHMINACA was the most commonly seen synthetic cannabinoid. There was a high representation of relatively older decedents and of older males in particular. While acute toxicity was the most common cause of death, cardiovascular disease was prominent.

The widespread recreational use of synthetic cannabinoids (SCBs) represents a major public health issue, as reports of intoxications and deaths following SCB use rapidly mount up. Specifically, a direct link between SCB use and acute kidney injury (AKI) has been established, although the pathophysiologic mechanisms remain undefined. Here we assessed the in vitro nephrotoxicity of 3 commonly detected and structurally distinct SCBs-AB-FUBINACA, JWH-122, and THJ-2201-in human proximal tubule cells (HK-2), to ascertain potential similarities and/or differences regarding their nephrotoxicity signatures. We showed that 2 of the 3 SCBs tested, namely JWH-122 and THJ-2201, at in vivo relevant concentrations (1 nM-1 μM), triggered apoptotic cell death pathways, mainly through a shared mechanism involving the deregulation of mitochondrial function (ie, with mitochondrial membrane hyperpolarization and increased intracellular ATP levels), as the primary molecular signature of nephrotoxicity mechanism. Noteworthy, no SCB affected cell viability (MTT reduction, lactate dehydrogenase release, Neutral Red inclusion). Use of the cannabinoid receptor (CBR) antagonists SR141716A and SR144528, as well as HEK293T cells, which do not express CBRs, confirmed the involvement of these receptors in SCB-mediated mitochondrial membrane hyperpolarization but not on other events, suggesting an off-target action regulating SCB-induced kidney cell death. Our results further strengthen the relevance of the endocannabinoid system in maintaining mitochondrial function in kidney cells, as we demonstrate that HK-2 incubation with CBR antagonists or inhibitors of endocannabinoid biosynthesis (ie, methyl arachydonyl fluorophosphonate, tetrahydrolipstatin) alone produced deleterious effects similar to those now reported for SCBs. Overall, SCB-induced nephrotoxicity seems to be mainly regulated at the mitochondrial level, but the specific mechanisms involved require further clarification., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Synthetic cannabinoids were introduced into market in early 2000s; since these "legal highs" are dramatically popular among youth, it becomes a deadly problem. Synthetic cannabinoids have high affinity to cannabinoid receptors; leading to various clinical symptoms. AKB48 (Apinaca) has been classified as a third-generation synthetic cannabinoid for the first time in 2014. The toxicity profile of AKB48 is unclear due to little information that mainly obtained from clinical and forensic cases; however, it is believed to be similar with other psychoactive substances. Thus, we aimed to investigate the possible toxicity mechanisms of AKB48 in SH-SY5Y (human bone marrow neuroblastoma) cell line. IC 50 value of AKB48 was calculated as 160.91 μM by MTT assay. AKB48 treatment enhanced (≥1.2-fold) the fluorescence intensity indicating increased reactive oxygen species production; however, glutathione levels did not changed in the range of 25-200 μM exposure concentrations. Cannabinoid type-1 receptor (CB1) expression was increased ≥15-fold in the range of 25-50 μM of AKB48, while cannabinoid type-2 receptor (CB2) did not expressed in SH-SY5Y cells. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α) were up-regulated with a dose-dependent manner, and the profiles were almost identical; however, mitogen-activated protein kinase 8 (MAPK 8) was only upregulated with 25 μM of AKB48 and nuclear factor kappa B (NF-ĸB) did not change. Our results should raise the concerns about the safety associated with synthetic cannabinoids uses., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Based on some common structural features of known compounds interfering with p53 pathways and our previously synthesized benzamides, we synthesized new ethyl 5-(4-substituted benzamido)-1-phenyl-1H-pyrazole-4-carboxylates 26a-c, ethyl 5-(4-substituted benzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-carboxylates 27a-c and N-(1H-indazol-6-yl)-4-substituted benzamides 31a,b bearing in the 4 position of the benzamido moiety the 2-phenylpropanamido or 2-phenoxyacetamido or cinnamamido groups. A preliminary test to evaluate the antiproliferative activity against human lung carcinoma H292 cells highlighted how compound 26c showed the best activity. This last was therefore selected for further studies with the aim to find the mechanism of action. Compound 26c induces intrinsic apoptotic pathway by activating p53 and is also able to activate TRAIL-inducing death pathway by promoting increase of DR4 and DR5 death receptors, downregulation of c-FLIP L and caspase-8 activation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Convulsant effects of abused synthetic cannabinoid (SCB) drugs have been reported in humans and laboratory animals, but the mechanism of these effects is not known. We compared convulsant effects of partial CB1R agonist ∆ 9 -tetrahydrocannabinol (THC), full CB1R agonist SCBs JWH-018 and 5F-AB-PINACA, and classic chemical convulsant pentylenetetrazol (PTZ) using an observational rating scale in mice. THC did not elicit convulsions, but both SCBs did so as effectively as and more potently than PTZ. SCB-elicited convulsions were attenuated by the CB1R antagonist rimonabant or by THC, or by dose regimens of THC and JWH-018, which downregulate and desensitize CB1Rs. None of these treatments altered the convulsant effects of PTZ, although diazepam attenuated PTZ-elicited convulsions without altering SCB-induced convulsant effects. Repeated administration of a subthreshold dose of PTZ kindled convulsant effects, but this was not observed with the SCBs, and no cross-kindling was observed. Repeated administration of the SCBs resulted in tolerance to convulsant effects, but no cross-tolerance to PTZ was observed. Inhibition on Phase I metabolism via nonselective inhibition of CYP450s with 1-aminobenzotriazole potentiated the hypothermic effects of the SCBs and protected against the convulsant effects of JWH-018, but not those of 5F-AB-PINACA or PTZ. Incubation of human liver microsomes with the SCBs showed that JWH-018 is eliminated via oxidation, whereas 5F-AB-PINACA is not. These studies suggest that SCB-elicited convulsions are mediated by high intrinsic efficacy at CB1Rs and that benzodiazepines may not be effective treatments. Finally, drug metabolism may dramatically modulate the convulsant effects of some, but not all, SCBs., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Chagas disease, a neglected tropical disease caused by infection with the protozoan parasite Trypanosoma cruzi, is a potentially life-threatening illness that affects 5-8 million people in Latin America, and more than 10 million people worldwide. It is characterized by an acute phase, which is partly resolved by the immune system, but then develops as a chronic disease without an effective treatment. There is an urgent need for new antiprotozoal agents, as the current standard therapeutic options based on benznidazole and nifurtimox are characterized by limited efficacy, toxicity, and frequent failures in treatment. In vitro and in vivo assays were used to identify some new low-cost 5-nitroindazoles as a potential antichagasic therapeutic alternative. Compound 16 (3-benzyloxy-5-nitro-1-vinyl-1H-indazole) showed improved efficiency and lower toxicity than benznidazole in both in vitro and in vivo experiments, and its trypanocidal activity seems to be related to its effect at the mitochondrial level. Therefore, compound 16 is a promising candidate for the development of a new anti-Chagas agent, and further preclinical evaluation should be considered., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Three different series of new 5-nitroindazole derivatives-1-(ω-aminoalkyl)-2-benzylindazolin-3-ones (series A; ten compounds), 3-(ω-aminoalkoxy)-2-benzylindazoles (series B; four compounds) and 3-alkylamino-2-benzylindazoles (series C; five compounds)-have been synthesized and evaluated against the protozoan parasites Trypanosoma cruzi, Leishmania amazonensis, and Trichomonas vaginalis: etiological agents of Chagas disease, cutaneous leishmaniasis, and trichomoniasis, respectively. Many indazoles of series A, B, and C were efficient against T. cruzi. Some compounds in series A, after successfully passing the preliminary screening for epimastigotes, exhibited activity values against amastigotes of several T. cruzi strains that were better than or similar to those shown by the reference drug benznidazole and displayed low nonspecific toxicity against mammalian cells. On the other hand, preliminary studies against promastigotes of L. amazonensis showed high leishmanicidal activity for some derivatives of series A and C. With regard to activity against T. vaginalis, some indazoles of series B and C were rather efficient against trophozoites of a metronidazole-sensitive isolate and showed low nonspecific toxicities toward Vero cell cultures. Additionally, some of these compounds displayed similar activity against metronidazole-sensitive and resistant isolates, showing the absence of cross-resistance between these derivatives and the reference drug., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Both histone deacetylase (HDAC) and fibroblast growth factor receptor (FGFR) are important targets for cancer therapy. Although combining dual HDAC pharmacophore with tyrosine kinase inhibitors (TKIs) had achieved a successful progress, dual HDAC/FGFR1 inhibitors haven't been reported yet. Herein, we designed a series of hybrids bearing 1H-indazol-3-amine and benzohydroxamic acids scaffold with scaffold hopping and molecular hybridization strategies. Among them, compound 7j showed the most potent inhibitory activity against HDAC6 with IC 50 of 34 nM and exhibited the great inhibitory activities against a human breast cancer cell line MCF-7 with IC 50 of 9 μM in vitro. Meanwhile, the compound also exhibited moderate FGFR1 inhibitory activities. This study provides new tool compounds for further exploration of dual HDAC/FGFR1 inhibition., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: The emergence of novel psychoactive substances (NPS), particularly synthetic cannabinoid receptor agonists (SCRA), has involved hundreds of potentially harmful chemicals in a highly dynamic international market challenging users', clinicians', and regulators' understanding of what circulating substances are causing harm. We describe a toxicovigilance system for NPS that predicted the UK emergence and identified the clinical toxicity caused by novel indole and indazole carboxylate SCRA., Methods: To assist early accurate identification, we synthesized 5 examples of commercially unavailable indole and indazole carboxylate SCRA (FUB-NPB-22, 5F-NPB-22, 5F-SDB-005, FUB-PB-22, NM-2201). We analyzed plasma and urine samples from 160 patients presenting to emergency departments with severe toxicity after suspected NPS use during 2015 to 2016 for these and other NPS using data-independent LC-MS/MS., Results: We successfully synthesized 5 carboxylate SCRAs using established synthetic and analytical chemistry methodologies. We identified at least 1 SCRA in samples from 49 patients, including an indole or indazole carboxylate SCRA in 17 (35%), specifically 5F-PB-22 (14%), FUB PB-22 (6%), BB-22 (2%), 5F NPB-22 (20%), FUB NPB-22 (2%), and 5F-SDB-005 (4%). In these 17 patients, there was analytical evidence of other substances in 16. Clinical features included agitation and aggression (82%), reduced consciousness (76%), acidosis (47%), hallucinations and paranoid features (41%), tachycardia (35%), hypertension (29%), raised creatine kinase (24%), and seizures (12%)., Conclusions: This toxicovigilance system predicted the emergence of misuse of indole and indazole carboxylate SCRA, documented associated clinical harms, and notified relevant agencies. Toxicity appears consistent with other SCRA, including mental state disturbances and reduced consciousness., (© 2017 American Association for Clinical Chemistry.)

Background and Purpose: Several anti-angiogenic cancer drugs that inhibit VEGF receptor (VEGFR) signalling for efficacy are associated with a 15-60% incidence of hypertension. Tyrosine kinase inhibitors (TKIs) that have off-target activity at VEGFR-2 may also cause blood pressure elevation as an undesirable side effect. Therefore, the ability to translate VEGFR-2 off-target potency into blood pressure elevation would be useful in development of novel TKIs. Here, we have sought to quantify the relationship between VEGFR-2 inhibition and blood pressure elevation for a range of kinase inhibitors., Experimental Approach: Porcine aortic endothelial cells overexpressing VEGFR-2 (PAE) were used to determine IC 50 for VEGFR-2 phosphorylation. These IC 50 values were compared with published reports of exposure attained during clinical use and the corresponding incidence of all-grade hypertension. Unbound average plasma concentration (C av,u ) was selected to be the most appropriate pharmacokinetic parameter. The pharmacokinetic-pharmacodynamic (PKPD) relationship for blood pressure elevation was investigated for selected kinase inhibitors, using data derived either from clinical papers or from rat telemetry experiments., Key Results: All-grade hypertension was predominantly observed when the C av,u was >0.1-fold of the VEGFR-2 (PAE) IC 50 . Furthermore, based on the PKPD analysis, an exposure-dependent blood pressure elevation >1 mmHg was observed only when the C av,u was >0.1-fold of the VEGFR-2 (PAE) IC 50 ., Conclusions and Implications: Taken together, these data show that the risk of blood pressure elevation is proportional to the amount of VEGFR-2 inhibition, and a margin of >10-fold between VEGFR-2 IC 50 and C av,u appears to confer a minimal risk of hypertension., (© 2017 The British Pharmacological Society.)

Novel hybrids with MAO and Aβ (1-42) self-aggregation inhibitory activities were designed and synthesized with the employment of indazole moiety and resveratrol. The biological screening results indicated that most compounds displayed potent inhibitory activity for Aβ (1-42) self-aggregation, and obvious selective inhibition to MAO-B. Among these compounds, compound 6e was the most potent inhibitor not only for hMAO-B (IC 50  = 1.14 μM) but also for Aβ (1-42) self-aggregation (58.9% at 20 μM). Molecular modeling and kinetic studies revealed that compound 6e was a competitive MAO-B inhibitor, which can occupy the active site of MAO-B, and interact with Aβ (1-42) via π-π and cation-π stacking interactions. In addition, compound 6e had no toxicity on PC12 cells and could cross the BBB. Collectively, all these results suggested that compound 6e might be a promising multi-target lead compound worthy of further investigation., (Copyright © 2017. Published by Elsevier Inc.)

Background: Cancer metabolic reprogramming rekindles enthusiasm for the research of metabolic regulation in cancer drug resistance. A growing number of metabolic modifiers combined with cancer drugs obtain the expected efficacy in in vitro or in vivo studies, also in clinical trial studies, indicating a good potential of enhancing efficacy and reducing resistance. Hence, a comprehensive review on the attenuations of metabolic modifiers in cancer drug resistance is necessary for rational drug design and clinical cancer drug research., Methods: Cancer drug resistance and cancer metabolic reprogramming were used as the key words to collect publications with reference value in bibliographic databases. Specifically, the focused question is the advances of metabolic modifiers on cancer resistance improvement. Figures and tables were applied to analyze the interventions in accordance with the inclusion criteria., Results: This review summarized the advances of metabolic modifiers combined with cancer drugs in in vitro, in vivo and clinical trial studies, especially for cancer resistance improvement. The relationship between metabolic regulation and cancer resistance was elaborated, and the potential metabolic modifiers were embraced. Metabolic targets were also visualized in categorization in 4 figures and expatiated in 4 tables. Three typical metabolic modifiers, namely lonidamine, 2-DG and 3-BrPA, conferring attenuation to cancer resistance were elucidated systematically., Conclusion: Metabolic regulation is an intervention with targeted perturbation in a modest manner and reflects homeostasis balance. When combined with cancer drugs, the metabolic modifiers always show exciting potential with practical significance, enhancing activity or exerting synergism., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Since 2005, the approved first-line treatment of metastatic renal cell carcinoma consists in tyrosine kinase inhibitors (TKIs) targeting the vascular endothelial growth factor receptors (VEGFRs). Axitinib is an oral second-generation TKI and a potent VEGFR inhibitor with a half maximal inhibitory concentration for the VEGF family receptors 10-fold lower than other TKIs. Axitinib activity in renal cell carcinoma (RCC) patients has been studied in various settings and particularly as second-line treatment. In this setting, axitinib with clinically based dose escalation compared to sorafenib has demonstrated an improvement in progression-free survival in a randomized Phase III trial leading to US Food and Drug Administration approval. In the first-line setting, axitinib failed to demonstrate improved efficacy over sorafenib, but the field of RCC treatment is rapidly changing with novel TKIs as cabozantinib or the emergence of check point inhibitors as nivolumab and the place of axitinib in therapy is therefore challenged. In this review, we focus on axitinib pharmacological and clinical properties in RCC patients and discuss its place in the treatment of patients with RCC., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The toxicities of azole pollutants that have widespread agricultural and industrial uses are either poorly understood or unknown, particularly with respect to how infaunal organisms are impacted by this class of persistent organic pollutant. To identify a molecular basis by which azole compounds may have unforeseen toxicity on marine annelids, we examine here their impact on the multifunctional dehaloperoxidase (DHP) hemoglobin from the terebellid polychaete Amphitrite ornata. Ultraviolet-visible and resonance Raman spectroscopic studies showed an increase in the six-coordinate low-spin heme population in DHP isoenzyme B upon binding of imidazole, benzotriazole, and benzimidazole (K d values of 52, 82, and 110 μM, respectively), suggestive of their direct binding to the heme-Fe. Accordingly, atomic-resolution X-ray crystal structures, supported by computational studies, of the DHP B complexes of benzotriazole (1.14 Å), benzimidazole (1.08 Å), imidazole (1.08 Å), and indazole (1.12 Å) revealed two ligand binding motifs, one with direct ligand binding to the heme-Fe, and another in which the ligand binds in the hydrophobic distal pocket without coordinating the heme-Fe. Taken together, the results demonstrate a new mechanism by which azole pollutants can potentially disrupt hemoglobin function, thereby improving our understanding of their impact on infaunal organisms in marine and aquatic environments.

Introduction: 5F-ADBINACA, AB-FUBINACA, and STS-135 are 3 novel third-generation fluorinate synthetic cannabinoids that are illegally marketed as incense, herbal preparations, or research chemicals for their psychoactive cannabis-like effects., Methods: The present study aims at investigating the in vitro and in vivo pharmacological activity of 5F-ADBINACA, AB-FUBINACA, and STS-135 in male CD-1 mice, comparing their in vivo effects with those caused by the administration of Δ 9 -THC and JWH-018. In vitro competition binding experiments revealed a nanomolar affinity and potency of the 5F-ADBINACA, AB-FUBINACA, and STS-135 on mouse and human CB 1 and CB 2 receptors. Moreover, these synthetic cannabinoids induced neurotoxicity in murine neuro-2a cells., Results: In vivo studies showed that 5F-ADBINACA, AB-FUBINACA, and STS-135 induced hypothermia; increased pain threshold to both noxious mechanical and thermal stimuli; caused catalepsy; reduced motor activity; impaired sensorimotor responses (visual, acoustic, and tactile); caused seizures, myoclonia, and hyperreflexia; and promoted aggressiveness in mice. Behavioral and neurological effects were fully prevented by the selective CB 1 receptor antagonist/inverse agonist AM 251. Differently, the visual sensory response induced by STS-135 was only partly prevented by the AM 251, suggesting a CB 1 -independent mechanism., Conclusions: For the first time, the present study demonstrates the pharmaco-toxicological effects induced by the administration of 5F-ADBINACA, AB-FUBINACA, and STS-135 in mice and suggests their possible detrimental effects on human health., (Copyright © 2017 John Wiley & Sons, Ltd.)

A facile and convenient approach has been developed for the synthesis of functionalized indazoles via solid state melt reaction using easily accessible starting materials under catalyst-free conditions. This transformation involves electrocyclization via a conjugated nitrene intermediate obtained under thermal conditions. Further anti-tubercular activity screening of the molecules was undertaken, among the compounds 3a-3x screened for in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv, compound 3u (MIC: 4.20μM) was found to be most active and are superior over existing standard drugs ciprofloxacin and ethambutol. Compounds 3c and 3x were found to equally potent as ethambutol. Among most potent compounds in the series, four compounds (3n, 3o, 3p and 3u) showed lower cytotoxicity which could be promising drug candidates for further development., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Foci of altered hepatocytes (FAH) are considered putative, pre-neoplastic lesions that can occur spontaneously in aging rodents, but can also be induced by chemicals or drugs. Progression of FAH to hepatocellular neoplasms has been reported repeatedly but increases in foci in rodents do not necessarily lead to tumors in carcinogenicity studies and the relevance for humans often remains unclear. Here we present the case of RG3487, a molecule which induced FAH and, later on, tumors in rats. Because the molecule was negative in genotoxicity assays it was classified as a non-genotoxic carcinogen. In order to assess the potential for liver tumor formation in humans, we analyzed treatment-induced changes in vivo to establish a possible mode of action (MoA). In vivo and in vitro gene expression analysis revealed that nuclear receptor signaling was unlikely to be the relevant MoA and no other known mechanism could be established. We therefore took an approach comparing phenotypic markers, including mRNA changes, proliferation and glycogen accumulation, in vitro using cells of different species to assess the human relevance of this finding. Since the alterations observed in rats were not seen in the liver of mice or dogs in vivo, we could validate the relevance of the cell models chosen by use of hepatocytes from these species in vitro. This ultimately allowed for a cross-species comparison, which suggested that the formation of FAH and liver tumors was rat specific and unlikely to translate to human. Our work showed that phenotypic species comparison in vitro is a useful approach for assessment of the human relevance of pre-clinical findings where no known mechanism can be established., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Angiogenesis plays a major role in the normal embryonic development and in diseases such as cancer. Drugs that control angiogenesis are an alternative way to tackle this disease. The polyphenols usnic acid (3), genistein (5), and daidzein (6) were tested for antiangiogenic and unwanted effects in zebrafish embryos whose blood vessel system resembles that of mammals. The established tyrosine kinase inhibitors axitinib (1) and tyrphostin AG490 (2) were included for comparison. All compounds except 6 caused distinct antiangiogenic effects such as a concentration-dependent reduction of intersegmental vessels, dorsal longitudinal anastomotic vessels, subintestinal veins and secondary sprouts. As side effects, pericardial oedema and the impairment of blood flow were observed. Usnic acid (3), genistein (5) and Cu(II)-genisteinate (7) gave rise to a curvature of the spine. Compounds 5 and 7 also induced cell death in the head of the embryos at higher doses. All effects were more pronounced when the compounds had been applied at an early stage (24 hpf) rather than at 48 hpf. The copper complexes 4 and 7 showed a stronger antiangiogenic effect than the free ligands 3 and 5. The genistein complex 7 was antiangiogenic at doses so low that side effects were tolerable, and thus it may be a potential anticancer drug candidate., (© 2017 Wiley-VHCA AG, Zurich, Switzerland.)

By use of a structure-based computational method for identification of structurally novel Janus kinase (JAK) inhibitors predicted to bind beyond the ATP binding site, a potent series of indazoles was identified as selective pan-JAK inhibitors with a type 1.5 binding mode. Optimization of the series for potency and increased duration of action commensurate with inhaled or topical delivery resulted in potent pan-JAK inhibitor 2 (PF-06263276), which was advanced into clinical studies.

Combination studies of neurokinin 1 (NK1) receptor antagonists and serotonin-selective reuptake inhibitors (SSRIs) have shown promise in preclinical models of depression. Such a combination may offer important advantages over the current standard of care. Herein we describe the discovery and optimization of an indazole-based chemotype to provide a series of potent dual NK1 receptor antagonists/serotonin transporter (SERT) inhibitors to overcome issues of ion channel blockade. This effort culminated in the identification of compound 9, an analogue that demonstrated favorable oral bioavailability, excellent brain uptake, and robust in vivo efficacy in a validated depression model. Over the course of this work, a novel heterocycle-directed asymmetric hydrogenation was developed to facilitate installation of the key stereogenic center.

Lonidamine (LND) was initially introduced as an antispermatogenic agent. It was later found to have anticancer activity sensitizing tumors to chemo-, radio-, and photodynamic-therapy and hyperthermia. Although the mechanism of action remained unclear, LND treatment has been known to target metabolic pathways in cancer cells. It has been reported to alter the bioenergetics of tumor cells by inhibiting glycolysis and mitochondrial respiration, while indirect evidence suggested that it also inhibited l-lactic acid efflux from cells mediated by members of the proton-linked monocarboxylate transporter (MCT) family and also pyruvate uptake into the mitochondria by the mitochondrial pyruvate carrier (MPC). Recent studies have demonstrated that LND potently inhibits MPC activity in isolated rat liver mitochondria (K i 2.5μM) and cooperatively inhibits l-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevis oocytes with K 0.5 and Hill coefficient values of 36-40μM and 1.65-1.85, respectively. In rat heart mitochondria LND inhibited the MPC with similar potency and uncoupled oxidation of pyruvate was inhibited more effectively (IC 50 ~7μM) than other substrates including glutamate (IC 50 ~20μM). LND inhibits the succinate-ubiquinone reductase activity of respiratory Complex II without fully blocking succinate dehydrogenase activity. LND also induces cellular reactive oxygen species through Complex II and has been reported to promote cell death by suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. We conclude that MPC inhibition is the most sensitive anti-tumour target for LND, with additional inhibitory effects on MCT-mediated l-lactic acid efflux, Complex II and glutamine/glutamate oxidation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Retinal arterial tortuosity and venous dilation are hallmarks of plus disease, which is a severe form of retinopathy of prematurity (ROP). In this study, we examined whether short-term interruption of vascular endothelial growth factor (VEGF) signals leads to the formation of severe ROP-like abnormal retinal blood vessels. Neonatal rats were treated subcutaneously with the VEGF receptor (VEGFR) tyrosine kinase inhibitors, KRN633 (1, 5, or 10 mg/kg) or axitinib (10 mg/kg), on postnatal day (P) 7 and P8. The retinal vasculatures were examined on P9, P14, or P21 in retinal whole-mounts stained with an endothelial cell marker. Prevention of vascular growth and regression of some preformed capillaries were observed on P9 in retinas of rats treated with KRN633. However, on P14 and P21, density of capillaries, tortuosity index of arterioles, and diameter of veins significantly increased in KRN633-treated rats, compared to vehicle (0.5% methylcellulose)-treated animals. Similar observations were made with axitinib-treated rats. Expressions of VEGF and VEGFR-2 were enhanced on P14 in KRN633-treated rat retinas. The second round of KRN633 treatment on P11 and P12 completely blocked abnormal retinal vascular growth on P14, but thereafter induced ROP-like abnormal retinal blood vessels by P21. These results suggest that an interruption of normal retinal vascular development in neonatal rats as a result of short-term VEGFR inhibition causes severe ROP-like abnormal retinal vascular growth in a VEGF-dependent manner. Rats treated postnatally with VEGFR inhibitors could serve as an animal model for studying the mechanisms underlying the development of plus disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Synthetic cannabinoids (SCs) cause similar effects as cannabis and are sold in herbal mixtures. Recent investigations indicate that some of these drugs possess genotoxic properties. Therefore, we tested representatives of two groups, namely, aminoalkylindoles (AM-2201 and UR-144) and 1-alkylindazoles (5F-AKB-48 and AM-2201-IC) in single cell gel electrophoresis and micronucleus (MN) assays with human lymphocytes and in Salmonella/microsome assays. All drugs except AM-2201 caused DNA-migration, the LOELs were between 50 and 75 µM. Furthermore, all SCs caused inhibition of cell division and significant induction of MN which reflect structural and numerical chromosomal aberrations. The LOEL values were 50 µM for UR-144 and 5-AKB-48 and 75 µM for the other drugs. Also the levels of nucleoplasmatic bridges which are formed from dicentric chromosomes were elevated under identical conditions while the frequencies of nuclear buds were not affected. These findings show that representatives of both groups cause chromosomal damage while the negative results in Salmonella assays (in strains TA98, TA100, TA1535, TA1537 and TA102) in absence and presence of metabolic activation indicate that they do not induce gene mutations. Taken together, these findings indicate that SCs may cause adverse health effects in users as a consequence of damage of the genetic material., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The medicinal chemistry community has directed considerable efforts toward the discovery of selective inhibitors of interleukin-2 inducible T-cell kinase (ITK), given its role in T-cell signaling downstream of the T-cell receptor (TCR) and the implications of this target for inflammatory disorders such as asthma. We have previously disclosed a structure- and property-guided lead optimization effort which resulted in the discovery of a new series of tetrahydroindazole-containing selective ITK inhibitors. Herein we disclose further optimization of this series that resulted in further potency improvements, reduced off-target receptor binding liabilities, and reduced cytotoxicity. Specifically, we have identified a correlation between the basicity of solubilizing elements in the ITK inhibitors and off-target antiproliferative effects, which was exploited to reduce cytotoxicity while maintaining kinase selectivity. Optimized analogues were shown to reduce IL-2 and IL-13 production in vivo following oral or intraperitoneal dosing in mice.

Background: Synthetic cannabinoid containing products are a public health threat as reflected by a number of outbreaks of serious adverse health effects over the past 4 years. The designer drug epidemic is characterized by the rapid turnover of synthetic cannabinoid compounds on the market which creates a challenge in identifying the particular etiology of an outbreak, confirming exposure in cases, and providing current information to law enforcement., Results: Between 28 May 2014 and 8 June 2014, 35 patients were evaluated and treated at the University of Florida Health Medical Center in Gainesville following reported exposure to a synthetic cannabinoid containing product obtained from a common source. Patients demonstrated acute delirium (24) and seizures (14), and five required ventilator support and ICU-level care; none died. The presence of N-[(1S)-1-(aminocarbonyl)-2-methylpropyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (AB-CHMINACA), or one of its predicted metabolites was confirmed in 15 of 21 cases. A rapid public health response and aggressive public messaging prevented further morbidity, identified the source, and led to law enforcement seizure of the implicated product., Discussion: The significance of this outbreak lies as much in the rapid occurrence of unpredictable, life-threatening adverse health effects from a newly identified synthetic cannabinoid compound as it does in the multidisciplinary investigation and novel partnership between local public health, the laboratory, and the chemical industry, resulting in termination of the outbreak., Conclusion: A coordinated response and collaboration between law enforcement, the local public health, emergency medical services and Health Center staff, were all key interventions in preventing a more substantial public health outbreak resulting from use of a novel synthetic cannabinoid compound. Real time collaborations between toxicology laboratories, suppliers of analytical standards and the public health system may be useful in the face of future novel chemical exposures.

Objective: Venous thromboembolism is a common complication in patients with cancer, resulting in significant morbidity and mortality. Clinical studies suggest that the incidence of venous thromboembolic events increased after treatment of these patients with antiangiogenic agents. Thrombi resolve through a process of remodeling, involving the formation of microvascular channels within the thrombus. Our aim was to determine whether inhibiting angiogenesis affects venous thrombus resolution., Approach and Results: Thrombus was induced in the inferior vena cava of mice. These mice were treated with axitinib (50 mg/kg per day), 2-methoxyestradiol (2ME, 150 mg/kg per day), or vehicle control. Thrombus size, recanalization, neovascularization, inflammatory cell content, and collagen content were assessed after axitinib (days 3, 10, 17) and 2ME (day 10 only) treatment (n=6/group). Axitinib treatment resulted in reduced thrombus resolution (P<0.002) and vein recanalization (P<0.001) compared with vehicle-treated controls. This was associated with inhibition of organization as seen through reduced thrombus neovascularization (P<0.0001) and collagen (P<0.0001) content, as well as reduced macrophage accumulation in the thrombus (P<0.001). Treatment with a second antiangiogenic agent, 2ME, mirrored these findings, with a similar order of magnitude of effect of treatment over vehicle control in all of the parameters measured, with the exception of neutrophil content, which was significantly reduced after 2ME treatment but not affected by axitinib., Conclusions: Antiangiogenic therapy (using axitinib and 2ME) inhibits the resolution of venous thrombi, which could lead to persistent venous obstruction and the possibility of thrombus extension. This potential prolongation of venous occlusion by antiangiogenic agents should therefore be taken into consideration in trials of these agents and when managing the complications of venous thromboembolic events in patients with cancer.

We have previously reported that treatment of newborn mice with KRN633, a vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor, delayed retinal vascularization leading to abnormal retinal vascular growth and patterns. To determine whether similar abnormalities are observed in newborn mice treated with other VEGF receptor tyrosine kinase inhibitors, we administered axitinib to mice on the day of birth and on the following day. When compared with control pups, a significant delay in retinal vascularization was observed in pups treated with axitinib (5 mg/kg). Axitinib-treated pups had a very dense capillary network on postnatal day (P) 6 and fewer central arteries and veins on P8 and P12. Central veins, but not arteries, were significantly enlarged on P8. These abnormalities were similar to those observed in KRN633-treated pups and probably represent a common phenotype induced by short-term treatment with VEGF receptor inhibitors in newborn mice. Therefore, mice treated postnatally with VEGFR inhibitors could serve as an animal model for studying the mechanisms of retinal vascular formation and patterning.

The aim of this study was to investigate the long-term effects of 7-nitroindazole on the heart, kidneys, thoracic aorta, and carotid arteries from the progeny of mothers that had been treated with 7-nitroindazole (7NI) (10 mg/kg/day in drinking water) during gestation and nursing. The offspring were also treated with 7NI (10 mg/kg/day in drinking water) until 10 weeks of age. Mean arterial pressure (BP) was measured by tail-cuff plethysmography starting at 4 weeks of age. After perfusion fixation with glutaraldehyde at 120 mmHg, the heart and kidneys were weighed and the thoracic aorta and carotid arteries were processed for morphological investigation. The BP and body weight of treated rats did not differ from age-matched control rats during the course of the experiment. In the experimental group, at the end of the experiment, the heart weight/body weight and kidney weight/body weight ratios were decreased. In addition, the wall thickness (intima + media), cross sectional area (intima + media), and wall thickness/inner diameter ratio were significantly decreased in both the thoracic aorta and carotid arteries without a change in the inner vessel diameter. Circumferential wall tension was increased in both arteries. The data clearly indicate that long-term inhibition of neuronal nitric oxide (NO) synthase with the specific inhibitor 7NI evokes BP-independent hypotrophy of the heart, kidneys, and conduit arterial walls in normotensive Wistar rats.

Nitric oxide (NO) deficiency contributes to hypertension and end-organ damage. Three nitric oxide synthase (NOS) isoforms have been identified: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). Whether selective nNOS or iNOS inhibition exacerbates kidney damage in spontaneously hypertensive rats (SHRs) remains unclear. Seven-week-old SHRs were randomly assigned to 4 groups (n=8 for each group): group 1, SHRs receiving no treatment; group 2 (SHR+7-NI), SHRs given 7-nitroindazole (7-NI, nNOS inhibitor) in their drinking water (10mg/kg/day); group 3 (SHR+salt), SHRs given 1% NaCl; and group 4 (SHR+AG), SHRs given 0.1% aminoguanidine (AG; iNOS inhibitor) in drinking water. The mean arterial pressure of SHRs treated with salt was significantly elevated compared with untreated controls. While AG caused a decrease of mean arterial pressure at 8 and 12 weeks of age in SHRs, both 7-NI and salt exacerbated kidney injury. In addition, AG significantly increased l-arginine levels and the l-arginine-to-asymmetric dimethylarginine (ADMA) ratio in the kidney. Salt treatment decreased renal nNOS-α protein levels and reduced dimethylarginine dimethylaminohydrolase (DDAH) activity. Salt and AG treatment increased nNOS-β and l-citrulline levels in SHR kidneys. AG attenuates hypertension development by upregulation of l-citrulline-to- l-arginine conversion and an increase in the l-arginine-to-ADMA ratio in SHR kidneys. 7-NI impairs renal function but has no effect on blood pressure, suggesting reno-protective role for the nNOS. Salt exacerbates kidney damage mainly through decreasing renal nNOS-α protein levels and DDAH activity. Our findings highlight the protective role of the nNOS/NO pathway in the development of kidney damage in SHRs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A novel, easy method for the syntheses of richly diversified 2H-indazoles and indazolones, called the Davis-Beirut reaction, and other recent 2H-indazole synthetic routes are briefly reviewed. An update on the biological activity of indazoles is also surveyed.

A series of fused tri- and tetracyclic indazoles and analogues compounds (NID) with potential antiparasitic effects were studied using voltamperometric and spectroscopic techniques. Nitroanion radicals generated by cyclic voltammetry were characterized by electron spin resonance spectroscopy (ESR) and their spectral lines were explained and analyzed using simulated spectra. In addition, we examined the interaction between radical species generated from nitroindazole derivatives and glutathione (GSH). Biological assays such as activity against Trypanosoma cruzi and cytotoxicity against macrophages were carried out. Finally, spin trapping and molecular modeling studies were also done in order to elucidate the potentials action mechanisms involved in the trypanocidal activity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Adjudin is a derivative of 1H-indazole-3-carboxylic acid that was shown to have potent anti-spermatogenic activity in rats, rabbits, and dogs. It exerts its effects most notably locally in the apical compartment of the seminiferous epithelium, behind the blood-testis barrier, by disrupting adhesion of germ cells, most notably spermatids to the Sertoli cells, thereby inducing release of immature spermatids from the epithelium that leads to infertility. After adjudin is metabolized, the remaining spermatogonial stem cells and spermatogonia repopulate the seminiferous epithelium gradually via spermatogonial self-renewal and differentiation, to be followed by meiosis and spermiogenesis, and thus fertility rebounds. Recent studies in rats have demonstrated unequivocally that the primary and initial cellular target of adjudin in the testis is the apical ectoplasmic specialization, a testis-specific anchoring junction type restricted to the interface between Sertoli cells and elongating spermatids (from step 8 to 19 spermatids). In this review, we highlight some of the recent advances and obstacles regarding the possible use of adjudin as a male contraceptive.

The objective of this study was to determine the effects of 7-nitroindazole (7NI--a preferential neuronal nitric oxide synthase (NOS) inhibitor) and NG-nitro-L-arginine (NNA--a non-selective NOS inhibitor) on the anticonvulsant action of pregabalin (PGB--a third-generation antiepileptic drug) in the maximal electroshock (MES)-induced seizure model in mice. Electroconvulsions were produced in mice by means of an alternating current (50 Hz, 500 V, 25 mA, ear-clip electrodes, 0.2 s stimulus duration, tonic hindlimb extension taken as the endpoint). The anticonvulsant action of PGB in the MES test was expressed as median effective doses (ED50 values) of the drug, protecting 50% of animals tested against MES-induced seizures. The acute adverse-effect potentials of PGB in combination with 7NI and NNA were evaluated in the chimney test (motor coordination), step-through passive avoidance task (long-term memory) and grip-strength test (skeletal muscular strength) in mice. 7NI (50 mg/kg, ip) significantly enhanced the anticonvulsant action of PGB by reducing the ED50 value of PGB from 145.0 mg/kg to 74.4 mg/kg (p<0.01). Similarly, 7NI at the lower dose of 25 mg/kg also potentiated the anticonvulsant action of PGB by lowering the ED50 value of PGB from 145.0 mg/kg to 117.9 mg/kg, although the results did not attain statistical significance. In contrast, NNA (40 mg/kg, ip) had no impact on the anticonvulsant effects of PGB. Moreover, none of the examined combinations of PGB with 7NI and NNA affected motor coordination, long-term memory and skeletal muscular strength in mice. Based on this preclinical study, one can conclude that 7NI significantly enhanced and NNA had no effect on the anticonvulsant activity of PGB against MES-induced seizures in mice.

Several new N-phenyl-1H-indazole-1-carboxamides 1c-h and 4l,m were prepared by reacting phenyl isocyanate derivatives 3a,b with 3-amino-1H-indazole derivatives 2c,e,g or 1H-indazole 2l respectively. Chemical transformations of compounds 1a,b and 1g,h gave 3-acetamido-N-phenyl-1H-indazole-1-carboxamide derivatives 5a,b, and 3,5-diamino-N-phenyl-1H-indazole-1-carboxamide derivatives 4i,j respectively. Finally, 3,5-diacetamido-N-phenyl-1H-indazole-1-carboxamide derivatives 6a,b were prepared by acetylation of 4i,j. Some of synthesized compounds were evaluated for their in vitro antiproliferative activity against the full NCI tumor cell lines panel derived from nine clinically isolated cancer types (leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast). Compound 1c, the most active of the series, was able to inhibit cell growth showing GI(50) values in the 0.041-33.6 μM range, mean GI(50) 1.90 μM, being very effective against colon and melanoma cell lines. Cell cycle analysis in K562 cells showed that 1c causes a marked increase of cells in G0-G1 phase. Moreover, it increases the ratio between hypophosphorylated pRb and total pRb., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

A commonly activated signaling cascade in many human malignancies, including glioblastoma multiforme, is the Akt pathway. This pathway can be activated via numerous upstream alterations including genomic amplification of epidermal growth factor receptor, PTEN deletion, or PIK3CA mutations. In this study, we screened phosphatidylinositol 3-kinase/Akt small-molecule inhibitors in an isogenic cell culture system with an activated Akt pathway secondary to a PIK3CA mutation. One small molecule, A-443654, showed the greatest selective inhibition of cells with the mutant phenotype. Based on these findings, this inhibitor was screened in vitro against a panel of glioblastoma multiforme cell lines. All cell lines tested were sensitive to A-443654 with a mean IC(50) of approximately 150 nmol/L. An analogue of A-443654, methylated at a region that blocks Akt binding, was on average 36-fold less active. Caspase assays and dual flow cytometric analysis showed an apoptotic mechanism of cell death. A-443654 was further tested in a rat intracranial model of glioblastoma multiforme. Animals treated intracranially with polymers containing A-443654 had significantly extended survival compared with control animals; animals survived 79% and 43% longer than controls when A-443654-containing polymers were implanted simultaneously or in a delayed fashion, respectively. This small molecule also inhibited glioblastoma multiforme stem-like cells with similar efficacy compared with traditionally cultured glioblastoma multiforme cell lines. These results suggest that local delivery of an Akt small-molecule inhibitor is effective against experimental intracranial glioma, with no observed resistance to glioblastoma multiforme cells grown in stem cell conditions.

Adjudin (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide; formerly called AF-2364) has been shown to inhibit spermatogenesis by disrupting anchoring junctions at the Sertoligerm cell interface. This, in turn, leads to germ cell loss from the seminiferous epithelium, and transient infertility. Adjudin's efficacyin inhibiting spermatogenesis, the recovery of spermatogenesis after cessation of the drug, and side effects were examined in adult male Japanese rabbits. The pharmacokinetics profiles of adjudin in rabbits after oral administration and after intravenous injection were compared. Rabbits received 25 mg/kg adjudin once weekly for 4 consecutive weeks either by intravenous injection or by gavage. Vehicle-treated rabbits were used as controls. At 1, 2, 3, 4, and 8 weeks after treatment, testes were removed for microscopic examination to assess the status of spermatogenesis. Four weeks after intravenous cessation of adjudin, the recovery of spermatogenesis also was monitored. Blood was withdrawn after first administration to measure plasma concentrations of adjudin by high-performance liquid chromatography. Four weeks after intravenous treatment, examination of testis sections showed rapid exfoliation of elongated/elongating spermatids and the presence of large multinucleated cells; more than 95% of germ cells were absent from the seminiferous epithelium. Intravenous treatment showed a more severe disturbance of spermatogenesis compared with gavage treatment, which was correlated with bioavailability of the drug. The areas under the curve for intravenous injection and gavage were 20.11 +/- 1.90 and 2.23 +/- 0.45 mg x h x L(-1), respectively. These results illustrate the potential of adjudin as a male contraceptive, and the efficacy is associated with the bioavailability of the drug.

Purpose: Cyclin-dependent kinases (CDKs) play a significant role in the control of cell-cycle progression and exhibit aberrant regulation in various neoplastic diseases. AG-024322 is a potent inhibitor of CDK1, CDK2, and CDK4 that produces cell-cycle arrest and antitumor activity in preclinical models. This study evaluated the toxicity of AG-024322 when given by intravenous (IV) infusion to cynomolgus monkeys, including reversibility of effects., Methods: Male and female monkeys received AG-024322 by 30-min IV infusion once daily for 5 days at doses of 2, 6, and 10 mg/kg (24, 72, and 120 mg/m(2), respectively). Controls received vehicle alone which was aqueous 5% dextrose, pH 3.8. Three animals/sex/group were necropsied on day 6, and two animals/sex/group at 6 and 10 mg/kg were necropsied on day 22 (reversal cohort). Doses were based upon the results of a dose range-finding study in monkeys; decreased white blood cells occurred at > or =3 mg/kg and 12 mg/kg produced central nervous system effects and was above the maximum-tolerated dose., Results: No deaths occurred and clinical signs of toxicity, including swelling at the IV administration site, were seen at > or =6 mg/kg. AG-024322 at > or =6 mg/kg produced pancytic bone marrow hypocellularity, lymphoid depletion, and vascular injury at the injection site. Renal tubular degeneration occurred at 10 mg/kg. These changes were either reversible or in a process of repair following the 17-day recovery period. Hematology changes included decreases in reticulocytes and/or granulocytes at > or =6 mg/kg, which were reversible and consistent with changes in the bone marrow. Lymphoid and bone marrow depletion are consistent with pharmacologic inhibition of CDKs by AG-024322 and were expected findings. On day 22, vacuolar degeneration of pancreatic acinar cells with increased serum amylase and lipase levels occurred in one female at 10 mg/kg. Neither sex-related differences in toxicokinetics nor plasma accumulation over 5 days of dosing were seen. Terminal phase overall mean half-life on day 5 ranged from 6.69 to 8.87 h (across dose levels) and was not dose dependent., Conclusion: The no-adverse-effect dose of AG-024322 was 2 mg/kg and associated with overall mean plasma AUC(0-24.5) of 2.11 microg h/mL.

It is well established that low level Pb-exposure is associated with a wide range of cognitive and neurobehavioral dysfunctions in children. In fact, Pb-induced damage occurs preferentially in the prefrontal cerebral cortex, hippocampus and cerebellum - the anatomical sites which are crucial in modulating emotional response, memory and learning. Previously it was also shown that nitric oxide (NO) signaling pathway as well as glutamatergic neurotransmission are both involved in brain development, neurotoxicity and neurodegeneration processes whereas Pb(2+) interfere with both. For this reason we investigated the effect of ontogenetic Pb(2+) exposure on dopaminergic neurotransmission in the medial prefrontal cortex (mPFC) of rats after amphetamine (AMPH) and/or 7-nitroindazole (7-NI) administration. Furthermore, the possible role of oxidative stress in Pb(2+)-induced neurotoxicity in prenatally Pb(2+)-treated rats was explored in the content of hydroxyl radical (HO) species in mPFC after AMPH and/or 7-NI injection, assessed by HPLC analysis of 2.3-dihydroxybenzoic acid (2.3-DHBA) - spin trap product of salicylate. As shown, the results of this study suggest that Pb(2+) exposure during intrauterine life did not substantially affect cortical dopaminergic neurotransmission in adult offspring rats evaluated by means of microdialysis of mPFC and the content of the cortical HO. It is likely that striatum, nucleus accumbens or other dopamine rich brain areas are more intricately associated with Pb(2+) precipitated behavioral, dopamine - dependent impairments observed in mammalians.