Randomised controlled trial of intravenous nafamostat mesylate in COVID pneumonitis: Phase 1b/2a experimental study to investigate safety, Pharmacokinetics and Pharmacodynamics.

BACKGROUND: Many repurposed drugs have progressed rapidly to Phase 2 and 3 trials in COVID19 without characterisation of Pharmacokinetics /Pharmacodynamics including safety data. One such drug is nafamostat mesylate. METHODS: We present the findings of a phase Ib/IIa open label, platform randomised controlled trial of intravenous nafamostat in hospitalised patients with confirmed COVID-19 pneumonitis. Patients were assigned randomly to standard of care (SoC), nafamostat or an alternative therapy. Nafamostat was administered as an intravenous infusion at a dose of 0.2 mg/kg/h for a maximum of seven days. The analysis population included those who received any dose of the trial drug and all patients randomised to SoC. The primary outcomes of our trial were the safety and tolerability of intravenous nafamostat as an add on therapy for patients hospitalised with COVID-19 pneumonitis. FINDINGS: Data is reported from 42 patients, 21 of which were randomly assigned to receive intravenous nafamostat. 86% of nafamostat-treated patients experienced at least one AE compared to 57% of the SoC group. The nafamostat group were significantly more likely to experience at least one AE (posterior mean odds ratio 5.17, 95% credible interval (CI) 1.10 - 26.05) and developed significantly higher plasma creatinine levels (posterior mean difference 10.57 micromol/L, 95% CI 2.43-18.92). An average longer hospital stay was observed in nafamostat patients, alongside a lower rate of oxygen free days (rate ratio 0.55-95% CI 0.31-0.99, respectively). There were no other statistically significant differences in endpoints between nafamostat and SoC. PK data demonstrated that intravenous nafamostat was rapidly broken down to inactive metabolites. We observed no significant anticoagulant effects in thromboelastometry. INTERPRETATION: In hospitalised patients with COVID-19, we did not observe evidence of anti-inflammatory, anticoagulant or antiviral activity with intravenous nafamostat, and there were additional adverse events. FUNDING: DEFINE was funded by LifeArc (an independent medical research charity) under the STOPCOVID award to the University of Edinburgh. We also thank the Oxford University COVID-19 Research Response Fund (BRD00230).

Novel [18F]-Labeled Meta-Bromobenzylguanidine Derivatives: Potential Positron Emission Tomography Imaging Probes for the Norepinephrine Transporter.

To develop novel norepinephrine transporter (NET)-targeting positron emission tomography (PET) probes with optimal pharmacokinetic properties, a series of meta-bromobenzylguanidine derivatives was synthesized. 4-Fluorodiethoxyethane-3-bromobenzylguanidine (compound 12) showed relatively good affinity for the NET (IC50 = 1.00 ± 0.04 µM). The corresponding radiotracer 18F-12 was prepared in high radiochemical purity (>98%) via a three-step method. The in vitro cellular uptake results demonstrated that 18F-12 was specifically taken up by NET-expressing SK-N-SH cells by the uptake-1 mechanism. Biodistribution studies in mice showed that 18F-12 exhibited high cardiac uptake (10.45 ± 0.66 %ID/g at 5 min p.i. and 6.44 ± 0.40 %ID/g at 120 min p.i.), faster liver clearance, and a lower dose of absorbed radiation than [123I]-labeled meta-iodobenzylguanidine ([123I]MIBG). Small animal PET imaging confirmed the high heart-to-background ratio of 18F-12 and the uptake-1 mechanism specific for the NET in rats, indicating its potential as a promising PET radiotracer for cardiac sympathetic nerve imaging.

A phase I study of high dose camostat mesylate in healthy adults provides a rationale to repurpose the TMPRSS2 inhibitor for the treatment of COVID-19.

Camostat mesylate, an oral serine protease inhibitor, is used to treat chronic pancreatitis and reflux esophagitis. Recently, camostat mesylate and its active metabolite 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) were reported to inhibit the infection of cells by severe acute respiratory syndrome coronavirus 2 by inhibiting type II transmembrane serine protease. We conducted a phase I study to investigate high-dose camostat mesylate as a treatment for coronavirus disease 2019. Camostat mesylate was orally administered to healthy adults at 600 mg 4 times daily under either of the following conditions: fasted state, after a meal, 30 min before a meal, or 1 h before a meal, and the pharmacokinetics and safety profiles were evaluated. In addition, the time of plasma GBPA concentration exceeding the effective concentration was estimated as the time above half-maximal effective concentration (EC50 ) by using pharmacokinetic/pharmacodynamic modeling and simulation. Camostat mesylate was safe and tolerated at all dosages. Compared with the fasted state, the exposure of GBPA after a meal and 30 min before a meal was significantly lower; however, no significant difference was observed at 1 h before a meal. The time above EC50 was 11.5 h when camostat mesylate 600 mg was administered 4 times daily in the fasted state or 1 h before a meal. Based on the results of this phase I study, we are currently conducting a phase III study.

Discovery of a novel series of guanidinebenzoates as gut-restricted enteropeptidase and trypsin dual inhibitors for the treatment of metabolic syndrome.

A novel series of guanidinebenzoate enteropeptidase and trypsin dual inhibitors has been discovered and SAR studies were conducted. Optimization was focused on improving properties for gut restriction, including increased aqueous solubility, lower cellular permeability, and reduced oral bioavailability. Lead compounds were identified with efficacy in a mouse fecal protein excretion study.

Pharmacokinetic behavior of peramivir in the plasma and lungs of rats after trans-nasal aerosol inhalation and intravenous injection.

Peramivir, a neuraminidase inhibitor, was approved globally and is indicated for the treatment of uncomplicated influenza in adults and children. However, the only approved intravenous formulation of peramivir limits its clinical application due to the need for the specialized dosing techniques and increases the risk of contracting influenza virus infection among healthcare professionals when dosing within a short distance to the patient. The purpose of this study was to investigate the pharmacokinetic profile of peramivir in plasma and the lung of rats and to compare the profiles following administration through trans-nasal aerosol inhalation (0.0888, 0.1776, and 0.3552 mg/kg) and intravenous injection (30 mg/kg). The plasma concentration reached the Cmax within 1.0 h (upon inhalation) and decreased at a t1/2 of 6.71 and 10.9 h after inhalation and injection, respectively. The absolute bioavailability of peramivir after inhalation was 78.2 %. Overall, the pharmacokinetic exposure of peramivir in the lungs was higher than that in the plasma after aerosol inhalation. After inhalation, the Cmax of peramivir in the lung was achieved within 1.0 h, and the elimination of the drug was slower than in the case of intravenous injection with t1/2 values 1.81 h for injection and 5.72, 53.5, and 32.1 h for low, middle, and high doses administered through inhalation. The Cmax and AUC0-t values for peramivir in the lungs increased linearly with the increased inhalation dose. The results elucidate the pharmacokinetic process of peramivir after trans-nasal aerosol inhalation to rats and provide useful information for further rational application of this drug formulation.

Quantitative elucidation of maternal-to-fetal transfer of neonicotinoid pesticide clothianidin and its metabolites in mice.

Neonicotinoids (NNs), a widely used class of systemic pesticides, are regarded as exhibiting selective toxicity in insects. However, NNs are suspected of exerting adverse effects on mammals as well, including humans. To date, only adult male animal models have been subjected to general toxicity studies of NNs; fetuses have yet to be considered in this context. Here, we focused on the NN clothianidin (CLO) for the first quantitative LC-MS/MS analysis of maternal-to-fetal transfer and residual property of once-daily (single or multiple days), orally administered CLO and its metabolites in mice. The results revealed the presence of CLO and its five metabolites at approximately the same respective blood levels in both dams and fetuses. In the dams, CLO showed a peak value 1 h after administration, after which levels rapidly decreased at 3 and 6 h. In the fetuses of each group, levels of CLO were almost the same as those observed in the corresponding dams. The present results clearly demonstrated rapid passage of CLO through the placental barrier. However, metabolite-dependent differences observed in blood pharmacokinetics and residual levels. This is the first quantitative demonstration of the presence of CLO and its metabolites in fetal mouse blood.

Bioanalytical method development and validation of MES207, a neuropeptide FF receptor antagonist, and its application in preclinical pharmacokinetics.

The nonpeptide small molecule, MES207, exhibits 17-fold preferential binding to the neuropeptide FF receptor 1 (NPFFR1) over NPFFR2 and shows antagonist functionality at NPFF receptors. In order to further the development of MES207 as a NPFFR1 probe, an UPLC-MS/MS bioanalytical method was developed and validated to quantify MES207 in rat plasma for a linearity range of 3-200 ng/mL. The method was applied in the analysis of the plasma, brain, and urine samples collected during pharmacokinetic studies in healthy male and female Sprague Dawley rats. The animals were dosed through oral gavage (50 mg/kg) and intravenously (2.5 mg/kg). Test samples were analyzed using the validated bioanalytical method to generate plasma concentration-time profiles. The results were further subjected to non-compartmental analysis using Phoenix 6.4®. MES207 exhibits a large volume of distribution (1.2 ±â€¯0.6 L), high clearance (0.8 ±â€¯0.1 L/h), and a poor oral bioavailability (1.7 ±â€¯0.4%). The compound also showed a multiple peak phenomenon with a very short absorption phase. It appears that gender does not significantly influence the differences in pharmacokinetic parameters of this NPFF probe.

Ventricular Distribution Pattern of the Novel Sympathetic Nerve PET Radiotracer 18F-LMI1195 in Rabbit Hearts.

We aimed to determine a detailed regional ventricular distribution pattern of the novel cardiac nerve PET radiotracer 18F-LMI1195 in healthy rabbits. Ex-vivo high resolution autoradiographic imaging was conducted to identify accurate ventricular distribution of 18F-LMI1195. In healthy rabbits, 18F-LMI1195 was administered followed by the reference perfusion marker 201Tl for a dual-radiotracer analysis. After 20 min of 18F-LMI1195 distribution time, the rabbits were euthanized, the hearts were extracted, frozen, and cut into 20-µm short axis slices. Subsequently, the short axis sections were exposed to a phosphor imaging plate to determine 18F-LMI1195 distribution (exposure for 3 h). After complete 18F decay, sections were re-exposed to determine 201Tl distribution (exposure for 7 days). For quantitative analysis, segmental regions of Interest (ROIs) were divided into four left ventricular (LV) and a right ventricular (RV) segment on mid-ventricular short axis sections. Subendocardial, mid-portion, and subepicardial ROIs were placed on the LV lateral wall. 18F-LMI1195 distribution was almost homogeneous throughout the LV wall without any significant differences in all four LV ROIs (anterior, posterior, septal and lateral wall, 99 ± 2, 94 ± 5, 94 ± 4 and 97 ± 3%LV, respectively, n.s.). Subepicardial 201Tl uptake was significantly lower compared to the subendocardial portion (subendocardial, mid-portion, and subepicardial activity: 90 ± 3, 96 ± 2 and *80 ± 5%LV, respectively, *p < 0.01 vs. mid-portion). This was in contradistinction to the transmural wall profile of 18F-LMI1195 (90 ± 4, 96 ± 5 and 84 ± 4%LV, n.s.). A slight but significant discrepant transmural radiotracer distribution pattern of 201Tl in comparison to 18F-LMI1195 may be a reflection of physiological sympathetic innervation and perfusion in rabbit hearts.

Peramivir for Influenza A and B Viral Infections: A Pharmacokinetic Case Series.

OBJECTIVE: To describe the peramivir (PRV) pharmacokinetics in critically ill children treated for influenza A or B viral infections. DESIGN: Retrospective electronic medical record review of prospectively collected data from critically ill children receiving peramivir for influenza A or B viral infections in the pediatric intensive care unit (PICU). SETTING: A 189-bed, freestanding children's tertiary care teaching hospital in Philadelphia, PA. PATIENTS: Critically ill children admitted to the PICU who were infected with influenza between January 1, 2016 and March 31, 2018. INTERVENTIONS: None. RESULTS: Eleven patients, two females (18%) and nine males (82%), accounted for 24 peramivir samples for therapeutic drug management. The median age was 5 years (interquartile range 1.5-6.5 yrs) with a median weight of 16.4 kg (interquartile range 14-24 kg). Ten (91%) patients demonstrated a larger volume of distribution, 11 (100%) patients demonstrated an increase in clearance, and 11 (100%) patients demonstrated a shorter half-life estimate as compared with the package insert and previous pediatric trial data for peramivir. Eight (73%) patients tested positive for a strain of influenza A and 3 (27%) patients tested positive for influenza B; 4 of 11 (36%) patients tested positive for multiple viruses. All patients had adjustments made to their dosing interval to a more frequent interval. Ten (91%) patients were adjusted to an every-12-hour regimen and 1 (9%) patient was adjusted to an every-8-hour regimen. No adverse events were associated with peramivir treatment. CONCLUSION: The pharmacokinetics of PRV demonstrated in this PICU cohort differs in comparison to healthy pediatric and adult patients, and alterations to dosing regimens may be needed in PICU patients to achieve pharmacodynamic exposures. Additional investigations in the PICU population are needed to confirm these findings.

Whole Body PET Imaging with a Norepinephrine Transporter Probe 4-[18F]Fluorobenzylguanidine: Biodistribution and Radiation Dosimetry.

PURPOSE: 4-[18F]Fluorobenzylguanidine ([18F]PFBG) is a positron emission tomography (PET) probe for non-invasive targeting of the norepinephrine transporter. The aim of this study was to assess uptake and distribution characteristics of this PET probe. PROCEDURES: Three cynomolgus monkeys were injected with 269 ± 51 MBq (7.3 ± 1.4 mCi) of [18F]PFBG and 21 whole body PET scans were acquired over 165 min. s around organs to generate time-activity curves. The absorbed doses to individual organs and the effective dose to the whole body were estimated. RESULTS: Favorable distribution of [18F]PFBG was noted with a fast wash-in and wash-out of radioactivity from several tissues. [18F]PFBG rapidly distributed in the heart, liver, kidneys, and adrenal glands. The uptake presented as %ID in the brain, lung, and spleen was 1.06 ± 0.45, 6.28 ± 0.33, and 1.39 ± 0.35 at 1 min and decreased to 0.29 ± 0.02, 1.78 ± 0.31, and 0.66 ± 0.22 by 112 min. In general, a two- to fourfold reduction was noted from peak radioactivity levels. Rapid uptake and significant retention of radioactivity was noted in the heart and the septal wall was distinctly visible by 20 min. Fast wash-in and washout kinetics for [18F]PFBG resulted in shorter residence times. The residence time for the liver, lungs, kidneys, and spleen were 28.01 ± 7.73 min, 2.97 ± 0.56 min, 6.04 ± 3.41 min, and 1.09 ± 0.33 min, respectively. The mean effective dose for the 70-kg male was 0.04 ± 0.00 mSv/MBq. The organs receiving the highest radiation dose in the 70-kg male model were the testes (0.11 ± 0.02 mGy/MBq), adrenals (0.08 ± 0.01 mGy/MBq), and urinary bladder wall (0.08 ± 0.01 mGy/MBq). CONCLUSIONS: [18F]PFBG shows a favorable biodistribution pattern. Rapid and persistent uptake was noted in innervated organs. Renal clearance was the major path for elimination of [18F]PFBG. The estimated radiation burden from [18F]PFBG was significantly lower than that from [124I]MIBG.

First-in-Human Studies of [18F] Fluorohydroxyphenethylguanidines.

BACKGROUND: Disease-induced damage to cardiac autonomic nerve populations is associated with an increased risk of sudden cardiac death. The extent of cardiac sympathetic denervation, assessed using planar scintigraphy or positron emission tomography, has been shown to predict the risk of arrhythmic events in heart failure patients staged for implantable cardioverter defibrillator therapy. The goal of this study was to perform first-in-human evaluations of 4-[18F]fluoro-meta-hydroxyphenethylguanidine and 3-[18F]fluoro-para-hydroxyphenethylguanidine, 2 new positron emission tomography radiotracers developed for quantifying regional cardiac sympathetic nerve density. METHODS AND RESULTS: Cardiac positron emission tomography studies with 4-[18F]fluoro-meta-hydroxyphenethylguanidine and 3-[18F]fluoro-para-hydroxyphenethylguanidine were performed in normal subjects (n=4 each) to assess their imaging properties and organ kinetics. Patlak graphical analysis of their myocardial kinetics was evaluated as a technique for generating nerve density metrics. Whole-body biodistribution studies (n=4 each) were acquired and used to calculate human radiation dosimetry estimates. Patlak analysis proved to be an effective approach for quantifying regional nerve density. Using 960 left ventricular volumes of interest, across-subject Patlak slopes averaged 0.107±0.010 mL/min per gram for 4-[18F]fluoro-meta-hydroxyphenethylguanidine and 0.116±0.010 mL/min per gram for 3-[18F]fluoro-para-hydroxyphenethylguanidine. Tracer uptake was highest in heart, liver, kidneys, and salivary glands. Urinary excretion was the main elimination pathway. CONCLUSIONS: 4-[18F]fluoro-meta-hydroxyphenethylguanidine and 3-[18F]fluoro-para-hydroxyphenethylguanidine each produce high-quality positron emission tomography images of the distribution of sympathetic nerves in human heart. Patlak analysis provides reproducible measurements of regional cardiac sympathetic nerve density at high spatial resolution. Further studies of these tracers in heart failure patients will be performed to identify the best agent for clinical development. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT02385877.

Peramivir: A Review in Uncomplicated Influenza.

Intravenous peramivir (Alpivab™; Rapivab®; Rapiacta®; PeramiFlu®), the most recent globally approved inhibitor of influenza neuraminidase, is indicated for the treatment of uncomplicated influenza in adults and children from the age of 2 years. This article, written from an EU perspective, reviews the clinical use of peramivir in this indication and summarizes its pharmacological properties. In large, randomized, double-blind, multicentre trials in previously healthy adults with uncomplicated influenza, a single infusion of peramivir 600 mg significantly shortened the median time to resolution of influenza symptoms compared with placebo and was noninferior to the recommended oseltamivir regimen in terms of this primary outcome. Albeit data are limited, results from a noncomparative phase 3 trial in paediatric patients (≈ 95% of whom were aged ≥ 2 years) with acute uncomplicated influenza receiving the recommended dose of peramivir were generally consistent with those in adults. Peramivir was generally well tolerated in children and adults participating in these clinical trials, with most adverse events of mild to moderate intensity. Given its simple single-dose regimen and with intravenous administration offering a potential advantage over oral administration in individuals with nausea, vomiting or having difficulty in swallowing, peramivir provides an additional option for treating uncomplicated influenza infection in adults and children from the age of 2 years.

Feasibility of myocardial PET imaging using a benzylguanidine analog: meta-(3-[18F]fluoropropyl)benzylguanidine ([18F]mFPBG).

INTRODUCTION: Global and regional sympathetic activity in the heart can be evaluated using [123I]meta-iodobenzylguanidine ([123I]mIBG) imaging. However, [123I]mIBG is associated with low image spatial resolution and sensitivity in cardiac imaging. We investigated the capability of an F-18-labeled mIBG derivative, meta-(3-[18F]fluoropropyl)benzylguanidine ([18F]mFPBG), for identifying ischemic and viable myocardium in a rat model of myocardial infarction. MATERIALS AND METHODS: The ex vivo biodistribution and in vivo metabolic stability of [18F]mFPBG were investigated in Sprague-Dawley rats. Selective cardiac adrenergic activation was confirmed via a blocking experiment involving pretreatment with desipramine (2 mg kg-1), followed by the administration of [18F]mFPBG. Imaging properties of [18F]mFPBG were compared with those of traditional cardiac imaging radiotracers ([123I]mIBG and [99mTc]MIBI) in a rat model of myocardial infarction. Non-invasive image-based measurements of infarct sizes were then compared with histological findings by using Bland-Altman analysis. RESULTS: The differences in infarct sizes determined using histological analysis and [18F]mFPBG PET were -2.55 ±â€¯4.99% (range: -12.33 to 7.22), -2.35 ±â€¯3.32% (range: -8.87 to 4.16), and -3.15 ±â€¯6.16% (range: -15.24 to 8.93) at 5, 20, and 40 min, respectively. Furthermore, [18F]mFPBG PET was superior to traditional imaging methods in assessing the degree of ischemia in areas of myocardial infarction, as well as the actual infarct size. CONCLUSION: Compared to [123I]mIBG, [18F]mFPBG showed improved spatial resolution and sensitivity in a rat model of myocardial infarction. This result suggested that [18F]mFPBG is a promising cardiac PET imaging agent for potential diagnostic application in PET cardiology.

Enantioselective Bioaccumulation and Toxicity of the Neonicotinoid Insecticide Dinotefuran in Earthworms ( Eisenia fetida).

The enantioselective bioaccumulation and toxicity of dinotefuran in earthworms were studied in this study. The results showed that S-dinotefuran accumulated faster than Rac-dinotefuran and R-dinotefuran in earthworms. The acute toxicity of S-dinotefuran was 1.49 and 2.67 times that of the Rac-dinotefuran and R-dinotefuran in artificial soil during 14 days of exposure. At 1.0 mg/kg, the three tested chemicals inhibited the growth and reproduction as well as induced oxidative stress effects in earthworms; however, the toxic effects induced by S-dinotefuran were the most serious. The transcriptome sequencing results showed that S-dinotefuran had stronger interactions to biomacromolecules and influences on the endoplasmic reticulum (ER) than R-dinotefuran, which may be the main reason for enantioselectivities between the two enantiomers. The present results indicated that the risk of S-dinotefuran was higher than that of Rac-dinotefuran and R-dinotefuran in the soil environment to earthworms. Risk assessment of dinotefuran should be evaluated at the enantiomer level.

A novel in vitro allometric scaling methodology for aldehyde oxidase substrates to enable selection of appropriate species for traditional allometry.

1. Failure to predict human pharmacokinetics of aldehyde oxidase (AO) substrates using traditional allometry has been attributed to species differences in AO metabolism. 2. To identify appropriate species for predicting human in vivo clearance by single-species scaling (SSS) or multispecies allometry (MA), we scaled in vitro intrinsic clearance (CLint) of five AO substrates obtained from hepatic S9 of mouse, rat, guinea pig, monkey and minipig to human in vitro CLint. 3. When predicting human in vitro CLint, average absolute fold-error was ≤2.0 by SSS with monkey, minipig and guinea pig (rat/mouse >3.0) and was <3.0 by most MA species combinations (including rat/mouse combinations). 4. Interspecies variables, including fraction metabolized by AO (Fm,AO) and hepatic extraction ratios (E) were estimated in vitro. SSS prediction fold-errors correlated with the animal:human ratio of E (r2 = 0.6488), but not Fm,AO (r2 = 0.0051). 5. Using plasma clearance (CLp) from the literature, SSS with monkey was superior to rat or mouse at predicting human CLp of BIBX1382 and zoniporide, consistent with in vitro SSS assessments. 6. Evaluation of in vitro allometry, Fm,AO and E may prove useful to guide selection of suitable species for traditional allometry and prediction of human pharmacokinetics of AO substrates.

Lethal effects of Cr(III) alone and in combination with propiconazole and clothianidin in honey bees.

Several anthropogenic contaminants, including pesticides and heavy metals, can affect honey bee health. The effects of mixtures of heavy metals and pesticides are rarely studied in bees, even though bees are likely to be exposed to these contaminants in both agricultural and urban environments. In this study, the lethal toxicity of Cr alone and in combination with the neonicotinoid insecticide clothianidin and the ergosterol-biosynthesis-inhibiting fungicide propiconazole was assessed in Apis mellifera adults. The LD50 and lowest benchmark dose of Cr as Cr(NO3)3, revealed a low acute oral toxicity on honey bee foragers (2049 and 379 mg L-1, respectively) and the Cr retention (i.e. bee ability to retain the heavy metal in the body) was generally low compared to other metals. A modified method based on the binomial proportion test was developed to analyse synergistic and antagonistic interactions between the three tested contaminants. The combination of an ecologically-relevant field concentration of chromium with clothianidin and propiconazole did not increase bee mortality. On the contrary, the presence of Cr in mixture with propiconazole elicited a slight antagonistic effect.

Characterization of the metabolic transformation of thiamethoxam to clothianidin in Helicoverpa armigera larvae by SPE combined UPLC-MS/MS and its relationship with the toxicity of thiamethoxam to Helicoverpa armigera larvae.

In order to characterize the metabolic transformation of thiamethoxam (TMX) to clothianidin (CLO) in Helicoverpa armigera larvae and clarify its relationship with the insecticidal toxicity of TMX, method for determination of TMX and its metabolite clothianidin (CLO) residues in H. armigera larvae by solid phase extraction (SPE) combined UPLC-MS/MS was established. Following acetonitrile extraction and purification by SPE on florisil cartridge and C18 cartridge sequently, and cleanup by PSA adsorption, TMX and CLO residues in H. armigera larvae were successfully determined by UPLC-MS/MS. By using the established method, the concentration-time curves of TMX and its metabolite CLO in H. armigera larvae in vivo and metabolism of TMX by microsome of H. armigera larvae midguts in vitro were studied. TMX was quickly eliminated from H. armigera larvae with the elimination half-life as 4.2h. Meanwhile, only a small amount of CLO was formed from TMX metabolism, with the maximum CLO level in H. armigera larvae only accounts for the metabolic transformation of 7.99% of TMX, at 10h after intravenous TMX administration. Our results suggested that the low insecticidal efficacy of TMX against H. armigera larvae was related with the rapidly elimination of TMX from H. armigera larvae, meanwhile, CLO as TMX metabolite at a very low level in vivo didn't contribute to TMX toxicity to H. armigera larvae. In H. armigera larvae, TMX didn't act as proinsecticide for CLO in insecticidal efficacy of TMX.

Synthesis, radiolabeling and preclinical evaluation of a [11C]GMOM derivative as PET radiotracer for the ion channel of the N-methyl-D-aspartate receptor.

INTRODUCTION: Presently available PET ligands for the NMDAr ion channel generally suffer from fast metabolism. The purpose of this study was to develop a metabolically more stable ligand for the NMDAr ion channel, taking [11C]GMOM ([11C]1) as the lead compound. METHODS: [11C]1, its fluoralkyl analogue [18F]PK209 ([18F]2) and the newly synthesized fluorovinyloxy analogue [11C]7b were evaluated ex vivo in male Wistar rats for metabolic stability. In addition, [11C]7b was subjected to a biodistribution study and its affinity (Ki) and lipophilicity (logD7.4) values were determined. RESULTS: The addition of a vinyl chain in the fluoromethoxy moiety did not negatively alter the affinity of [11C]7b for the NMDAr, while lipophilicity was increased. Biodistribution studies showed higher uptake of [11C]7b in forebrain regions compared with cerebellum. Pre-treatment with MK-801 decreased the overall brain uptake significantly, but not in a region-specific manner. 45min after injection 78, 90 and 87% of activity in the brain was due to parent compound for [11C]1, [18F]2 and [11C]7b, respectively. In plasma, 26-31% of activity was due to parent compound. CONCLUSION: Complete substitution of the alpha-carbon increased lipophilicity to more favorable values. Substitution of one or more hydrogens of the alpha-carbon atom in the methoxy moiety improved metabolic stability. In plasma, more parent compound was found for [18F]2 and [11C]7b then for [11C]1, although differences were not significant. At 45min, significantly more parent [18F]2 and [11C]7b was measured in the brain compared with [11C]1.

The sigma-1 receptor modulates dopamine transporter conformation and cocaine binding and may thereby potentiate cocaine self-administration in rats.

The dopamine transporter (DAT) regulates dopamine (DA) neurotransmission by recapturing DA into the presynaptic terminals and is a principal target of the psychostimulant cocaine. The sigma-1 receptor (σ1R) is a molecular chaperone, and its ligands have been shown to modulate DA neuronal signaling, although their effects on DAT activity are unclear. Here, we report that the prototypical σ1R agonist (+)-pentazocine potentiated the dose response of cocaine self-administration in rats, consistent with the effects of the σR agonists PRE-084 and DTG (1,3-di-o-tolylguanidine) reported previously. These behavioral effects appeared to be correlated with functional changes of DAT. Preincubation with (+)-pentazocine or PRE-084 increased the Bmax values of [3H]WIN35428 binding to DAT in rat striatal synaptosomes and transfected cells. A specific interaction between σ1R and DAT was detected by co-immunoprecipitation and bioluminescence resonance energy transfer assays. Mutational analyses indicated that the transmembrane domain of σ1R likely mediated this interaction. Furthermore, cysteine accessibility assays showed that σ1R agonist preincubation potentiated cocaine-induced changes in DAT conformation, which were blocked by the specific σ1R antagonist CM304. Moreover, σ1R ligands had distinct effects on σ1R multimerization. CM304 increased the proportion of multimeric σ1Rs, whereas (+)-pentazocine increased monomeric σ1Rs. Together these results support the hypothesis that σ1R agonists promote dissociation of σ1R multimers into monomers, which then interact with DAT to stabilize an outward-facing DAT conformation and enhance cocaine binding. We propose that this novel molecular mechanism underlies the behavioral potentiation of cocaine self-administration by σ1R agonists in animal models.