Immunotoxicity of N-butylbenzenesulfonamide: impacts on immune function in adult mice and developmentally exposed rats.

N-butylbenzenesulfonamide (NBBS) is a high-production volume plasticizer that is an emerging contaminant of concern for environmental and human health. To understand the risks and health effects of exposure to NBBS, studies were conducted in adult-exposed mice and developmentally exposed rats to evaluate the potential for NBBS to modulate the immune system. Beginning between 8 and 9 weeks of age, dosed feed containing NBBS at concentrations of 0, 313, 625, 1250, 2500, and 5000 ppm was continuously provided to B6C3F1/N female mice for 28 days. Dosed feed was also continuously provided to time-mated Harlan Sprague Dawley (Sprague Dawley SD) rats at concentrations of 0-, 250-, 500-, and 1000-ppm NBBS from gestation day 6 to postnatal day 28 and in F1 rats until 11-14 weeks of age. Functional assessments of innate, humoral, and cell-mediated immunity were conducted in adult female mice and F1 rats following exposure to NBBS. In female mice, NBBS treatment suppressed the antibody-forming cell (AFC) response to SRBC with small increases in T-cell responses and natural killer (NK)-cell activity. In developmentally exposed rats, NBBS treatment-related immune effects were sex dependent. A positive trend in NK-cell activity occurred in male F1 rats while a negative trend occurred in female F1 rats. The AFC response to SRBC was decreased in female F1 rats but not in male F1 rats. These data provide evidence that oral exposure to NBBS has the potential to produce immunomodulatory effects on both innate and adaptive immune responses, and these effects appear to have some dependence on species, sex, and period of exposure (developmental vs adult).

Do Storage Conditions Affect Collected Cookstove Emission Samples? Implications for Field Studies.

Cookstove emissions are a significant source of indoor air pollution in developing countries and rural communities world-wide. Considering that many research sites for evaluating cookstove emissions and interventions are remote and require potentially lengthy periods of particulate matter (PM) filter sample storage in sub-optimal conditions (e.g., lack of cold storage), an important question is whether samples collected in the field are stable over time. To investigate this, red oak was burned in a natural-draft stove, and fine PM (PM2.5) was collected on polytetrafluoroethylene filters. Filters were stored at either ambient temperature or more optimal conditions (-20°C or -80°C) for up to 3 months and extracted. The effects of storage temperature and length on stability were evaluated for measurements of extractable organic matter (EOM), PM2.5, and polycyclic aromatic compound (PAC) levels in the filter extracts. A parallel, controlled laboratory condition was also evaluated to further explore sources of variability. In general, PM2.5 and EOM in both simulated field and laboratory samples were similar regardless of the storage condition or duration. The extracts were also analyzed by gas chromatography to quantify 22 PACs and determine similarities and/or differences between the conditions. PAC levels were a more sensitive stability measure in differentiating between storage conditions. The findings suggest that measurements are relatively consistent across storage duration/temperatures for filter samples with relatively low EOM levels. This study aims to inform protocols and filter storage procedures for exposure and intervention research conducted in low- and middle-income countries where studies may be budget- and infrastructure-limited.

Quantitation of Phenolic Benzotriazole Class Compounds in Plasma by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Phenolic benzotriazoles are used as UV stabilizers in consumer products and have been detected in the environment suggesting potential human exposure. Phenolic benzotriazoles were nominated to the Division of National Toxicology Program for testing based on their potential widespread human exposure and lack of adequate toxicity data. Nine chemicals were selected for toxicological evaluation, representing unsubstituted (2-(2H-benzotriazole-2-yl)phenol, (P-BZT)), monosubstituted (drometrizole; 2-(2H-benzotriazol-2-yl)-4-tert-butylphenol (tBu-BZT); octrizole), disubstituted (2-(2H-benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol (diMeEtPh-BZT), 2-(2H-benzotriazol-2-yl)-4,6-bis(1,1-dimethylpropyl)phenol (ditPe-BZT); 3-(2H-benzotriazol-2-yl)-5-(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, octylester (tBuPrOcEst-BZT) and halogenated trisubstituted (bumetrizole; 2-(5-chloro-2H-benzotriazol-2-yl)-4,6-bis(1,1-dimethylethyl)phenol (ditBuCl-BZT)) compounds. Different extraction methods were utilized and methods were developed to analyze phenolic benzotriazoles by quantitating free (unconjugated parent) and total (free and conjugated parent) analyte levels in plasma of rats to aid in interpretation of toxicity data, understanding of absorption, distribution, metabolism, and excretion differences. The calibration standard range was 1-500 ng/mL for free analytes and 1-1000 ng/mL for total analytes. The methods were linear (r2 ≥ 0.99). The accuracy was determined as relative error (RE) and ranged from -18.2 to +17.8, and precision was determined as relative standard deviation (RSD) and ranged from 0.0 to 20.1% for both free and total plasma calibration standards, respectively. The limit of quantitation was ≤ 5.0 and 10.0 ng/mL and limit of detection was ≤ 1.2 and 2.0 ng/mL, for free and total analytes, respectively. These data demonstrate that the methods are suitable for quantitation of free and total analytes in rat plasma.

Development and Validation of an Analytical Method to Quantitate Hydroxycitric Acid, the Key Constituent in Garcinia cambogia Extract, in Rodent Plasma and Fetus.

Garcinia cambogia extract (GCE) is a popular botanical supplement used in weight loss products. Hydroxycitric acid (HCA) is the principal component in GCE. Due to lack of adequate toxicity data to assess the safe use of GCE, the National Toxicology Program is testing GCE in Hsd:Sprague Dawley® SD® rats following perinatal exposure and in adult B6C3F1/N mice. We report a validated method utilizing sample clean up with ultrafiltration followed by liquid chromatography-tandem mass spectrometry analysis to quantify HCA in rat plasma over the concentration range of 20 to 800 ng/mL. The method was linear (r2 ≥ 0.99) with the limits of quantitation (LOQ) and detection (LOD) of 20.0 and 3.9 ng/mL plasma, respectively. The accuracy (determined as relative error, RE) and precision (determined as relative standard deviation, RSD) using Quality Control standards analyzed over multiple days were ≤ ± 7.5% and ≤ 9.5%, respectively. The method can be applied to quantify HCA in study matrices (RE ≤ ± 23.0%; RSD ≤ 6.0) except gestational day (GD)18 fetus. The method was partially validated in GD18 fetal homogenate over the concentration range 60-3000 ng/g (r2 ≥ 0.99, RE ≤ ± 11.9%, and RSD ≤ 5.5%; LOQ 60.0 ng/g; LOD 7.77 ng/g). The standards as high as 20,000 ng/mL (plasma) and 502,000 ng/g (fetus) can successfully be quantified after diluting into the validated range (RE ≤ ± 2.6%; RSD ≤ 5.2%). These data demonstrate that the method is suitable to quantify HCA in rodent matrices and can be adapted to other biological matrices.

Working with the natural complexity: Selection and characterization of black cohosh root extract for use in toxicology testing.

Black cohosh (Actaea racemosa L.) is a botanical supplement marketed to women of all ages. Due to paucity of data to assess the safe use, the National Toxicology Program (NTP) is evaluating the toxicity of black cohosh. The use of an authentic, quality material is imperative to generate robust data. Because botanical materials are complex mixtures with variable composition, the selection of a material is challenging. We describe selection and phytochemical characterization of an unformulated black cohosh root extract (i.e., an extract that serves as source material for a formulated product) to be used in the NTP assessments. A material was selected using a combination of non-targeted and targeted chemical analyses, including confirmation of authenticity, absence of contaminants and adulterants, and similarity to a popular black cohosh product used by consumers. Thirty-nine constituents covering three major classes, triterpene glycosides, phenolic acids, and alkaloids were identified. Among constituents quantified, triterpene glycosides made up approximately 4.7% (w/w) with total constituents quantified making up 5.8% (w/w) of the extract. Non-targeted chemical analysis followed by chemometric analysis of various materials sold as black cohosh, and reference materials for black cohosh and other Actaea species further confirmed the suitability of the selected extract for use.

Whole-body inhalation exposure to 2-ethyltoluene for two weeks produced nasal lesions in rats and mice.

OBJECTIVE: Ethyltoluenes are isolated during crude oil refinement for use in gasoline and commercial products and are ubiquitous in the environment. However, minimal toxicity data are available. Previously, we identified 2-ethyltoluene (2-ET) as the most potent isomer via nose-only inhalation exposure in rodents. Here, we expanded the hazard characterization of 2-ET in two rodent models using whole-body inhalation exposure and evaluated the role of prenatal exposure. METHODS: Time-mated Hsd:Sprague Dawley® SD® rats were exposed to 0, 150, 300, 600, 900, or 1200 ppm 2-ET via inhalation starting on gestation day 6 until parturition. Rat offspring (n = 8/exposure/sex) were exposed to the same concentrations as the respective dams for 2 weeks after weaning. Adult male and female B6C3F1/N mice (n = 5/exposure/sex) were exposed to the same concentrations for 2 weeks. RESULTS AND DISCUSSION: Exposure to ≥600 ppm 2-ET produced acute toxicity in rats and mice characterized by large decreases in survival, body weight, adverse clinical observations, and diffuse nasal olfactory epithelium degeneration (rats) or necrosis (mice). Due to the early removal of groups ≥600 ppm, most endpoint evaluations focused on lower exposure groups. In 150 and 300 ppm exposure groups, reproductive performance and littering were not significantly changed and body weights in exposed rats and mice were 9-18% lower than controls. Atrophy of the olfactory epithelium and nerves was observed in all animals exposed to 150 and 300 ppm. These lesions were more severe in mice than in rats. CONCLUSION: Nasal lesions were observed in all animals after whole-body exposure up to 600 ppm 2-ET for 2 weeks. Future studies should focus on 2-ET metabolism and distribution to better understand species differences and refine hazard characterization of this understudied environmental contaminant.

Phenolic benzotriazoles: a class comparison of toxicokinetics of ultraviolet-light absorbers in male rats.

Phenolic benzotriazoles are ultraviolet-light absorbers used in a variety of industrial and consumer applications. We investigated the toxicokinetic behaviour of 9 compounds, covering unsubstituted, monosubstituted, disubstituted, and trisubstituted compounds, following a single gavage (30 and 300 mg/kg) and intravenous (IV) (2.25 mg/kg) administration in male rats.Following IV administration, no distinct pattern in plasma elimination was observed for the compounds with half-lives ranging from 15.4-84.8 h. Systemic exposure parameters, maximum concentration (Cmax) and area under the concentration time curve (AUC), generally increased with the degree of substitution.Following gavage administration, Cmax and AUC of unsubstituted compound were lower compared to the substituted compounds. Cmax and AUC increased ≤7-fold with a 10-fold increase in the dose except for the AUC of the unsubstituted compound where the increase was 30-fold. Plasma elimination half-lives for the class ranged from 1.57 to 192 h with the exception of 30 mg/kg drometrizole.Oral bioavailability was low with ∼ 6% estimated for unsubstituted compound and 12.8-23% for others at 30 mg/kg dose. Bioavailability was lower following administration of the higher dose.Taken collectively, these data point to low oral absorption of phenolic benzotriazoles. The absorption decreased with increasing dose. Substituted compounds may be less metabolized compared to the unsubstituted.

Toxicokinetics of the plasticizer, N-butylbenzenesulfonamide, in plasma and brain following oral exposure in rodents: Route, species, and sex comparison.

N-Butylbenzenesulfonamide (NBBS) is a widely used plasticizer and hence there is potential for human exposure via oral routes. This work investigates the toxicokinetic behavior of NBBS in rodents following a single gavage (20, 60, and 200 mg/kg body weight) or multi-day feed administration (500, 1000, and 2000 ppm). In male and female rats following gavage administration, maximum plasma NBBS concentration, Cmax, was reached at ≤0.539 h. Cmax increased proportionally to the dose. Area under the curve (AUC) increased more than proportionally to the dose and was 4- to 5-fold higher in females than in males. In mice, plasma Cmax was reached at ≤0.136 h and increased proportionally to the dose in female mice and more than proportionally to the dose in males. AUC increased more than proportionally to the dose with no apparent sex difference. Elimination of NBBS in plasma was faster in mice (half-life (h); mice ≤0.432, rat ≤3.55). Oral bioavailability was higher in female rats (≥60%) than males (23-52%) with apparent saturation of clearance at ∼200 mg/kg body weight in females. In mice, bioavailability (5-14%) was lower with no apparent sex difference. NBBS was detected in brains of rats and mice but with low brain:plasma ratios (rats, ≤5; mice, ≤1) suggesting low potential to cross the blood brain barrier. Systemic exposure in male rats and mice following a single gavage administration was ≥48-fold higher than multi-day feed exposure. These data demonstrate potential species, sex, dose- and route-related difference in toxicokinetics of NBBS in rodents.

Comparative toxicokinetics of Trans-resveratrol and its major metabolites in Harlan Sprague Dawley rats and B6C3F1/N mice following oral and intravenous administration.

Trans-resveratrol (RES) is a naturally occurring stilbene found in numerous plants and foods. Due to its widespread human exposure and lack of toxicity and carcinogenicity data, RES was nominated to the National Toxicology Program for testing. To aid the toxicology studies, the dose, sex, and species differences in RES toxicokinetics was investigated in Harlan Sprague Dawley rats and B6C3F1/N mice following single intravenous (IV) (10 mg/kg) or oral gavage administration (312.5, 625, and 1250 mg/kg and 625, 1250, and 2500 mg/kg in rats and mice, respectively). Following IV and gavage administration, systemic exposure of RES based on AUC was trans-resveratrol-3-O-ß-D-glucuronide (R3G)> > trans-resveratrol-3-sulfate (R3S) > RES in both species. Following gavage administration Tmax_predicted values were ≤ 263 min for both species and sexes. RES elimination half-life was longer in rats than mice, and shortest in male mice. Clearance was slower in mice with no apparent sex difference in both species. In both rats and mice, following gavage administration AUC increased proportionally to the dose. After gavage administration, enterohepatic recirculation of RES was observed in both rats and mice with secondary peaks occurring around 640 min in the concentration-time profiles. RES was rapidly metabolized to R3S and R3G in both species. Extensive first pass conjugation and metabolism resulted in low levels of the parent compound RES which was confirmed by the low estimates for bioavailability. The bioavailability of RES was low, ~12-31% and ~2-6% for rats and mice, respectively, with no apparent difference between sexes.

A strategy for test article selection and phytochemical characterization of Echinacea purpurea extract for safety testing.

Botanical dietary supplements (BDS) are used around the world for many purported therapeutic properties. The selection of an authentic product and it's phytochemical characterization is critical to generate robust safety data. Because botanicals are complex mixtures with variable quality, identification of a representative product for testing has been challenging. Echinacea is used for its purported immune stimulant properties and was listed as the 2nd top-selling BDS in 2018. However, there are limited safety data for Echinacea. Hence, the National Toxicology Program (NTP) has selected Echinacea for safety testing using rodent models. Here, we describe selection and comprehensive characterization of an Echinacea purpurea root extract to be used in the NTP testing program. Using non-targeted chemical analyses combined with chemometric analysis, a potential unfinished product (i.e., an extract that serves as source material for finished products) of Echinacea purpurea was selected. The product was then authenticated using chemical and DNA techniques and characterized, including the phytochemical composition. Among numerous constituents identified, caftaric acid, chicoric acid, chlorogenic acid and dodeca-2(E),4(E),8(Z),10(E/Z)-tetraenoic acid isobutylamide made up a small fraction of the extract. Based on these analyses, an approach is proposed for test article selection for Echinacea research which can be adapted to other botanicals.

Systemic exposure to Ginkgo biloba extract in male F344/NCrl rats: Relevance to humans.

Ginkgo biloba extract (GBE) is a popular botanical dietary supplement used worldwide and the safety of use is a public health concern. While GBE is a complex mixture, the terpene trilactones and flavonol glycosides are believed to elicit the pharmacological and/or toxicological effects of GBE. In a National Toxicology Program (NTP) 2-year rodent bioassay with GBE, hepatotoxicity was observed in rodents (≥100 mg/kg in rats, ≥ 200 mg/kg in mice). Subsequently, questions arose about whether or not the GBE used in NTP studies was representative of other GBE products and how rodent doses are related to human doses. To address these, we generated systemic exposure data for terpene trilactones in male rats following oral administration of 30, 100, and 300 mg/kg GBE test article from the 2-year bioassay. Dose-normalized Cmax and AUC∞ for terpene trilactones from the current study were within 5-fold of published rodent studies using a standardized GBE preparation. Comparison of our rat systemic exposure data at 100 mg/kg GBE to published human data following ingestion of 240 mg GBE-containing product showed that the rat/human exposure multiple was 3-22, for terpene trilactones. These data demonstrate the relevance of NTP rodent toxicity data to humans.

Systemic exposure of vinpocetine in pregnant Sprague Dawley rats following repeated oral exposure: An investigation of fetal transfer.

Vinpocetine is being used worldwide by people of all ages, including pregnant women, for its purported multiple health benefits. However, limited data is available addressing the safety/toxicity of vinpocetine. The National Toxicology Program conducted studies to examine potential effects of vinpocetine on the developing rat. Disposition data is helpful to put the fetal findings into context and provide information on the potential risk for humans. The current study reports the systemic exposure and toxicokinetic (TK) parameters of vinpocetine and metabolite, apovincaminic acid (AVA), in pregnant Harlan Sprague Dawley rats, fetuses and amniotic fluid following oral gavage exposure of dams to 5 and 20mg/kg vinpocetine from gestational day 6 to 18. Vinpocetine was absorbed rapidly in dams with a maximum plasma concentration (Cmax) reaching ≤1.37h. Predicted Cmax and area under the concentration versus time curve (AUC) increased less than proportionally to the dose. Vinpocetine was rapidly distributed to the peripheral compartment. More importantly, significant transfer of vinpocetine from dam to fetuses was observed with fetal Cmax and AUC≥55% of dams. Vinpocetine was cleared rapidly from dam plasma with an elimination half-life of ≤4.02h with no apparent dose-related effect. Vinpocetine was rapidly and highly metabolized to AVA with AVA plasma levels in dams ≥2.7-fold higher than vinpocetine, although in the fetuses, AVA levels were much lower than vinpocetine. Comparison of current rat data with literature human data demonstrates that systemic exposure to vinpocetine in rats following repeated exposure to 5mg/kg is similar to that following a single human relevant dose of 10mg suggesting that the findings from the toxicology study may be relevant to humans.

Simultaneous Quantitation of 2-Hydroxy-4-Methoxybenzophenone, a Sunscreen Ingredient, and its Metabolites in Harlan Sprague Dawley Rat Plasma Following Perinatal Dietary Exposure.

2-Hydroxy-4-methoxybenzophenone (HMB) is a common ingredient in sunscreens and other personal care products and thus significant potential exists for human exposure. HMB was nominated to the National Toxicology Program (NTP) for testing due to its high exposure through consumer products and inadequate toxicological data at the time, which also included increasing concern for the potential effects of HMB on reproduction and development. HMB is metabolized to numerous metabolites in vivo and in vitro including 2,4-dihydroxybenzophenone (DHB), 2,3,4-trihydroxybenzophenone (THB) and 2,5-dihydroxy-4-methoxybenzophenone (2,5-DHMB) as well as their corresponding glucuronide and/or sulfate conjugates. In this study, we have developed and validated a liquid chromatography-tandem mass spectrometry method to quantitate free (unconjugated) HMB and DHB, and total (combined conjugated and unconjugated) HMB, DHB, THB and 2,5-DHMB. The method was successfully applied to quantitate these analytes in plasma from postnatal day 28 and 56 male and female Harlan Sprague Dawley rat pups following perinatal dietary exposure to 0 (control), 3,000, 10,000 and 30,000 ppm HMB beginning on gestational Day 6. All determined analyte concentrations increased with increasing dose and were significantly higher than the controls at both timepoints. All the total analytes were quantified in all plasma samples and total concentrations were considerably higher than free, suggesting extensive conjugation. Mean concentrations of total HMB and DHB were higher (~100-300-fold) than the free HMB and DHB concentrations, and total concentrations in plasma were approximately HMB≈DHB > 2,5-DHMB¼THB. Free and total analyte plasma concentrations were not sex-dependent and in general, both free and total analytes were detected in the control samples. Comparison of our rat data, using the internal dose, with human data available in the literature suggests that the rat doses used in our studies were within 4-fold of the human dose.

Dynamic Response of Mycobacterium vanbaalenii PYR-1 to BP Deepwater Horizon Crude Oil.

We investigated the response of the hydrocarbon-degrading Mycobacterium vanbaalenii PYR-1 to crude oil from the BP Deepwater Horizon (DWH) spill, using substrate depletion, genomic, and proteome analyses. M. vanbaalenii PYR-1 cultures were incubated with BP DWH crude oil, and proteomes and degradation of alkanes and polycyclic aromatic hydrocarbons (PAHs) were analyzed at four time points over 30 days. Gas chromatography-mass spectrometry (GC-MS) analysis showed a chain length-dependent pattern of alkane degradation, with C12 and C13 being degraded at the highest rate, although alkanes up to C28 were degraded. Whereas phenanthrene and pyrene were completely degraded, a significantly smaller amount of fluoranthene was degraded. Proteome analysis identified 3,948 proteins, with 876 and 1,859 proteins up- and downregulated, respectively. We observed dynamic changes in protein expression during BP crude oil incubation, including transcriptional factors and transporters potentially involved in adaptation to crude oil. The proteome also provided a molecular basis for the metabolism of the aliphatic and aromatic hydrocarbon components in the BP DWH crude oil, which included upregulation of AlkB alkane hydroxylase and an expression pattern of PAH-metabolizing enzymes different from those in previous proteome expression studies of strain PYR-1 incubated with pure or mixed PAHs, particularly the ring-hydroxylating oxygenase (RHO) responsible for the initial oxidation of aromatic hydrocarbons. Based on these results, a comprehensive cellular response of M. vanbaalenii PYR-1 to BP crude oil was proposed. This study increases our fundamental understanding of the impact of crude oil on the cellular response of bacteria and provides data needed for development of practical bioremediation applications.

Pharmacokinetics of MMB4 DMS in rats, rabbits, and dogs following a single IV administration.

Organophosphorus (OP) nerve agents pose tremendous threats to both military and civilian populations. The substance 1,1'-methylenebis[4-[(hydroxyimino)methyl]-pyridinium] (MMB4) is being developed as a replacement for the currently fielded 2-pyridine aldoxime, or pralidoxime (2-PAM) as a treatment for OP nerve agent-induced toxicity. The present study characterized pharmacokinetic (PK) profiles of MMB4 in male and female Sprague-Dawley rats, New Zealand White rabbits, and beagle dogs given a single intravenous (IV) administration of MMB4 dimethanesulfonate (DMS) at 55, 25, and 15 mg/kg dose, respectively. The plasma MMB4 concentration versus time profiles were biphasic for all species tested and fit a 2-compartment model with first-order elimination. There were no overt sex-related differences in the calculated PK parameters. For the rat, rabbit, and dog, the average systemic exposure parameters predicted Cmax (µg/mL) and AUC∞ (µg·h/mL) were 273 and 71.0, 115 and 48.1, and 87.4 and 39.6; the average volume of distribution (mL/kg) values to the central and peripheral compartments were 207 and 143, 242 and 172, and 198 and 213; and the average elimination half-life (hour) and clearance (mL/h/kg) values were 0.18 and 778, 0.29 and 577, and 0.32 and 430, respectively, when the PK parameters for males and females were combined. The current study revealed a similarity in the volume of distribution to the central compartment for MMB4 among the 3 species tested while demonstrating species-related differences in the elimination half-life and clearance of MMB4.

Comparative toxicokinetics of MMB4 DMS in rats, rabbits, dogs, and monkeys following single and repeated intramuscular administration.

1,1'-Methylenebis[4-[(hydroxyimino)methyl]-pyridinium] (MMB4) dimethanesulfonate (DMS) is a bisquaternary pyridinium aldoxime that reactivates acetylcholinesterase inhibited by organophosphorus nerve agent. Time courses of MMB4 concentrations in plasma were characterized following 7-day repeated intramuscular (IM) administrations of MMB4 DMS to male and female Sprague-Dawley rats, New Zealand White rabbits, beagle dogs (single dose only), and rhesus monkeys at drug dose levels used in earlier toxicology studies. In general, there were no significant differences in MMB4 toxicokinetic (TK) parameters between males and females for all the species tested in these studies. After a single IM administration to rats, rabbits, dogs, and monkeys, MMB4 DMS was rapidly absorbed, resulting in average T max values ranging from 5 to 30 minutes. Although C max values did not increase dose proportionally, the overall exposure to MMB4 in these preclinical species, as indicated by area under the curve (AUC) extrapolated to the infinity (AUC∞) values, increased in an approximately dose-proportional manner. The MMB4 DMS was extensively absorbed into the systemic circulation after IM administration as demonstrated by greater than 80% absolute bioavailability values for rats, rabbits, and dogs. Repeated administrations of MMB4 DMS for 7 days did not overtly alter TK parameters for MMB4 in rats, rabbits, and monkeys (150 and 300 mg/kg/d dose groups only). However, C max and AUC values decreased in monkeys given 450 and 600 mg/kg IM doses of MMB4 DMS following repeated administrations for 7 days. Based on the TK results obtained from the current study and published investigations, it was found that the apparent volume of distribution and clearance values were similar among various preclinical species, except for the rat.

MMB4 DMS: cardiovascular and pulmonary effects on dogs and neurobehavioral effects on rats.

The objectives of these studies were to determine the cardiopulmonary effects of a single intramuscular administration of 1,1'-methylenebis[4-[(hydroxyimino)methyl]-pyridinium] dimethanesulfonate (MMB4 DMS) on dogs and on the central nervous system in rats. On days 1, 8, 15, and 22, male and female dogs received either vehicle (water for injection/0.5% benzyl alcohol/methane sulfonic acid) or MMB4 DMS (20, 50, or 100 mg/kg). Pulmonary function was evaluated for the first 5 hours after concurrent dosing with cardiovascular monitoring; then cardiovascular monitoring continued for 72 hours after dosing. Rats were dosed once by intramuscular injection with vehicle (water for injection/0.5% benzyl alcohol/methane sulfonic acid) or MMB4 DMS (60, 170, or 340 mg/kg). In dogs, 100 mg/kg MMB4 DMS resulted in increased blood pressure, slightly increased heart rate, slightly prolonged corrected QT, and moderately increased respiratory rate. There were no toxicological effects of MMB4 DMS on neurobehavioral function in rats administered up to 340 mg/kg MMB4 DMS.

Comparative toxicology studies in Sprague-Dawley rats, rhesus monkeys, and New Zealand White rabbits to determine a no observed adverse effect level for 1,1'-methylenebis[4-[(hydroxyimino)methyl]-pyridinium] dimethanesulfonate.

Studies were conducted in Sprague-Dawley rats, New Zealand White (NZW) rabbits, and rhesus monkeys to characterize the toxicity of 1,1'-methylenebis[4-[(hydroxyimino)methyl]-pyridinium] dimethanesulfonate (MMB4 DMS) following intramuscular administration. Rats received MMB4 DMS once daily for 7 days at 100, 200, 400, and 800 mg/kg/d; rabbits received a range of dose levels in 3 separate 7-day studies from 3 to 800 mg/kg/d and in a single-dose study from 50 to 200 mg/kg; and monkeys received MMB4 DMS at 150 to 600 mg/kg/d. Mortality was noted in rats and rabbits administered ≥ 200 mg/kg. All monkeys survived until scheduled termination. Adverse clinical observations were noted in the rats at ≥ 400 mg/kg/d and in rabbits administered ≥ 200 mg/kg; no adverse findings were noted in the monkeys. Clinical pathology changes were noted in the rabbit related to cardiac and renal function. In the rabbit and monkey, elevations in myoglobin, alanine aminotransferase/aspartate aminotransferase, platelets, creatine kinase, and coagulation factors were related to local inflammation at the intramuscular administration site. Light microscopic examination at the injection sites revealed acute skeletal muscle necrosis in vehicle control and treated groups. Target tissues in the rabbit studies were identified as kidney, heart, and lungs at ≥ 100 mg/kg/d. All changes noted in all the species demonstrated partial to complete recovery comparable to control values or to a clinically irrelevant level of effect. The NZW rabbit was the most sensitive species, and the no observed adverse effect level (NOAEL) was determined as 50 mg/kg/d; the NOAEL in the rat was 100 mg/kg/d; and the NOAEL in rhesus monkeys was >600 mg/kg/d.

Evaluations of in vitro metabolism, drug-drug interactions mediated by reversible and time-dependent inhibition of CYPs, and plasma protein binding of MMB4 DMS.

1,1'-Methylenebis[4-[(hydroxyimino)methyl]-pyridinium] (MMB4) dimethanesulfonate (DMS) is a bisquaternary pyridinium aldoxime that reactivates acetylcholinesterase inhibited by organophosphorus nerve agent. Drug metabolism and plasma protein binding for MMB4 DMS were examined using various techniques and a wide range of species. When (14)C-MMB4 DMS was incubated in liver microsomes, 4-pyridine aldoxime (4-PA) and an additional metabolite were detected in all species tested. Identity of the additional metabolite was postulated to be isonicotinic acid (INA) based on liquid chromatography with a tandem mass spectrometry analysis, which was confirmed by comparison with authentic INA. Formation of INA was dependent on species, with the highest level found in monkey liver microsomes. The MMB4 DMS exhibited reversible inhibition in a concentration-dependent manner toward cytochrome P450 1A2 (CYP1A2), CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in human liver microsomes showing the highest inhibition for CYP2D6. Human recombinant CYPs were used to evaluate inhibitory curves more adequately and determine detailed kinetic constants for reversible inhibition and potential time-dependent inhibition (TDI). The MMB4 DMS exhibited reversible inhibition toward human-recombinant CYP2D6 with an inhibition constant (K i) value of 66.6 µmol/L. Based on the k inact/K I values, MMB4 DMS was found to exhibit the most potent TDI toward CYP2D6. The MMB4 DMS at 5 different concentrations was incubated in plasma for 5 hours using an equilibrium dialysis device. For all species tested, there were no concentration-dependent changes in plasma protein binding, ranging from 10% to 17%. These results suggest that MMB4 was not extensively bound to plasma protein, and there were no overt species-related differences in the extent of MMB4 bound to plasma protein.