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.

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.

Absorption, distribution, metabolism, and excretion of 14C-MMB4 DMS administered intramuscularly to Sprague-Dawley rats and New Zealand White rabbits.

1,1'-Methylenebis[4-[(hydroxyimino)methyl]-pyridinium] dimethanesulfonate (MMB4 DMS) is currently under development for the treatment of chemical warfare organophosphorus nerve agent poisoning. The present study evaluates the absorption, distribution, metabolism, and excretion of (14)C-MMB4 DMS administered intramuscularly to rats and rabbits. The formulated mixture of radiolabeled and nonradiolabeled MMB4 DMS was administered as a single or 7-day repeated dose. Rat doses were 55 or 220 mg/kg (100 µCi/kg), and rabbit doses were 25 or 100 mg/kg (31.25 and 62.5 µCi/kg, respectively). Urine, bile (rats only), feces, blood, and tissues were collected for up to 72 hours. Metabolic profiling using high-performance liquid chromatography with radiodetection was performed on selected urine samples. For both animal species, the majority of the total radioactivity was excreted in the urine (74%-94%) by 72 hours after dosing with greater than 90% of the radioactivity measured in the urine within 8 to 12 hours after dosing. There were no apparent species or dose differences in the urine excretion pattern. The distribution of (14)C-MMB4 DMS-derived radioactivity was rapid and generally reached the highest concentration by the first collection time point (0.25 hours). The tissue-blood concentration ratios were highest at the injection sites and in the kidneys and gastrointestinal tract contents for both the species. Two metabolites of MMB4 DMS were detected in rat and rabbit urine; their structure was confirmed by liquid chromatography with tandem mass spectrometry as 4-pyridine aldoxime and isonicotinic acid (pyridine-4-carboxylic acid).

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.

Human safety and pharmacokinetic study of intramuscular midazolam administered by autoinjector.

Midazolam in an autoinjector was evaluated in an open-label dose escalation study involving 39 healthy participants. Safety and pharmacokinetic parameters were determined for doses ranging from 5 to 30 mg. No serious adverse events were noted during the study. Two participants (30 mg) experienced changes in their electrocardiogram (trigeminy and prolongation of QRS complex) that met the criteria for dose-limiting adverse events. No significant respiratory depression was noted during the study. The midazolam doses studied exhibited a median t(max) of 0.5 hours with a geometric mean terminal elimination half-life value of 4.1 hours (range, 2.9-4.5 hours). The extent of systemic exposure, assessed by area under the curve (AUC) and maximum concentration (C(max)), tended to increase proportionally with increasing doses from 5 to 30 mg; however, for the male 30-mg group, there was evidence of a larger than proportional increase in AUC.