Derivation of occupational exposure limits based on target blood concentrations in humans.

An approach for deriving occupational exposure limits (OEL) for pharmaceutical compounds is the application of safety factors to the most appropriate pre-clinical toxicity endpoint or the lowest therapeutic dose (LTD) in humans. Use of this methodology can be limited when there are inadequate pre-clinical toxicity data or lack of a well-defined therapeutic dose, and does not include pharmacokinetic considerations. Although some methods have been developed that incorporate pharmacokinetics, these methods do not take into consideration variability in response. The purpose of this study was to investigate how application of compartmental pharmacokinetic modeling could be used to assist in the derivation of OELs based on target blood concentrations in humans. Quinidine was used as the sample compound for the development of this methodology though the intent was not to set an OEL for quinidine but rather to develop an alternative approach for the determination of OELs. The parameters for the model include body weight, breathing rate, and chemical-specific pharmacokinetic constants in humans, data typically available for pharmaceutical agents prior to large scale manufacturing. The model is used to simulate exposure concentrations that would result in levels below those that may result in any undesirable pharmacological effect, taking into account the variability in parameters through incorporation of Monte Carlo sampling. Application of this methodology may decrease some uncertainty that is inherent in default approaches by eliminating the use of safety factors and extrapolation from animals to humans. This methodology provides a biologically based approach by taking into consideration the pharmacokinetics in humans and reported therapeutic or toxic blood concentrations to guide in the selection of the internal dose-metric.

Genetic polymorphisms in ethanol metabolism: issues and goals for physiologically based pharmacokinetic modeling.

Chronic exposure to excessive ethanol consumption has adverse effects on virtually all organs and tissues in the body, including but not limited to the liver, pancreas, reproductive organs, central nervous system, and the fetus. Exposure to ethanol can also enhance the toxicity of other chemicals. Not all persons exposed to the same amount of ethanol experience the same degree of adverse effects. For example, only 12% to 13% of alcohol abusers develop cirrhosis. Possible factors which may alter susceptibility include age, sex, nutritional status, health status (i.e., smokers) and race. Some of these factors affect susceptibility because they alter ethanol metabolism, which occurs primarily in the liver by alcohol dehydrogenase (ADH). Genetic polymorphisms for ADH partially account for the observed differences in ethanol elimination rates among various populations but the relative contribution to susceptibility is not completely understood. Incorporation of the kinetic parameters associated with ADH polymorphisms into a physiologically based pharmacokinetic (PBPK) model for ethanol will aid in assessing the relative contribution to susceptibility. The specific information required to develop this model includes Km and Kcat values for each ADH isoform and the amount of each isoform present in the liver. Blood ethanol concentrations (BEC) from various populations with known ADH phenotypes are also necessary to validate the model. The impact of inclusion of these data on PBPK model predictions was examined using available information from adult white and African American males.

Development and application of a physiologically based pharmacokinetic model for ethanol in the mouse.

The purpose of the present study was to develop a physiologically based pharmacokinetic (PBPK) model in the mouse and to utilize it to evaluate the relative contribution, if any, of gastric alcohol dehydrogenase (ADH) to the bioavailability of ethanol. The PBPK model developed in Swiss Webster male mice accurately simulated blood and brain ethanol concentrations following an intraperitoneal administration of 0.82 and 3.2 g of ethanol/kg body weight. Application of the model illustrated that inclusion of gastric ADH into the model provided a less accurate fit to the experimental data, and therefore gastric ADH did not contribute to the overall disposition of an orally administered ethanol dose of 0.75 g/kg. Furthermore, the model also indicated that changes in percentage cardiac output to the liver had a minimal effect on the blood ethanol concentration (BEC) time curve. The results illustrate the validity of the PBPK model developed for ethanol and demonstrate that in the Swiss Webster male mouse the bioavailability of ethanol is minimally affected, if at all, by metabolism by gastric ADH.

2,3-Butanedione monoxime protects mice against the convulsant effect of picrotoxin by facilitating GABA-activated currents.

While adult mice receiving picrotoxin (PTX) alone responded with clonic and tonic-clonic seizures, this response was greatly suppressed for mice simultaneously injected with 2,3-butanedione monoxime (BDM). For example, 60% and 10% of the mice convulsed when injected (i.p.) with 3.0 mg/kg PTX alone or PTX plus 205 mg/kg of BDM, respectively. In contrast, a non-oxime analogue of BDM, 2,3-butanedione (BTD), did not have this anticonvulsant effect. In order to explore the basis for the anticonvulsant effect of BDM, we recorded GABA-activated currents (IGABA) of frontal cortical as well as ventromedial hypothalamic neurons before, during and after exposure to this oxime. BDM had a biphasic effect on IGABA. That is, high concentrations (100 microM-40 mM) decreased and lower concentrations (0.01 microM-0.001 microM) potentiated IGABA; these effects of BDM reversed upon washout of the oxime. In contrast, BTD had no effect on IGABA. Finally, when 0.001 microM BDM, 10-30 microM PTX and GABA were co-applied the inhibitory effect of the toxin on IGABA was markedly suppressed. These data suggest that the anticonvulsant effect of oximes involves facilitation of the inhibitory action of GABA.

Pharmacologic doses of ascorbic acid prolong the effects of pentobarbital anesthesia.

Saline or ascorbic acid (AA), 1000 mg/kg and 1400 mg/kg was administered intraperitoneally (IP) to 3 groups of female Swiss-Webster mice after they had been anesthetized with sodium pentobarbital (SP). Both doses of AA given IP after SP anesthesia significantly prolonged sleeping time. An additional 50 mice were injected IP with 0.3 ml SP containing 6 mg/ml unlabeled SP and 0.6 ng C14 labeled SP. Immediately 25 mice received 40 mg AA in 0.4 ml saline, IP, and the remainder received saline. The serum half life of the radioactively labeled SP was more than 3 times longer in AA treated mice than in the controls.

Deposition of low dose benzo(a)pyrene into fetal tissue: influence of protein binding.

The influence of maternal binding of benzo(a)pyrene (BaP) on its disposition into fetal tissue was investigated in pregnant Swiss-Webster mice. Low doses (10 ng/mouse) of radiolabeled BaP were administered by intravenous injection on day 15 of gestation. BaP was administered along with normal rabbit serum (NRS) (low binding paradigm) or anti-BaP antiserum (high binding paradigm) and animals killed at various time points. Total radioactivity in the fetus increased with time to peak concentrations in whole fetal homogenates at 12 hours. In contrast, maternal serum, liver, and lung showed a decrease in total radioactivity over the same time period. Total radioactivity/gram of fetal tissue was significantly higher in NRS-treated animals compared to anti-BaP-antiserum-treated animals. Since the levels of the parent compound, BaP, in fetal tissue fell over time similar to maternal liver and lung, the increase in total radioactivity in the fetus was due to an increased concentration of a BaP metabolite fraction in both the low binding and high binding groups. Significantly, a lower level of this metabolite fraction was found in fetal tissue from the anti-BaP-antiserum-treated animals. The present study shows that maternal exposure to this environmental pollutant, even at low doses, results in an accumulation of a metabolite-rich fraction in the fetal compartment, which may contribute to the teratogenic potential of BaP. The data also demonstrate that the amount of this accumulation can be diminished by increasing maternal binding proteins, such as by treatment with anti-BaP antiserum.

Pharmacologic response to pentobarbital in passively immunized mice.

Rabbits were actively immunized using a barbiturate--BGG conjugate as the immunogen. The antiserum obtained from actively immunized rabbits was administered intravenously to mice to accomplish passive immunization. The antibody binding capacity for 3H-phenobarbital was shown to be sustained in passively immunized mice for periods of up to three weeks. Serum levels of 3H-phenobarbital in passively immunized mice and control mice were compared following drug administration and found to be altered in the antibody-containing mice. There was a 4-fold higher amount of 3H-phenobarbital present in the serum of passively immunized mice compared to control animals. The higher barbiturate levels were due to binding of 3H-phenobarbital to globulin fraction of serum in passively immunized mice. Additionally, decreased pentobarbital-induced ataxia was demonstrated in passively immunized mice. The decreased responsiveness was selective for barbiturates in passively immunized mice and did not modify the ataxia produced in these animals by another depressant agent, ethanol.

Pharmacologic response to pentobarbital in actively immunized mice.

Mice were placed on an immunization schedule known to result in the production of antibodies directed against a variety of barbiturates (antibody binding capacity = 2.71 pmole 3H-phenobarbital bound/ml undiluted serum). The pharmacologic response to barbiturate in these actively immunized mice and suitably treated controls was investigates using rotarod apparatus for monitoring CNS depression. It was found that the response to pentobarbital in actively immunized mice was decreased, as reflected by an increase in the time the mice remained on the rotarod and a shift of the dose--response curve to the right. The decreased pharmacologic response to pentobarbital was not the result of changes in levels of the hepatic drug metabolism components. Furthermore, the alteration of pharmacologic response in actively immunized mice was selective for barbiturate and did not modify the ataxia produced by administration of another depressant agent, ethanol.

Barbiturates: radioimmunoassay.

The development of a radioimmunoassay for barbiturate is described. The barbiturate is made antigenic by coupling it to a protein, bovine gamma globulin. The radioimmunoassay can measure as little as 5 nanograms of barbiturate.