Exploration of the African green monkey as a preclinical pharmacokinetic model: oral pharmacokinetic parameters and drug-drug interactions.

African green monkeys (vervets) have been proposed as an alternate species that might allow improved access and provide high-quality pharmacokinetic results comparable with other primates. However, no oral data are available in vervets to evaluate cross-species predictive performance. Therefore, this study was conducted to evaluate the use of the vervet to predict human oral pharmacokinetics and drug interactions. Oral pharmacokinetic studies were conducted in the vervet for eight compounds: phenytoin, moxifloxacin, erythromycin, lidocaine, propranolol, ciprofloxacin, metroprolol, and prednisolone. To assess drug-drug interactions, co-administration experiments were conducted with ketoconazole and either propranolol or erythromycin. In general, the vervet provided similar predictivity for human oral exposure as cynomolgus or rhesus monkeys. In all non-human primates, human exposure to phenytoin would be over-predicted, and erythromycin, lidocaine, and propranolol under-predicted, with good predictivity for the other compounds studied. Furthermore, in the vervet, ketoconazole co-administration resulted in a six-fold increase in exposure to erythromycin, demonstrating proof of concept for drug-drug interaction screening. These data support further exploration of the vervet as an alternate primate species for use in preclinical pharmacokinetic screening.

A pharmacokinetic model of inhaled methanol in humans and comparison to methanol disposition in mice and rats.

We estimated kinetic parameters associated with methanol disposition in humans from data reported in the literature. Michaelis-Menten elimination parameters (Vmax = 115 mg/L/hr; Km = 460 mg/L) were selected for input into a semi-physiologic pharmacokinetic model. We used reported literature values for blood or urine methanol concentrations in humans and nonhuman primates after methanol inhalation as input to an inhalation disposition model that evaluated the absorption of methanol, expressed as the fraction of inhaled methanol concentration that was absorbed (phi). Values of phi for nonexercising subjects typically varied between 0.64 and 0.75; 0.80 was observed to be a reasonable upper boundary for fractional absorption. Absorption efficiency in exercising subjects was lower than that in resting individuals. Incorporation of the kinetic parameters and phi into a pharmacokinetic model of human exposure to methanol, compared to a similar analysis in rodents, indicated that following an 8-hr exposure to 5000 ppm of methanol vapor, blood methanol concentrations in the mouse would be 13- to 18-fold higher than in humans exposed to the same methanol vapor concentration; blood methanol concentrations in the rat under similar conditions would be 5-fold higher than in humans. These results demonstrate the importance in the risk assessment for methanol of basing extrapolations from rodents to humans on actual blood concentrations rather than on methanol vapor exposure concentrations.

Comparative pathogenesis of haloacetic acid and protein kinase inhibitor embryotoxicity in mouse whole embryo culture.

Haloacetic acids (HAs) are embryotoxic contaminants commonly found in drinking water. The mechanism of HA embryotoxicity has not been defined, but may be mediated in part by protein kinase C (PKC) inhibition. This study was conducted to evaluate the pathogenesis of HA embryotoxicity, and to compare these data with those from specific (Bis I) and non-specific (staurosporine) inhibitors of PKC. Embryos were incubated for varying times with several HAs, Bis I, staurosporine, or Bis V (a negative control). Cell cycle analysis was performed by flow cytometry following nuclear staining with propidium iodide; apoptosis was evaluated by fluorescence microscopy following LysoTracker staining. At concentrations producing 100% embryotoxicity with no embryolethality, only staurosporine perturbed the cell cycle. However, flow cytometry revealed accumulation of sub-G1 events (an apoptotic indicator) across time with bromochloroacetic acid, dichloroacetic acid, and staurosporine, but not dibromoacetic acid, Bis I, or Bis V. Sub-G1 events were particularly prominent in the head region, and remained at control levels in the heart. LysoTracker staining confirmed a similar pattern of apoptosis in the intact embryo; BCA and DCA produced intense staining in the prosencephalon, with virtually no staining in the heart. These data indicate that while cell-cycle perturbation may not mediate the pathogenesis of HA embryotoxicity, these agents do induce embryonic apoptosis. In addition, the lack of Bis I-induced apoptosis indicates that PKC inhibition is unlikely to be the sole mediator of HA embryotoxicity.

Dysmorphogenic effects of a specific protein kinase C inhibitor during neurulation.

Protein kinase C (PKC) plays a key role in signal transduction and is an important mediator of events throughout development. However, no information exists regarding the effect of a specific PKC inhibitor on mammalian embryogenesis during neurulation. This investigation was undertaken to examine the effects of a specific inhibitor of PKC, as well as inhibitors of other important kinases, on cultured mouse embryos. CD-1 mouse embryos (3 to 6 somite stage) were exposed to bisindolylmaleimide I (a specific PKC inhibitor) as well as specific inhibitors of PKA, PKG, and MAP kinase kinase for 24 h. The PKC inhibitor was a potent embryotoxicant and elicited malformations at concentrations as low as 0.01 microM. Inhibitors of other kinases also produced malformations but at much higher concentrations than those required to produce similar defects with the PKC inhibitor. These data suggest that PKC plays an important role in mammalian neurulation. Further research is required to clarify the mechanism by which PKC inhibition at this developmental stage produces malformations and the potential effects of environmental toxicants with PKC inhibitory properties on this signal transduction pathway.

In vitro biocompatibility assessment of multipurpose contact lens solutions: effects on human corneal epithelial viability and barrier function.

PURPOSE: To explore the in vitro effects of multipurpose contact lens solutions (MPSs) on corneal epithelial barrier function and viability. METHODS: Human corneal epithelial cells (HCEpiC) were exposed to 50% MPSs A-G. Viability was determined using metabolic activity, protease release and caspase assays. Barrier function was evaluated using immunostaining for the tight junction protein zonnula occludens-1 (ZO-1) and resistance measurements. RESULTS: MPS A and G did not affect HCEpiC monolayer viability after 2 h, while MPSs B-F significantly decreased viability. There was a significant decrease in stratified HCEpiC viability after exposure to MPSs B-E for 2 h, while there was no effect of MPS A. After exposure of HCEpiC monolayers to MPS A, F or G for 30 min, ZO-1 staining appeared similar to control. HCEpiC exposed to MPSs B and C demonstrated tight junction breakdown. There was no significant change in HCEpiC monolayer resistance after exposure to MPS A or F for 2 h, while MPSs B-E and G reduced resistance. After exposure to MPS A-E, stratified HCEpiC resistance was significantly decreased after 2 or 4 h. The decrease in resistance was significantly less with MPS A as compared to the other MPSs. CONCLUSIONS: MPSs caused varying modifications to cell viability and barrier function in monolayer and stratified HCEpiC. MPS A did not alter monolayer HCEpiC viability or barrier function, while MPSs B-G caused significant decreases of at least one parameter. Furthermore, MPS A had significantly less effect than MPSs B-E on viability and barrier function of stratified HCEpiC.

Effect of a novel multipurpose contact lens solution on human corneal epithelial barrier function.

PURPOSE: To explore the effect of a novel multipurpose contact lens solution (MPS) on the junction protein distribution and barrier function of cultured human corneal epithelial cell monolayers. METHODS: Cultured human corneal epithelial cells (HCEpiC) were exposed to a novel MPS (MPS A; Biotrue™ multi-purpose solution, Bausch & Lomb Incorporated) at 50%, 75% and 100% for 10 or 30 min. Four commercially available MPS products, MPS B (AQuify, Ciba Vision), MPS C (COMPLETE MPS Easy Rub, AMO), MPS D (OPTI-FREE Express, Alcon) and MPS E (OPTI-FREE RepleniSH, Alcon) were tested in parallel. Tight junction structure and integrity were evaluated by confocal microscopy using ZO-1 antibody and scanning microscopy (SEM). Quantitative evaluation of MPSs on epithelial barrier function was determined by measuring transepithelial electrical resistance (TEER) across HCEpiC grown on Transwell Clear permeable supports and on electric cell-substrate impedance sensing (ECIS) electrode arrays. RESULTS: Overall after exposure to the three concentrations (50%, 75%, and 100%) of MPS A, ZO-1 distribution and fluorescent intensity on the cell surface appeared similar to the media control with continuous tight junctions and clear intercellular junctions. At all measured time points after exposure to MPS A (50% or 75%) there was also no effect on the TEER using both resistance methodologies, and SEM showed that MPS A appeared similar to the Hank's balanced salt solution (HBSS) control. In cells exposed to MPS D there was a dose-dependent change in the distribution of ZO-1, some cell detachment, and a decrease in monolayer resistance at all time points measured. Ultrastructurally, MPS D caused gross changes, including damage to cell junctions and plasma membranes. To a lesser extent, the remaining three commercial MPS products demonstrated some effects on tight junction ZO-1 distribution and/or TEER. CONCLUSIONS: Based on the in vitro measurements of tight junction protein expression, monolayer integrity, and transepithelial electrical resistance, the novel multipurpose contact lens solution (MPS A) did not alter corneal barrier function as compared to media, PBS or HBSS control. Clinical significance of the observed differences in epithelial barrier function among the MPSs tested needs further investigation.

Comparative evaluation of oral systemic exposure of 56 xenobiotics in rat, dog, monkey and human.

The prediction of human pharmacokinetics is often based on in vivo preclinical pharmacokinetic data. However, to date, no clear guidance has been available about the relative ability of the major preclinical species to estimate human oral exposure. The study was conducted to survey the literature on oral pharmacokinetic parameters in rat, dog, monkey and human, and to compare various methods for prediction of oral exposure in humans. Fifty-six non-peptide xenobiotics were identified with oral pharmacokinetic data in rat, dog, monkey and human, and comparison of the data from each species to humans was conducted along with an evaluation of the molecular features of these compounds. Monkey liver blood flow-based oral exposure was qualitatively and quantitatively more predictive of human oral exposure than rat or dog. Furthermore, generation of data in three versus two preclinical species did not always improve human predictivity. The use of molecular properties did not substantially improve the prediction of human oral exposure compared with the prediction from monkey alone. These observations confirm the continued importance of non-human primates in preclinical pharmacokinetics, and also have implications for pharmacokinetic lead optimization and for prediction of human pharmacokinetic parameters from in vivo preclinical data.

Use of intrauterine microdialysis to investigate methanol-induced alterations in uteroplacental blood flow.

Methanol is teratogenic in rodents; it has been postulated that this teratogenicity may be mediated in part by conceptal hypoxia. To construct a model to predict conceptal risk following maternal methanol exposure, conceptal disposition of methanol must be determined and any effects of such exposure on blood flow must be quantitated. In the present study, these toxicokinetic and toxico-dynamic parameters were evaluated by in vivo intrauterine microdialysis. Microdialysis probes were inserted into the uteri of Gestational Day (gd) 20 rats; methanol was administered as either an iv bolus (100 or 500 mg/kg) or infusion (100 or 1000 mg/kg/hr). In separate experiments, methanol (100 or 500 mg/kg) and 3H2O (20 microCi/kg) were administered iv to gd 20 and 14 rats and gd 18 mice. In both experiments, maternal blood and uterine microdialysate were collected and analyzed for methanol or 3H2O content. The methanol concentration-time data were consistent with saturable maternal elimination and apparent first-order transfer between maternal and conceptal compartments; at distribution equilibrium, conceptal methanol concentrations exceeded those in the dam by approximately 25%. The initial rate of conceptal permeation of methanol was proportional to the reciprocal of maternal blood methanol concentration (r2 = 0.910). Methanol also reduced significantly the rate of 3H2O uptake into the conceptus in a concentration-dependent fashion in gd 14 and 20 rats and gd 18 mice. These data indicate that methanol may decrease uteroplacental blood flow, decreasing methanol presentation to the conceptus and possibly producing conceptal hypoxia.

Comparative toxicokinetics of methanol in pregnant and nonpregnant rodents.

Methanol toxicokinetics were examined in pregnant Sprague-Dawley rats and CD-1 mice to explore the possibility of gestational-associated alterations in metabolism and disposition. In vitro biotransformation of methanol in rat and mouse fetal livers also was examined to assess the capability of the near-term rodent fetus to metabolize methanol. In the in vivo studies, rats received a single dose (100 or 2,500 mg/kg) of methanol either orally (by gavage) or intravenously; mice received a single oral or intravenous 2500-mg/kg dose. The maximal rate of methanol elimination (Vmax) in vivo decreased at term in both rodent species; Vmax in near-term rats and mice was only 65-80% of that in nonpregnant animals. Gestation also affected intercompartmental transfer rate constants, although there was no obvious relationship between these changes and gestational stage. In vitro metabolism studies supported the in vivo data; adult near-term rodent livers metabolized methanol with a Vmax of approximately 85% that in livers from nonpregnant rodents (p < 0.05). Fetal rodent liver was capable of metabolizing methanol in vitro, but only at a rate < 5% of respective adult livers. Data generated in these experiments demonstrate that alterations in methanol disposition associated with gestational stage must be accounted for in the development of a toxicokinetic model for methanol in pregnant mammals.

Methanol inhalation: site and other factors influencing absorption, and an inhalation toxicokinetic model for the rat.

PURPOSE: This investigation was conducted to identify the site and characteristics of methanol absorption and to develop an inhalation model relating methanol absorption, blood concentration, and elimination. METHODS: Rats were exposed to methanol in chambers that allowed measurement of methanol uptake, ventilation, and blood concentrations; anesthetized rats with a tracheal cannula were examined to determine tracheal concentrations. In separate experiments, methanol exposed rats received an iv methanol bolus to examine the effect of blood methanol on ventilation and absorption; ventilation also was manipulated by CO(2) or pentobarbital to assess the effect of ventilation rate on methanol absorption. These data were combined to construct a semi-physiologic model of methanol uptake. RESULTS: Only 1-3 percent of inhaled methanol reached the trachea, primarily from systemic methanol partitioning into the trachea; blood methanol did not alter methanol absorption. Manipulation of ventilation and application of the pharmacokinetic model indicated that ventilation was less significant than environmental methanol concentration in determining the fraction of inhaled methanol absorbed, although both parameters were important determinants of the total mass absorbed. CONCLUSIONS: These data indicate that methanol uptake is a complex process that depends upon several parameters. Despite these complexities, a relatively simple semi-physiologic model was capable of describing methanol uptake over a wide range of exposure concentrations in the rat.

Comparative toxicokinetics of inhaled methanol in the female CD-1 mouse and Sprague-Dawley rat.

Female CD-1 mice were exposed for 8 hr, both individually and in groups of eight to nine, to 2500, 5000, and 10,000 ppm methanol vapor in a flowthrough exposure chamber. The ventilation of individually exposed mice and the absorption of methanol from the chamber airstream were measured. The extraction of methanol from the airstream and the blood methanol concentration at various time points during and following exposure were determined for the group-exposed mice. The similarity of systemic kinetic parameters (volume of distribution; Michaelis-Menten elimination parameters, Vmax and KM) between inhalation exposure and iv and po routes of administration was verified. Total 8-hr ventilation decreased slightly with increasing exposure concentration. The fraction of inhaled methanol absorbed (0.85 +/- 0.14) did not vary statistically with exposure concentration. Measured ventilation, fractional absorption, and systemic kinetic parameters were combined in a semiphysiologic pharmacokinetic model that yielded accurate predictions of blood methanol concentrations during and after an 8-hr exposure. Model predictions for the mouse were compared to a previously developed inhalation toxicokinetic model for the rat. The comparison demonstrated that at similar methanol vapor concentrations, mice evidenced a two- to threefold higher blood methanol concentration than rats, despite the fact that the apparent Vmax for methanol elimination in the mouse is twofold larger than that in the rat. These data may have significant implications in understanding species differences in methanol-induced teratogenic effects.

Evaluation of the use of accelerated infusions for the determination of pharmacokinetic linearity.

Accelerated infusions are potentially useful in the investigation of pharmacokinetic linearity. However, little information exists to validate this technique or to demonstrate its limitations. This investigation was performed to determine whether accelerated infusion regimens reliably estimate the range of pharmacokinetic linearity for molecules of varying pharmacokinetic properties, to evaluate the ability of accelerated infusions to identify pharmacokinetic nonlinearity, and to validate the accelerated infusion technique using compounds with known pharmacokinetic parameters. Simulations incorporating accelerated infusion as the input function resulted in the anticipated concentration-time profiles that contained an initial lag phase before reaching a linear slope. This lag phase increased with increasing distributional volume and in some instances was sufficiently great to obscure or prevent the linear portion of the profile. These simulations also revealed that clearance estimated from the apparently linear portion of the concentration-time profile can be erroneous under some conditions, as for large-volume compounds. Simulations of structured nonlinearity produced the predicted profiles for compounds with low to moderate volumes of distribution while demonstrating that modeling of data derived from compounds with large volumes of distribution may be inaccurate. Finally, experiments using accelerated infusions with various test compounds further demonstrated the usefulness of this technique while presenting limits imposed on the interpretation of the data. The results of this investigation indicate that the accelerated infusion may be used to determine pharmacokinetic linearity for compounds within certain pharmacokinetic boundaries, but that appropriate caution should be exercised in the extent of interpretation that should be extracted from such studies.

Comparative toxicokinetics of methanol in the female mouse and rat.

The toxicokinetics of methanol in female CD-1 mice and Sprague-Dawley rats were examined to explore the possibility of species differences in the disposition of the compound. Mice received a single dose of 2.5 g/kg methanol either po (by gavage) or i.v. (as a 1-min infusion). Rats received a single oral dose of 2.5 g/kg methanol. As expected, the disposition of methanol was nonlinear in both species. Data obtained after i.v. administration of methanol to mice were well described by a one-compartment model with Michaelis-Menten elimination. Blood methanol concentration--time data after oral administration could be described by a one-compartment (mice) or two-compartment (rats) model with Michaelis-Menten elimination from the central compartment and biphasic absorption from the gastrointestinal tract. Kinetic parameters (Vmax for elimination, apparent volume of the central compartment [Vc], first-order rate constants for intercompartmental transfer [k12 and k21], and first-order absorption rate constants for fast [kAF] and slow [kAS] absorption processes) were compared between species. When normalized for body weight, mice evidenced a higher maximal elimination rate than rats (Vmax = 117 +/- 3 mg/hr/kg vs 60.7 +/- 1.4 mg/hr/kg for rats). The contribution of the fast absorption process to overall methanol absorption also was larger in the mouse than in the rat.

Apparent absolute oral bioavailability in excess of 100% for a vitronectin receptor antagonist (SB-265123) in rat. I. Investigation of potential experimental and mechanistic explanations.

1. SB-265123 is a novel alphavbeta3 (the vitronectin receptor) antagonist. Previous rat studies with it revealed an apparent absolute oral bioavailability (Fapp) of greater than 100%. The present studies were conducted to investigate the potential causes for this observation. 2. Of 49 SB-265123 analogues evaluated in rat using an identical experimental design, Fapp > 100% was observed for 22 of them, suggesting that the observed Fapp >100% with SB-265123 was not anomalous. All 22 compounds had clearances < 15 ml min(-1) kg(-1). However, Fapp>100% were not recorded for all low-clearance analogues. 3. Using SB-265123 as a model to investigate potential artefacts, it was demonstrated that using a chiral assay did not decrease Fapp. Additionally, qualitative sample analysis demonstrated that no metabolites were present in the plasma that could interfere with the liquid chromatography coupled with tandem mass spectrometry detection assay. The high Fapp was also dose-order-, delivery system- and vehicle-independent, and was not affected by the feeding status of the animals. Furthermore, a linearity experiment and an absorption study indicated that oral administration of SB-265123 does not result in hepatic portal vein concentrations that exceed the pharmacokinetic linearity of SB-265123. 4. These observations suggest that the observed Fapp > 100% for SB-265123 is not due to an experimental artefact or an obvious pharmacokinetic non-linearity. The mechanism(s) for this phenomenon is explored further in the second part of the present paper.

Development of an in vivo preclinical screen model to estimate absorption and bioavailability of xenobiotics.

The purpose of this study was to develop an in vivo screening method for rapid preclinical characterization of absorption and bioavailability of large numbers of compounds. This effort involved several steps. First, a pharmacokinetic characterization of a reference compound was conducted in the monkey. These data were used to verify theoretical calculations of a maximal portal dose-normalized area under the concentration-time curve. Next, a monkey screen was implemented using mixtures of up to five compounds each (i.e., cassettes) to estimate the bioavailability of approximately 200 compounds. Cassettes were administered as a single intraduodenal dose to a single monkey followed by simultaneous portal and systemic blood sampling. Definitive studies were then conducted to determine absolute bioavailability of 14 of these compounds. The studies with the reference compound demonstrated that the theoretical methodology based on a single intraduodenal dose with portal and systemic sampling provided consistent estimates of bioavailability. In the screen studies, approximately 75% of the test compounds were excluded from further evaluation due to poor absorption. Of the 14 compounds selected for follow-up evaluation from both well and poorly absorbed compounds, the absolute bioavailability of 10 of them were correctly classified from the screening data. The remaining 4 compounds were false positives, which showed low bioavailability; no false negatives were encountered. This approach allows for a rapid and reliable screen to evaluate absorption and bioavailability using a single dose in a preclinical model.

Development of a physiologically based pharmacokinetic model to describe the disposition of methanol in pregnant rats and mice.

Physiologically based pharmacokinetic (PBPK) models have been developed in recent years to describe the disposition of xenobiotics during gestation. These models can account for the dynamics of physiologic changes associated with pregnancy and represent a significant advantage in quantitatively assessing potential exposure of the conceptus. The PBPK approach was used to develop a model of methanol disposition during gestation in rats and mice. To validate this model, concentrations of methanol in the dam and the conceptus were determined after methanol exposure of rats on Gestational Day (gd) 14 and 20 and of mice on gd 18. At the developmental stages examined, the model provided a good description of methanol disposition in the maternal circulation and the conceptus of both species. Furthermore, the model was capable of providing good fits to methanol concentration-time data from the literature. In pregnant animals, conceptal/maternal AUC and Cmax ratios decreased with increasing dose at both gd 14 and gd 20 in the rat and at gd 18 in the mouse. Additionally, the conceptal/maternal diffusion constant ratio consistently decreased with increasing dose in pregnant rats and mice. These results are consistent with earlier observations that methanol limits its own delivery to the conceptus. Further experimentation is required to continue the process of developing a generalized PBPK model to describe the disposition of xenobiotics in pregnancy, to examine specific mechanisms of nonlinear conceptal methanol disposition, and to expand the model to extrapolate to low-dose human exposures.

Apparent absolute oral bioavailability in excess of 100% for a vitronectin receptor antagonist (SB-265123) in rat. II. Studies implicating transporter-mediated intestinal secretion.

1. Transporters have been increasingly identified as a factor in limiting the oral bioavailability of certain drugs. Previously, the present authors investigated a compound (SB-265123) with an apparent absolute oral bioavailability (Fapp) consistently > 100%, and excluded likely artefactual causes for this observation, as well as standard considerations of non-stationary or non-linear pharmacokinetics. The data led the authors to believe that SB-265123 might be a transporter substrate in the rat, and it was hypothesized that transporter interactions might be responsible for the observed Fapp > 100%. 2. In the present study, a model was proposed incorporating rapid and complete absorption and elimination by a saturable intestinal secretory pathway. Intestinal secretion was demonstrated for SB-265123 using a rat single-pass intestinal perfusion technique. In addition, in a study employing both independent and simultaneous intravenous and oral administration of SB-265123, exposure to SB-265123 was greater than additive on joint intravenous and oral administration, lending further support to the hypothesis of a saturable transporter. Furthermore, in a study with co-administration of GF120918A, a transporter inhibitor, the observed Fapp for SB-265123 was only 84 +/- 17%, providing additional evidence for transporter involvement in the >100% Fapp phenomenon. 3. Experience with SB-265123 illustrates a counterintuitive impact of transporters on oral bioavailability and highlights the importance of considering transporter interactions in the systemic disposition of xenobiotics, even those not demonstrating low oral bioavailability.

Practical strategies for detecting and confirming vancomycin-intermediate Staphylococcus aureus: a tertiary-care hospital laboratory's experience.

The clinical microbiology laboratory plays a critical role in the detection of Staphylococcus aureus with decreased susceptibility to vancomycin. Staff education and rapid laboratory response are of utmost importance. We report on our laboratory's experience and provide recommendations for the identification and confirmation of vancomycin-intermediate S. aureus.