We describe a general approach using Bayesian analysis for the estimation of parameters in physiological pharmacokinetic models. The chief statistical difficulty in estimation with these models is that any physiological model that is even approximately realistic will have a large number of parameters, often comparable to the number of observations in a typical pharmacokinetic experiment (e.g., 28 measurements and 15 parameters for each subject). In addition, the parameters are generally poorly identified, akin to the well-known ill-conditioned problem of estimating a mixture of declining exponentials. Our modeling includes (a) hierarchical population modeling, which allows partial pooling of information among different experimental subjects; (b) a pharmacokinetic model including compartments for well-perfused tissues, poorly perfused tissues, fat, and the liver; and (c) informative prior distributions for population parameters, which is possible because the parameters represent real physiological variables. We discuss how to estimate the models using Bayesian posterior simulation, a method that automatically includes the uncertainty inherent in estimating such a large number of parameters. We also discuss how to check model fit and sensitivity to the prior distribution using posterior predictive simulation. We illustrate the application to the toxicokinetics of tetrachloroethylene (perchloroethylene [PERC]), the problem that motivated this work.

Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.

Context β2*-nicotinic acetylcholine receptor (β2*-nAChR) availability is higher in recently abstinent smokers compared to never smokers. Variations in β2*-nAChR availability over the course of abstinence may be related to the urge to smoke, the extent of nicotine withdrawal and successful abstinence. Objective To examine changes in β2*-nAChR availability during acute and prolonged abstinence from tobacco smoking and to determine how changes in β2*-nAChR availability were related to clinical features of tobacco smoking. Design Tobacco smokers participated in up to 4 [123I]5-IA-85380 ([123I]5-IA) single photon emission computed tomography (SPECT) scans during abstinence: 1 day (n=7), 1 week (n=17), 2 weeks (n=7), 4 weeks (n=11), and 6-12 weeks (n=6). Age-matched nonsmokers participated in 1 [123I]5-IA SPECT scan. All subjects completed 1 magnetic resonance imaging study. Setting Academic imaging center. Participants Tobacco smokers (n=19) and an age-matched nonsmoker comparison group (n=20). Main Outcome Measure [123I]5-IA SPECT images were converted to distribution volume and were analyzed using regions of interest. Results Compared to nonsmokers, β2*-nAChR availability in the striatum, cortex and cerebellum of smokers was not different at one day of abstinence, significantly higher at 1week of abstinence and not different at 4 or 6-12 weeks of abstinence. In smokers, at 6-12 weeks of abstinence, β2*-nAChR availability was significantly lower in the cortex and cerebellum compared to 1 week of abstinence. Additionally, cerebellar β2*-nAChR availability at 4 weeks of abstinence was positively correlated with craving on the day of scan. Conclusions These data suggest higher β2*-nAChR availability persists up to 1 month of abstinence, and normalizes to nonsmoker levels by 6-12 weeks of abstinence from tobacco smoking. These marked and persistent changes in β2*-nAChR availability may contribute to difficulties with tobacco cessation.

Background Modulation of nicotinic acetylcholine receptors (nAChRs), specifically α4β2 subunit containing nAChRs, may be effective in the treatment of patients with major depressive disorder (MDD). Using [123I] 5-I-A-85380 single photon emission computed tomography (SPECT), we studied β2 subunit containing nAChR (β2*-nAChR) availability in patients with MDD. In order to understand the molecular basis of the change in receptor availability, we also studied β2*-nAChR binding in postmortem samples of human brains of MDD subjects. Methods 23 medication-free, early-onset, non-smoking subjects with familial MDD (8 acutely depressed (aMDD), 15 euthymic, recovered MDD subjects (rMDD)), and 23 age- and gender-matched, non-smoking controls had one [123I] 5-I-A-85380 SPECT scan and a magnetic resonance imaging (MRI) scan. β2*-nAChR availability was quantified as VT/fP. β2*-nAChR binding was analyzed in postmortem samples of the prefrontal cortex in 14 subjects with MDD and age-matched controls with [125I] 5-I-A-85380. Results β2*-nAChR availability in aMDD and rMDD subjects was significantly lower across all brain regions than in respective controls and lower in aMDD subjects than in rMDD subjects. MDD patients showed significant correlations between β2*-nAChR availability and lifetime number of depressive episodes, trauma and anxiety scores. There were no differences in β2*-nAChR number between groups in the human postmortem study. Conclusion β2*-nAChR availability is decreased in patients with MDD. The difference between β2*-nAChR availability in vivo and in postmortem samples may be analogous to data with dopaminergic PET ligands and dopamine receptor availability; lower receptor availability for the SPECT ligand could be caused by increased endogenous acetylcholine.

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A set of 1091 human skeletal muscle cDNA clone inserts representing more than 800 human gene transcripts were spotted as PCR products at high density on nylon membranes. Replicas of the filters were hybridized in stringent conditions with 33P-radiolabeled cDNA probes transcribed from skeletal muscle poly(A)+ RNA. Hybridization signals were collected on phosphor screens and processed using a software specifically adapted for this application to identify and quantitate each spot. Parameters likely to influence the hybridization signal intensity were assessed to eliminate artifacts. Each clone was assigned to one of four intensity classes reflecting the steady-state level of transcription of the corresponding gene in skeletal muscle. Differential expression of specific gene transcripts was detected using complex cDNA probes derived from nine different tissues, allowing assessment of their tissue specificity. This made it possible to identify 48 novel gene transcripts (including 7 homologous or related to known sequences) with a muscle-restricted pattern of expression. These results were validated through the analysis of known muscle-specific transcripts and by Northern analysis of a subset of the novel gene transcripts. All these genes have been registered in the Genexpress Index, such that sequence, map, and expression data can be used to decipher their role in the physiology and pathology of human muscles.

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