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Abstract: At the drug discovery stage, in vivo metabolic hepatic clearance (CLhep) is commonly predicted using in vitro parent compound disappearance data generated in liver microsomes or hepatocytes. Correction for the unbound fraction of a compound in the in vitro system and in plasma/serum is known to be critical for the accuracy of metabolic clearance predictions. Discrete generation of these required experimental parameters can be laborious. Herein, we describe a straightforward and direct approach to obtain the ratio of unbound fraction in plasma (fu p) to unbound fraction in the microsomal system (fu mic) of a small molecule compound using equilibrium dialysis. Experimental conditions were optimized with respect to incubation time, temperature, and plate shaking speed. Results obtained from this system were validated for a set of test compounds by comparison to individually measured fu p and fu mic data using ultracentrifugation. The correlation for fu p/fu mic between the two methods for a set of 23 data points was very good with R 2 of 0.94, slope of 1.05 and an intercept of 0.007. The impact of microsomal binding on predicted CLhep was illustrated for a tightly bound compound using a series of incubations with increasing concentration of monkey liver microsomal protein. Alteration of this experimental parameter profoundly affected calculated CLhep using the well-stirred model. Significant differences were observed in the prediction when the model was corrected for fu p only; in contrast, the model corrected for plasma protein and microsomal protein binding predicted clearance values independent of the microsomal protein concentration. [ABSTRACT FROM AUTHOR]

Although large animals, such as dogs and nonhuman primates, often are used for more than 1 pharmacokinetics study, common practice is to use only naive rodents for pharmacokinetics studies. We undertook a series of studies to validate whether surgically cannulated nonnaive rats could be used again after a 7-d washout. When vascular catheters are cared for appropriately, we find that they remain patent for more than 2 wk, with negligible drug carryover. Hematocrit decreased approximately 11% after pharmacokinetics studies but rebounded to prestudy levels after a 7-d washout. We empirically tested whether drugs known to alter drug disposition (1-aminobenzotriazole and quinidine) had residual effects on drug disposition after a 7-d washout and found that they did not. This finding suggests that after a 7-d washout, nonnaive rats likely would produce pharmacokinetics data similar to those of naive rats. We also tested reference compounds in naive and nonnaive rats and found no difference in pharmacokinetics parameters. Using surgically cannulated rats for a second study was feasible because of the relatively noninvasive nature of pharmacokinetics sampling (unrestrained rats attached to automated blood samplers). In addition, reusing surgically altered animals yields considerable cost savings. Our studies indicate that pharmacokinetics parameters did not differ significantly between naive and nonnaive rats. Cost-benefit analysis, monetary considerations, and validation studies support using rats for a second study after a 7-d washout period.

Levo-alpa-acetylmethadol (LAAM) is a methadone derivative used to treat the opiate addict. We previously reported on the kinetics for transplacental transfer of LAAM and its levels in the fetal circuit using the technique of dual perfusion of the placental lobule. The aim of this investigation was to identify the enzyme responsible for the biotransformation of LAAM and norLAAM and the metabolites formed in the term human placenta. Placental microsomes exhibited higher activities than the mitochondrial and cytosolic fractions in metabolizing LAAM to norLAAM. None of these subcellular fractions catalyzed the formation of dinorLAAM from either LAAM or norLAAM as determined by HPLC/UV. Evidence obtained from the effects of cytochrome P450 (CYP) inhibitors on the demethylation of LAAM to norLAAM by placental microsomes suggested that CYP 19/aromatase is the major enzyme involved. Out of 10 monoclonal antibodies raised against various CYP isoforms, only that for aromatase caused over 80% inhibition of norLAAM formation. The biotransformation of LAAM to norLAAM exhibited monophasic kinetics with apparent Km and Vmax values of 105 +/- 57 microM and 86.8 +/- 15.6 pmol mg(-1) protein min(-1), respectively. The kinetic profile determined for a cDNA-expressed CYP 19 metabolism of LAAM to norLAAM was similar to that determined for placental microsomes. Taken together, the above data indicate that CYP 19/aromatase is the enzyme responsible for the N-demethylation of LAAM to norLAAM in term human placentas obtained from healthy pregnant women.

The agonists buprenorphine and l-alpha-acetylmethadol (LAAM) were introduced as alternatives to methadone for treatment of the adult opiate addict. The direct and indirect effects of these drugs on normal fetal growth and development are currently under investigation in our laboratory. The goal of this report is to provide part of the data necessary to assess the safety of LAAM in treatment of the pregnant opiate addict. To achieve this goal, the technique of dual perfusion of placental lobule was utilized to determine the kinetics for transplacental transfer of LAAM and its effects on the viability and functional parameters of the tissue. LAAM is rapidly metabolized to the pharmacologically active norLAAM that was also included in this investigation. The two opiates were transfused at their plasma levels in patients under treatment, a concentration of 35 ng/ml. The drugs exhibited similar pharmacokinetic profiles, characterized by an initial phase of distribution into placental tissue followed by their low transfer to the fetal circuit. During the 4-h experimental period, the transfused tissue retained significant amounts of LAAM and norLAAM, and neither drug was metabolized. LAAM did not affect placental tissue viability and functional parameters. However, norLAAM caused a significant decrease in the release of human chorionic gonadotropin. At this time, it is unclear whether a similar effect for norLAAM may occur in vivo and, if so, what the consequences would be on its role in implantation and normal fetal growth and development.