Passive drug permeation through membranes and cellular distribution.

Although often overlooked, passive mechanisms can lead to significant accumulation or restriction of drugs to intracellular sites of drug action. These mechanisms include lipoidal diffusion of ionized species and pH partitioning according to the electrochemical potential and to pH gradients that exist across subcellular compartments, respectively. These mechanisms are increasingly being exploited in the design of safe and effective drugs for the treatment of a wide variety of diseases. In this work, the authors review these efforts and the associated passive mechanisms of cellular drug permeation. A generic mathematical model of the cell is provided and used to illustrate concepts relevant to steady-state intracellular distribution. Finally, the authors review methods for estimating determinant parameters and measuring the net effect at the level of unbound intracellular drug concentrations.

Comparison of in vitro BBMEC permeability and in vivo CNS uptake by microdialysis sampling.

The studies presented in this report were designed to assess the correlation of the bovine brain microvessel endothelial cell (BBMEC) apparent permeability coefficient (P(app)) and in vivo BBB penetration using microdialysis sampling. A mathematical model was developed to describe the relationship of brain extracellular fluid (ECF) concentration to free drug in plasma. The compounds studied have a broad range of physico-chemical characteristics and have widely varying in vitro and in vivo permeability across the blood-brain barrier (BBB). BBMEC permeability coefficients vary in magnitude from a low of 0.9 x 10(-5) cm/s to a high value of 7.5 x 10(-5) cm/s. Corresponding in vivo measurements of BBB permeability are represented by clearance (CL(in)) into the brain ECF and range from a low of 0.023 microl/min/g to a high of 12.9 microl/min/g. While it is apparent that in vitro data from the BBMEC model can be predictive of the in vivo permeability of a compound across the BBB, there are numerous factors both prior to and following entry into the brain which impact the ultimate uptake of a compound. Even in the presence of high BBB permeability, factors such as high plasma protein binding, active efflux across the BBB, and metabolism within the CNS can greatly limit the ultimate concentrations achieved. In addition, concentrations in the intracellular space may not be the same as concentrations in the extracellular space. While these data show that the BBMEC permeability is predictive of the in vivo BBB permeability, the complexity of the living system makes prediction of brain concentrations difficult, based solely on the in vitro measurement.

Evaluation of the BBMEC model for screening the CNS permeability of drugs.

Combinatorial synthesis and high-throughput pharmacology screening have greatly increased compound throughput in modern drug-discovery programs. For CNS drugs, it is also important to determine permeability to the blood--brain barrier. Yet, given the increased pace of discovery, it difficult to conduct this screen in a timely fashion. In this presentation, we describe several improvements to an existing CNS permeability screen, the bovine brain microvessel endothelial cell (BBMEC) model. By implementation of these incremental process improvements, we have achieved a robust, facile screen for determination of CNS permeability of multiple compounds.

Investigation of the CNS penetration of a potent 5-HT2a receptor antagonist (MDL 100,907) and an active metabolite (MDL 105,725) using in vivo microdialysis sampling in the rat.

MDL 100,907 is a selective 5-HT2a receptor antagonist which is currently being developed for the treatment of schizophrenia. Pharmacokinetic studies of MDL 100,907 in rats and dogs show that the drug is well absorbed but undergoes extensive first-pass metabolism to an active metabolite (MDL 105,725). The purpose of this study was to determine concentrations of MDL 100,907 and MDL 105,725 in the brain extracellular fluid (ECF) after administration of MDL 100,907. In vivo microdialysis sampling was used to determine the brain penetration of both parent (MDL 100,907) and metabolite (MDL 105,725). Animals (n = 3/dose) were given 5 i.v. and 50 mg kg-1 oral doses of MDL 100,907. Brain medial prefrontal cortex (mPFC) ECF concentrations were determined using microdialysis and plasma levels were determined by collecting blood samples through an indwelling cannula implanted in the jugular vein. Dialysate samples were analyzed using an LC/MS/MS assay. The data presented in this report show that the blood brain barrier (BBB) permeability of MDL 100,907 is more than four times (4x) that of MDL 105,725 and that MDL 100,907 does not undergo significant metabolism to MDL 105,725 in the brain. It appears, from the data presented, that MDL 100,907 is the predominant active species present in the brain at high doses.

Quantification of a dual angiotensin I-converting enzyme-neutral endopeptidase inhibitor and the active thiol metabolite in dog plasma by high-performance liquid chromatography with ultraviolet absorbance detection.

MDL 100,240 ([4S-[4 alpha,7 alpha(R*), 12b beta]]-7-[[2- (acetylthio)-1-oxo-3-phenylpropyl]amino]-1,2,3,4,6,7,8,12b-octahyd ro-6-oxo- pyrido[2,1-a][2]benzazepine-4-carboxylic acid, I) is the thioacetyl prodrug of the active thiol, MDL 100,173 (II), a dual inhibitor of angiotensin-I converting enzyme (ACE) and neutral endopeptidase (NEP). A drug which simultaneously inhibits both ACE and NEP may provide a unique therapy for hypertension and congestive heart failure. Methods based on high-performance liquid chromatography with UV absorbance detection at 200 nm were developed to support preclinical pharmacokinetic investigations. One method is used to measure unchanged I and free II, while the second method is used to quantify the total level of the thiol II after the plasma is incubated with the disulfide reducing agent, dithiothreitol. By either method, the analytes are quantified over the range of 25-1000 ng/ml with good accuracy and precision. The overall extraction efficiencies of unchanged I and free II in dog plasma were 79% and 86%, respectively, while the extraction efficiency of total II averaged 75%. Described in this report are the results obtained in validating the assay methods for measuring the compounds in plasma. Pharmacokinetic data are presented which were obtained by applying these methods to plasma collected from dogs dosed with I.

Urinary and biliary disposition of the lactone and carboxylate forms of 20(S)-camptothecin in rats.

Recently, analytical methods have become available for determination of both the lactone (active form) and the carboxylate (inactive form) forms of 20(S)-camptothecin in biological fluids. Studies in our laboratory have shown that there are significant differences in the in vivo behavior of the two forms of camptothecin and that much higher plasma levels of the lactone form are present in rats after dosing with camptothecin (lactone) than after dosing with the sodium salt of the ring-opened camptothecin (carboxylate form). The present studies show that there are significant differences in the urinary and biliary elimination of the two forms and that the urinary excretion of the carboxylate form appears to be pH dependent. This apparent pH dependence of the urinary elimination of the carboxylate form may provide a method of reducing the bladder toxicity associated with the use of camptothecin. After administration of a 1 mg/kg iv dose of camptothecin (lactone) to rats, 10.1 +/- 4.2% of the dose was excreted into the urine and 7.5 +/- 4.2% of the dose was excreted into the bile. Following an equivalent intravenous dose of the carboxylate form, 39.5 +/- 10.4% of the dose was excreted into the urine and 26.4 +/- 8.9% of the dose was excreted into the bile.

Plasma pharmacokinetics of lactone and carboxylate forms of 20(S)-camptothecin in anesthetized rats.

20(S)-Camptothecin exists in equilibrium between its lactone (CPT) and its carboxylate forms (Na-CPT) under stimulated physiological conditions, with the equilibrium favoring the carboxylate form. The rates of lactone hydrolysis were studied in plasma, serum albumin, and blood and were found to be faster than in aqueous buffers at equivalent pH values. From mechanistic information and in vivo activity data, the lactone appears to be the active form of the drug. It has been argued, therefore, that if an equilibrium existed between the lactone and the carboxylate, Na-CPT could be used to deliver the lactone effectively. In the present study, plasma pharmacokinetics were performed in sodium pentobarbital-anesthetized rats treated with both CPT (lactone) and the sodium salt of camptothecin (carboxylate, Na-CPT) and the lactone and carboxylate, as well as the total drug, concentration versus time profiles were assessed. It was found that plasma concentrations and AUC values for the lactone were significantly higher after dosing with CPT than after dosing with Na-CPT. After i.v. administration, the ratio of plasma lactone to carboxylate was skewed by the apparent rapid and extensive uptake of the lactone into tissues and the rapid clearance of both species. From our results, it appears that the lower in vivo activity of Na-CPT compared to that from CPT administration might be attributed to the altered conversion of carboxylate into lactone in vivo compared to that predicted from in vitro data.

In vivo microdialysis sampling in the bile, blood, and liver of rats to study the disposition of phenol.

Methods for continuous in vivo sampling in the bile, blood, and liver extracellular fluid are described. These methods are based on microdialysis sampling in anesthetized rats. A new flow-through microdialysis probe is described for sampling bile while maintaining normal bile flow. All three sites are simultaneously and continuously sampled to provide concentration-time profiles at multiple sites in a single experimental animal. This technique is demonstrated by studying the hepatic metabolism and biliary excretion of phenol in rats. Following an i.v. infusion of phenol, the major hepatic metabolite was found to be phenyl-glucuronide. Hydroquinone and 2-glutathionyl-hydroquinone were also detected but at lower concentrations. A similar pattern of metabolites was found in the bile and blood. For all of the metabolites, bile concentrations are higher than liver concentrations, indicating that the metabolites are actively excreted into the bile.

Enzymatic formation and electrochemical characterization of multiply substituted glutathione conjugates of hydroquinone.

1,4-Benzoquinone can undergo redox cycling in the presence of glutathione to produce multiply substituted products. It has previously been shown that the nephrotoxicity of the hydroquinone-glutathione conjugates increases with increasing substitution. However, based on chromatographically-assisted hydrodynamic voltammetry (CA-HDV), the oxidation potential was shown to apparently increase which, should lead to decreased toxicity. From the chemical formation of multiply substituted hydroquinone-glutathione conjugates from benzoquinone and glutathione, it is clear that the thermodynamic oxidation potential must decrease as substitution increases. This was confirmed by cyclic voltammetric (CV) characterization of the isolated conjugates. The discrepancy between the CV and CA-HDV data apparently results from kinetic factors arising from differences in the treatment of the electrode surface between the two experiments. The multiply substituted hydroquinone-glutathione conjugates were also produced in horseradish peroxidase incubations containing hydroquinone and glutathione. These products were identified chromatographically, spectrophotometrically, and electrochemically. The increasing ease of oxidation and the possible enzymatic formation of multiply substituted hydroquinone-glutathione conjugates indicates that this pathway may occur in vivo and contribute to the toxicity of quinones.

Intravenous microdialysis sampling in awake, freely-moving rats.

Intravenous microdialysis sampling in the awake, freely-moving rat for the determination of free drug concentrations in blood is described. Intravenous microdialysis was performed with a nonmetallic, flexible dialysis probe. The pharmacokinetics of theophylline were determined using both microdialysis sampling and collection of whole blood following an iv dose. There was no difference in the half-life of elimination of theophylline determined by microdialysis, 4.4 +/- 0.4 h, and whole blood sampling, 4.5 +/- 0.7 h. The kinetics of elimination were affected by removing blood samples and by using anesthesia. The half-life of elimination was 4.4 +/- 0.4 h when using simultaneous microdialysis and whole-blood sampling and only 3.0 +/- 0.4 h using microdialysis alone. The half-life of elimination was 17.0 +/- 7.1 h in chloral hydrate anesthesized rats. Microdialysis samples were continuously collected for over 7 h without fluid loss using a single experimental animal. Microdialysis sampling directly assesses the free drug concentration in blood. The extent of theophylline binding to blood proteins was determined in vitro in rat plasma and rat whole blood using both ultrafiltration and microdialysis. Theophylline was (47.3 +/- 1.3)% bound in rat plasma and (52.2 +/- 1.6)% bound in rat whole blood. Microdialysis sampling is a powerful tool for pharmacokinetic studies, providing accurate and precise pharmacokinetic data.

In vivo microdialysis sampling for pharmacokinetic investigations.

In vivo microdialysis sampling coupled to liquid chromatography was used to study acetaminophen disposition in anesthetized rats. The pharmacokinetics of acetaminophen and its sulfate and glucuronide metabolites were determined using both microdialysis sampling and collection of whole blood. For microdialysis, samples were continuously collected for over 5 hr without fluid loss using a single experimental animal. Microdialysis sampling directly assesses the free drug concentration in blood. The pharmacokinetic results obtained with microdialysis sampling were the same as those obtained from blood collection. The administration of heparin, necessary when collecting blood samples, was found to double the elimination half-life of acetaminophen. Microdialysis sampling is a powerful tool for pharmacokinetic studies, providing accurate and precise pharmacokinetic data.

Microdialysis sampling for determination of plasma protein binding of drugs.

The use of microdialysis sampling to study the binding of drugs to plasma proteins was evaluated. Microdialysis sampling is accomplished by placing a short length of dialysis fiber in the sample and perfusing the fiber with a vehicle. Small molecules in the sample, such as drugs, diffuse into the fiber and are transported to collection vials for analysis. Larger molecules, such as proteins and protein-bound drugs are excluded by the dialysis membrane. Microdialysis was found to give values for in vitro protein binding in plasma equivalent to those determined by ultrafiltration. Microdialysis offers advantages in terms of maintaining equilibria and experimental versatility. Microdialysis sampling also provides potential use for in vivo determinations of protein binding.

In vivo microdialysis sampling coupled to liquid chromatography for the study of acetaminophen metabolism.

In vivo microdialysis sampling coupled to liquid chromatography is a powerful tool for the study of drug metabolism. This technique is illustrated by investigating the pharmacokinetics of acetaminophen in the blood and liver of an anesthetized rat. The pharmacokinetics of the sulfate and glucuronide metabolites as well as the parent acetaminophen can be determined with high precision using microdialysis sampling. Microdialysis samples can be collected at a high rate from several sites without fluid loss with a single animal. Because the animal serves as its own control better data can be obtained. Liquid chromatography provides determination of multiple analytes per sample for metabolic profiling. This technique will provide more accurate and precise pharmacokinetic data while requiring fewer animals.

Identification of 9-hydroxylamine-1,2,3,4-tetrahydroacridine as a hepatic microsomal metabolite of tacrine by high-performance liquid chromatography and electrochemistry.

Amperometric detection using a dual-electrode thin-layer cell in the series configuration can aid in the identification of unknown components in complicated samples by voltammetric characterization. This is shown by studying the metabolism of tacrine by rat hepatic microsomes using high-performance liquid chromatography with electrochemical detection. The major metabolite detected in microsomal incubations did not co-elute with any standard acridine available and was produced in too small a quantity for mass spectral characterization. Tentative identification of this metabolite as 9-hydroxylamine-1,2,3,4-tetrahydroacridine was made by electrochemical characterization. The electrochemistry of the metabolite was compared to that of the hydroxylamine produced and studied by cyclic voltammetry.

Microdialysis-perfusion sampling for the investigation of phenol metabolism.

In vivo methods provide several advantages for the study of metabolism relative to the commonly used in vitro techniques. The integrity of the organism and actual physiological conditions are maintained to reflect more accurately the processes occurring on exposure to a xenobiotic compound. Experimental precision is improved because each animal serves as its own control and can be used to generate a complete pharmacokinetic experiment. This may result in the added benefit that fewer experimental animals will be needed for a metabolic investigation using in vivo techniques. The technique of microdialysis perfusion was characterized for the in vivo study of the hepatic metabolism of phenol and conjugation by glutathione. In this study, in vivo experiments were conducted by implanting a microdialysis probe into the intact, in-place liver of a killed rat. These results were compared to in vitro experiments using liver homogenate and liver-microsomal protein. Substantial differences were observed between the in situ experiments and those performed in vitro.