Microdialysis of Lipophilic Compounds: A Methodological Study

Abstract: The influence of perfusion medium on in vitro recovery of ¹⁴C-oleate to microdialysis probes was investigated. Four perfusion media were investigated: water, 4% bovine serum albumine (BSA) in Ringer solution, 2.25% glycerol in water and a 20% soybeanoil/eggphospholipid emulsion (Intralipid^®). The following order of recovery was found (highest recovery first): BSA = Intralipid^® > glycerol > water. The recoveries at 0.5 μl/min. perfusion rate of the dialysis probe were 4.8%, 4.4%, 2.6% and 1.2% respectively. It was attempted to measure ¹⁴C-oleate after intravenous infusion. The samples were extracted with hexane/isopropylalcohol in order to separate the free fatty acid from products of oxidation which are hydrophilic. Dialysis probes were implanted in interscapular adipose tissue, epididymal fat, muscle, liver and jugular vein of rats. We were not able to detect unbound fatty acids, neither basally nor after stimulation with 1 mg/kg norepinephrine intraperitoneally. However, products of oxidation from the infused oleate were released in response to norepinephrine stimulation. It is concluded that the recovery of lipophilic compounds in microdialysis devices can be improved by means of a lipophilic perfusion medium or by means of e.g. BSA to which the compound binds. The free fatty acid levels were too small to be measureable in vivo in the present study.     

A Composite Model for the Transport of Hydrophilic and Lipophilic Compounds Across the Skin: Steady-State Behavior

A porous pathway feature has been added to an existing skin diffusion model to extend the range of applicability to highly polar solutes that do not readily diffuse across the stratum corneum (SC) lipid/corneocyte matrix. The porous pathway consists of 2 components: Pathway A is appendageal and is implemented as an array of aqueous shunts (the macropores), which themselves have microporous walls with transient aqueous pores (the micropores). Two varieties of shunts are discussed, one representing a terminal hair follicle and the other representing an eccrine sweat duct; however, the focus here is on the hair follicle. Pathway B is transcellular, with lipid-phase transport accomplished through defects or breaks in the bilayer lipid structure. The composite model admits polar solutes into the skin in a size-selective manner with an effective micropore radius of 1.6 nm. Steady-state permeabilities, desorption rates from isolated SC, and SC/water partition coefficients of both polar and lipophilic solutes are effectively explained.     

Estimating the Fraction Dose Absorbed from Suspensions of Poorly Soluble Compounds in Humans: A Mathematical Model

A microscopic mass balance approach has been developed to predict the fraction dose absorbed of suspensions of poorly soluble compounds. The mathematical model includes four fundamental di-mensionless parameters to estimate the fraction dose absorbed: initial saturation (Is), absorption number (An), dose number (Do), and dissolution number (Dn). The fraction dose absorbed (F) increases with increasing Is, An, and Dn and with decreasing Do. At higher Dn and lower Do, the fraction dose absorbed reaches the maximal F, which depends only on An. The dissolution number limit on F can appear at both lower Do and lower Dn. Likewise, at higher Do and Dn, the fraction dose absorbed reaches a Do limit. Initial saturation makes a significant difference in F at lower Do and Dn. It is shown that the extent of drug absorption is expected to be highly variable when Dn and Do are approximately one. Furthermore, by calculating these dimensionless groups for a given compound, a formulation scientist can estimate not only the extent of drug absorption but also the effect, if any, of particle size reduction on the extent of drug absorption.     

Generic Hockey-Stick Model for Estimating Benchmark Dose and Potency: Performance Relative to BMDS and Application to Anthraquinone

Benchmark Dose Model software (BMDS), developed by the U.S. Environmental Protection Agency, involves a growing suite of models and decision rules now widely applied to assess noncancer and cancer risk, yet its statistical performance has never been examined systematically. As typically applied, BMDS also ignores the possibility of reduced risk at low doses ("hormesis"). A simpler, proposed Generic Hockey-Stick (GHS) model also estimates benchmark dose and potency, and additionally characterizes and tests objectively for hormetic trend. Using 100 simulated dichotomous-data sets (5 dose groups, 50 animals/group), sampled from each of seven risk functions, GHS estimators performed about as well or better than BMDS estimators, and a surprising observation was that BMDS mis-specified all of six non-hormetic sampled risk functions most or all of the time. When applied to data on rodent tumors induced by the genotoxic chemical carcinogen anthraquinone (AQ), the GHS model yielded significantly negative estimates of net potency exhibited by the combined rodent data, suggesting that-consistent with the anti-leukemogenic properties of AQ and structurally similar quinones-environmental AQ exposures do not likely increase net cancer risk. In addition to its simplicity and flexibility, the GHS approach offers a unified, consistent approach to quantifying environmental chemical risk.     

The Temporal Dynamics of Ozone-Induced FEV1 Changes in Humans: An Exposure-Response Model

Although ozone is known to induce reversible decrements in forced expiratory volume in 1 s (FEV₁), no exposure-response model has been identified that accurately describes the dynamics of response to the changing concentrations and activity patterns of normal ambient human exposure. The purpose of the current analysis was to identify and evaluate a dynamic model of FEV₁ response using a large existing data set (541 volunteers, 864 exposures, 3485 FEV₁ measures) with a wide range of exposure conditions (ozone = 0.0 to 0.4 ppm, activity level = rest to heavy exercise, duration = 1 to 7.6 h), including recovery in clean air. A previously described model containing a differential equation and a logistic function was modified to include a new between-subjects variance structure and was fitted to the data. The model described well the mean observed response data across the range of exposure conditions, including the periods of recovery in clean air. Predicted values of individual responses were distributed lognormally and appeared to accurately describe the distribution of observed responses. We observed that responsiveness to ozone decreased with age, that response was weakly related to body size, and that response was marginally more sensitive to changes in ozone concentration than to changes in minute ventilation. In summary, we have identified a dynamic ozone exposure-response model that accurately describes the temporal pattern of FEV₁ response to a wide range of changing exposure conditions and that may have utility for predicting population responses to ambient exposures.     

A Model with Separate Hepato-Portal Compartment ("First-Pass" Model): Fitting to Plasma Concentration-Time Profiles in Humans

Purpose. To demonstrate the value of "first-pass" pharmacokinetic models (FPMs) in which the hepato-portal (HP) system is kinetically separated from the central compartment in fitting pharmacokinetic data obtained after intravenous (IV) and oral administration. Methods. Plasma concentration-time profiles of an investigational drug obtained in six healthy subjects each received 4 mg as an intravenous (IV) bolus dose and 10 mg as an oral solution served as a real data example. The common three- and four-compartment models with the first-order absorption and lag time (3CM and 4CM, respectively) in which HP system is assumed to be part of the central compartment were used as alternative models. We tested also: (i) the sensitivity of the output of FPM to variations in its parameters assuming IV and oral administration; (ii) practical estimability of the FPM parameters by fitting it to 20 simulated noisy data sets; (iii) distinguishability of FPM, 3CM and 4CM by fitting them to the simulated data sets. Results. FPM was shown to give the best fit as compared to 3CM or 4CM in 5 subjects of 6. The sensitivity of FPM was sufficient for the sake of parameter estimation. The "individual" means of parameter estimates obtained after fitting simulated data did not differ significantly from the preselected values. The variance in "individual" estimates was dependent on the sampling frequency. FPM was demonstrated to be distinguishable among relevant models. Conclusions. FPM is preferable as compared to standard compartmen-tal models for drugs extensively taken up by the intestine and/or the liver, and may have a broad spectrum of applications.     

Dissolution Testing for Generic Drugs: An FDA Perspective

In vitro dissolution testing is an important tool used for development and approval of generic dosage forms. The objective of this article is to summarize how dissolution testing is used for the approval of safe and effective generic drug products in the United States (US). Dissolution testing is routinely used for stability and quality control purposes for both oral and non-oral dosage forms. The dissolution method should be developed using an appropriate validated method depending on the dosage form. There are several ways in which dissolution testing plays a pivotal role in regulatory decision-making. It may be used to waive in vivo bioequivalence (BE) study requirements, as BE documentation for Scale Up and Post Approval Changes (SUPAC), and to predict the potential for a modified-release (MR) drug product to dose-dump if co-administered with alcoholic beverages. Thus, in vitro dissolution testing plays a major role in FDA's efforts to reduce the regulatory burden and unnecessary human studies in generic drug development without sacrificing the quality of the drug products.     

A Transition Model for Ordinal Response Data with Random Dropout: An Application to the Fluvoxamine Data

Many methods are available to analyze incomplete longitudinal ordinal responses. In this paper a general transition model is proposed for longitudinal ordinal responses with random dropout. Maximum likelihood estimates are obtained for the transition probabilities when there are repeated observations. The likelihood function of the general model is partitioned to make possible the use of existing software to estimate model parameters. Some reduced forms of the model are also considered where for estimation of parameters in these models one has to use numerical optimization methods. The approach is applied to the well-known Fluvoxamine data. For these data, two important results, which have not been previously reported, are obtained: (1) some transition probabilities are estimated to be zero and (2) the model for current response, which conditions on previous response, removes the effects of some covariates that had previously been significant.     

Persistent abnormalities in the rat mammary gland following gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure during gestation has revealed reproductive anomalies in rat offspring, including inconclusive reports of stunted mammary development in females (Brown et al., 1998, Carcinogenesis 19, 1623-1629; Lewis et al., 2001, TOXICOL: Sci. 62, 46-53). The current studies were designed to examine mammary-gland development in female offspring exposed in utero and lactationally to TCDD, and to determine a critical exposure period and cellular source of these effects. Long-Evans rats were exposed to 1 microg TCDD/kg body weight (bw) or vehicle on gestation day (GD) 15. TCDD-exposed females sacrificed on postnatal days (PND) 4, 25, 33, 37, 45, and 68 weighed significantly less than control litter mates, and peripubertal animals exhibited delayed vaginal opening and persistent vaginal threads, yet did not display altered estrous cyclicity. Mammary glands taken from TCDD-exposed animals on PND 4 demonstrated reduced primary branches, decreased epithelial elongation, and significantly fewer alveolar buds and lateral branches. This phenomenon persisted through PND 68 when, unlike fully developed glands of controls, TCDD-exposed rats retained undifferentiated terminal structures. Glands of offspring exposed to TCDD or oil on gestation days 15 and 20 or lactation days 1, 3, 5, and 10 were examined on PND 4 or 25 to discern that GD 15 was a critical period for consistent inhibition of epithelial development. Experiments using mammary epithelial transplantation between control and TCDD-exposed females suggested that the stroma plays a major role in the retarded development of the mammary gland following TCDD exposure. Our data suggest that exposure to TCDD prior to migration of the mammary bud into the fat pad permanently alters mammary epithelial development in female rat offspring.     

Development of a Sensitive Radioimmunoassay for Fab Fragments: Application to Fab Pharmacokinetics in Humans

Anti-sheep Fab fragment antisera were produced in rabbits using sheep digoxin-specific Fab fragments (Digidot) as immunogen. These antisera were used for the development of a radioimmunoassay (RIA) of sheep Fab fragments in human plasma and urine using ¹²⁵I-labeled Fab fragments. Interference in the assays by digoxin, human proteins, and antibodies from different species was insignificant, but cross-reactivity between anti-sheep Fab antisera and goat IgG or Fab fragments was 22 to 67%. The limit of detection was 0.1 µg/mL and the assay was linear over a 0.6–28 µg/mL range of Fab fragments. Intra- and interassay coefficients of variation were less than 6.9 and 10.5%, respectively. Accuracy of plasma and urine assays at various Fab fragment levels ranged from 96 to 106%. RIA was applied to the pharmacokinetic study of sheep digoxin-specific Fab fragments in one patient acutely intoxicated by digitoxin and treated with Digidot. The Fab elimination half-life was 12.1 hr. Steady-state volume of distribution and total-body clearance were 10.8 L and 23.4 mL/min, respectively. Unchanged Fab fragments (50 kD) and degradation products (25 kD) isolated by gel filtration chromatography of a urine sample cross-reacted with the anti-Fab antiserum.     

A Physiologically Based Pharmacokinetic Model of Mitoxantrone in Mice and Scale-up to Humans: a Semi-Mechanistic Model Incorporating DNA and Protein Binding

We conducted a pharmacokinetic (PK) study of mitoxantrone (Novantrone®), a clinically well-established anticancer agent, in mice and developed a mechanism-based PBPK (physiologically based pharmacokinetic) model to describe its disposition. Mitoxantrone concentrations in plasma and six organs (lung, heart, liver, kidney, spleen, and brain) were determined after a 5 mg/kg i.v. dose. We evaluated three different PBPK models in order to characterize our experimental data: model 1 containing Kp values, model 2 incorporating a deep binding compartment, and model 3 incorporating binding of mitoxantrone to DNA and protein. Among the three models, only model 3 with DNA and protein binding captured all the experimental data well. The estimated binding affinity for DNA (K_DNA) and protein (K_macro) were 0.0013 and 1.44 μM, respectively. Predicted plasma and tissue AUC values differed from observed values by <19 %, except for heart (60 %). Model 3 was further used to simulate plasma mitoxantrone concentrations in humans for a 12-mg/m² dose, using human physiological parameters. The simulated results generally agreed with the observed time course of mitoxantrone plasma concentrations in patients after a standard dose of 12 mg/m². In summary, we reported for the first time a mechanism-based PBPK model of mitoxantrone incorporating macromolecule binding which may have clinical applicability in optimizing clinical therapy. Since mitoxantrone is a substrate of the efflux transporters ABCG2 and ABCB1, the incorporation of efflux transporters may also be necessary to characterize the data obtained in low-dose studies.KEY WORDS: DNA binding, human scale-up, mitoxantrone, physiologically based pharmacokinetic model     

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 CO₂ 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% 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.     

TCDD adsorbed on silica as a model for TCDD contaminated soils: Evidence for suppression of humoral immunity in mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the prototypical aryl hydrocarbon receptor (AhR) ligand, exhibits immune suppression in vivo and in vitro. Suppression of primary humoral immune responses in particular has been well characterized as one of the most sensitive functional immune endpoints in animals treated with TCDD. Previous studies have used purified TCDD to elucidate the mechanisms by which TCDD and dioxin-like compounds (DLC) impair IgM production by B cells, but did not represent the route by which animals and humans are likely to be exposed environmentally. In the studies reported here, mice were treated with TCDD adsorbed onto a well-defined synthetic silica phase of known purity and physical properties, followed by sensitization with sheep erythrocytes to initiate a humoral immune response. We found that surfactant-templated mesoporous forms of amorphous silica provided an ideal combination of purity, dispersibility and textural properties for immobilizing TCDD. TCDD-adsorbed silica distributed to the spleen and liver after oral administration as assessed by induction of cyp1a1 gene expression. Most notably, TCDD delivered in the adsorbed state on amorphous silica and as a solute in corn oil (CO) produced similar suppression of the anti-sheep red blood cell immunoglobulin M antibody forming cell (sRBC IgM AFC) response at equivalent doses of TCDD. These results suggest that TCDD immobilized on silicate particles found in soils distributes to the spleen and suppresses humoral immunity.     

A Compartment Model for the Membrane-Coated Fiber Technique Used for Determining the Absorption Parameters of Chemicals into Lipophilic Membranes

PURPOSE: The purpose of this work was to develop a compartment model for the membrane-coated fiber (MCF) technique for determining the absorption parameters of chemicals into lipophilic membranes. METHODS: A polymer membrane coated onto a section of inert fiber was used as a permeation membrane in the MCF technique. When MCFs were immersed into a donor solution, the compounds in the solution partitioned into the membrane. At a given permeation time, a fiber was removed from the solution and transferred into a gas chromatography injector for quantitative analysis. The permeation process of a given chemical from the donor phase into the membrane was described by a one-compartment model by assuming first-order kinetics. RESULTS: A mathematical model was obtained that describes the cumulative amount of a chemical permeated into the membrane as a function of the permeation time in an exponential equation. Two constants were introduced into the compartment model that were clearly defined by the physiochemical parameters of the system (a kinetic parameter and the equilibrium absorption amount) and were obtained by regression of the experimental data sampled over a limited time before equilibrium. The model adequately described the permeation kinetics of the MCF technique. All theoretical predictions were supported by the experimental results. The experimental data correlated well with the mathematical regression results. The partition coefficients, initial permeation rate, uptake, and elimination rate constants were calculated from the two constants. CONCLUSIONS: The compartment model can describe the absorption kinetics of the MCF technique. The regression method based on the model is a useful tool for the determination of the partition coefficients of lipophilic compounds when it takes too long for them to reach permeation equilibrium. The kinetic parameter and the initial permeation rate are unique parameters of the MCF technique that could be used in the development of quantitative structure-activity relationship models.     

The Use of Human Hepatocytes to Select Compounds Based on Their Expected Hepatic Extraction Ratios in Humans

Purpose. The present investigation retrospectively evaluates the use of human hepatocytes to classify compounds into low, intermediate or high hepatic extraction ratio in man. Methods. A simple approach was used to correlate the in vivo hepatic extraction ratio of a number of compounds in man (literature and in-house data) with the corresponding in vitro clearance which was determined in human hepatocytes. The present approach assumes that, for compounds eliminated mainly through liver metabolism, intrinsic clearance is the major determinant for their in vivo hepatic extraction ratio and subsequently their bioavailability in man. The test compounds were selected to represent a broad range of extraction ratios and a variety of metabolic pathways. Results. The present data show that in vitro clearances in human hepatocytes are predictive for the hepatic extraction ratios in vivo in man. Most of the test compounds (n = 19) were successfully classified based upon human hepatocyte data into low, intermediate or high hepatic extraction compounds, i.e. compounds with potential for high, intermediate or low bioavailabilities in humans. Conclusions. The present approach, validated so far with 19 test compounds, appears to be a valuable tool to screen for compounds with respect to liver first-pass metabolism at an early phase of drug discovery.     

Determining Intestinal Metabolism and Permeability for Several Compounds in Rats. Implications on Regional Bioavailability in Humans

Purpose. To investigate the regional differences in small intestinal (SI) metabolism and permeability for several compounds and to ascertain the potential significance of these differences on the reported reductions in regional bioavailability in humans. Methods. The regional SI metabolism and permeability of captopril, didanosine (ddl), mannitol, ofloxacin and zidovudine (ZDV) were investigated in rats using a Single Pass Intestinal Perfusion (SPIP) procedure or intestinal homogenates. Results. ddI was metabolized to a greater extent in the upper SI whereas captopril was metabolized to a greater extent in the lower SI. Relatively low homogenate concentrations resulted in significant degradation of captopril in the upper and lower SI. All other compounds were stable and changes in the buffer system or the initial concentration did not affect the results. The SI permeabilities of all compounds, with the exception of mannitol, decreased linearly with respect to SI location and the slopes of the corresponding normalized regression lines were not significantly different. Conclusions. It has been reported that captopril and ddl demonstrate regional intestinal bioavailability in several species including humans. The current results suggest that the reported reduction in the lower SI bioavailability of captopril may be a result of a reduction in permeability and an increase in intestinal metabolism whereas for ddl, the reduction in the lower SI bioavailability appears to be attributable to a reduction in intestinal permeability. Other factors such as luminal metabolism may also significantly effect regional differences in the intestinal bioavailability of ddl or captopril. Based on these results, a strong possibility exists that ofloxacin and ZDV may also demonstrate regional differences in intestinal bioavailability.     

Lipophilic Conjugates of Drugs: A Tool to Improve Drug Pharmacokinetic and Therapeutic Profiles

Pharmaceutical Research - Lipophilic conjugates (LCs) of small molecule drugs have been used widely in clinical and pre-clinical studies to achieve a number of pharmacokinetic and therapeutic...     

Persistent suppression of contact hypersensitivity, and altered T-cell parameters in F344 rats exposed perinatally to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The outcome of perinatal low-level TCDD exposure on the T-cell-mediated contact hypersensitivity (CHS) response in adult F344 rats was investigated. Suppression of the 2,4-dinitrofluorobenzene (DNFB)-specific contact hypersensitivity reponse occurred in mature offspring of dams dosed by gavage with 1microg or 3microg TCDD/kg on gestation day (GD) 14. To determine if this effect was correlated with altered distribution or activation of major T-cell subtypes, cells of the auricular lymph node draining the hapten-treated skin were evaluated by flow cytometry for expressed phenotype, including activation markers, 24h after challenge. Six-month-old female offspring with significantly decreased CHS and born to dams given 3microg TCDD/kg, had significantly greater proportion of CD4(+) T cells expressing a naive phenotype marker, CD45RC(hi), in their draining nodes. The greater relative frequency of this CD4(+) subset in peripheral lymphoid tissues associated with a reduced CHS in these rats may be attributed to a reduction in the proportion of CD4(+) T cells maintaining or recruited into an activated state. The CHS proved to be a valuable bioassay for investigating long-term immunotoxic effects of perinatal TCDD exposure in rats.