Percutaneous and Systemic Disposition of Hexamethylene Lauramide and Its Penetration Enhancement Effect on Hydrocortisone in a Rat Sandwich Skin-Flap Model

The percutaneous absorption and distribution profile of hexamethylene lauramide (hexahydro-1-lauroyl-lH-azepine) were examined using a rat skin-flap model. After a topical dose to the skin flap, the drug concentrations in the vasculature at the site of drug application and in the systemic blood were monitored simultaneously. Hexamethylene lauramide penetrated the skin and reached a steady state in stratum corneum, viable epidermis, dermis, and cutaneous blood in 3 hr. Its concentration in the skin was much higher than that in the blood. Its apparent concentration in the epidermis was 19 times that in the dermis and about 3000 times that in the cutaneous blood. The percutaneous absorption of ¹⁴C-hexamethylene lauramide resulted in ascending systemic blood concentrations throughout the experimental period, whereas the cutaneous blood levels remained steady. The topically absorbed hexamethylene lauramide was quantitatively recovered in urine (85%) and feces (13%). The half-lives of urinary and fecal excretion of ¹⁴C-hexamethylene lauramide were 17 and 30 hr, respectively. Hexamethylene lauramide, when topically coadministered in an experimental formulation, enhanced the skin penetration of hydrocortisone with increased drug contents in the stratum corneum (2-fold) and with increased hydrocortisone concentrations in the cutaneous blood (3.4-fold) and the systemic blood (3.5-fold). The results indicated that the high concentration and retention of hexamethylene lauramide in stratum corneum and viable epidermis may contribute to its penetration enhancement effect in the skin. A steady state in percutaneous tissues was observed before the drug reached distribution equilibrium systemically. The systemic blood concentration of a topically applied agent therefore may not reflect its percutaneous kinetic processes before a systemic distribution equilibrium is reached. Temporal profiles of a topical penetration enhancer in the skin and in the body are important information for the development of dematologic preparations for the treatment of skin disorders.     

Disposition and Metabolic Profiling of the Penetration Enhancer Azone. I. In Vivo Studies: Urinary Profiles of Hamster,Rat, Monkey,and Man

Chain-labeled ¹⁴C-Azone was intravenously administered to hamster, monkey, and rat, to compare its metabolic profile with that obtained previously in humans after dermal application. Azone-derived radioactivity was excreted predominantly in the urine for both hamster and monkey, which is similar to the disposition in humans. Metabolic profiling in urine revealed extensive systemic metabolism to occur in all species studied. The main fraction of the metabolites was most polar in man, followed by rat, monkey, and hamster. Traces of the parent compound were detectable only in hamster urine. Although some of the polar major human metabolites were also present in rat urine, the animals were unsuitable for collecting metabolites of Azone observed in humans. In rats, complete cleavage of the dodecyl side chain was ruled out by administering Azone that had been labeled at two distinct positions of the molecule. Additionally, oral administration of Azone to rats resulted in the same metabolic profile as intravenous administration, indicating that gastrointestinal metabolism does not occur or is similar to systemic metabolism.     

Phonophoresis of hydrocortisone with enhancers: an acoustically defined model

The aim of the study was to define an acoustic model to determine the effect of ultrasound on the penetration of hydrocortisone through whole rat skin. Methods: ultrasound dosimetry measurements were used to define an ultrasound source used to measure the phonophoretic enhancement of hydrocortisone transport through rat skin in vitro. The effect of conductive heating was also studied. Results: acoustic dosimetry measurements indicated that the skin barrier was exposed to ultrasound standing waves and this focused heat generation within the tissue. While sonication alone did not significantly enhance hydrocortisone permeation, a significant synergistic effect was observed with Azone but not with oleic acid. The ultrasound-Azone effect could be duplicated with conductive heating. Conclusion: synergism between phonophoresis and Azone treatment was observed in the enhancement of hydrocortisone percutaneous transport. It is probable that the thermal effects of ultrasound were responsible for accelerated Azone diffusion through the stratum corneum.     

Comparative Analysis of Percutaneous Absorption Enhancement by d-Limonene and Oleic Acid Based on a Skin Diffusion Model

Percutaneous absorption-enhancing effects of d-limonene and oleic acid were investigated using three model drugs with different lipophilicities in in vitro diffusion experiments with guinea pig skin. Pretreatment of the skin with d-limonene resulted in a large penetration enhancement for the lipophilic butylparaben (BP) and amphiphilic 6-mercaptopurine (6-MP) but had little effect on the hydrophilic mannitol (MT). Oleic acid caused a large effect only on 6-MP penetration. The penetration profiles were analyzed with a two-layer skin diffusion model consisting of stratum corneum with polar and nonpolar routes and viable epidermis plus dermis. Through curve-fitting, six parameters corresponding to drug diffusivity and partitioning in these three regions of the skin were obtained, and the mechanisms of enhancers were assessed in comparison with those of l-geranylazacycloheptan-2-one (GACH) reported previously. Increased penetration was caused mainly by modification of the barrier property of the nonpolar route in the stratum corneum in all cases. In the nonpolar route, d-limonene increased mainly drug diffusivity, while GACH enhanced predominately drug partitioning. On the other hand, oleic acid moderately increased both parameters.     

The Impact of Lag Time on the Estimation of Pharmacokinetic Parameters. I. One-Compartment Open Model

Lag time in pharmacokinetics corresponds to the finite time taken for a drug to appear in systemic circulation following extravascular administration. Lag time is a reflection of the processes associated with the absorption phase such as drug dissolution and/or release from the delivery system and drug migration to the absorbing surface. Failure to specify the lag time can lead to inappropriate or erroneous estimates of pharmacokinetic parameters. This has been demonstrated in the case of a one-compartment open model by the pharmacokinetic analysis of bioequivalence data from a study involving the administration of propoxyphene napsylate to human volunteers. Subsequently, pharmacokinetic and statistical analyses of data obtained from a series of 49 simulations involving a wide range of absorption and elimination rate constants (0.05 to 5.00 and 0.01 to 0.95 hr^–1, respectively) showed that lag time has a substantial effect on several primary and secondary pharmacokinetic parameters.     

Kinetic Analysis on the Skin Disposition of Cytotoxicity as an Index of Skin Irritation Produced by Cetylpyridinium Chloride: Comparison of In Vitro Data using a Three-Dimensional Cultured Human Skin Model with In Vivo Results in Hairless Mice

PURPOSE: The aim of this study was to kinetically and dynamically analyze in vitro cytotoxicity as an index of skin irritation by use of a three-dimensional cultured human skin model and to compare the in vitro assay data with data from living animals. METHODS: A cationic surfactant, cetylpyridinium chloride (CPC), was selected as a model irritant. Living skin equivalent-high (LSE-high) and hairless mice were used for the in vitro and in vivo tests, respectively. Skin irritation dermatodynamics was evaluated by calorimetric thiazoyl blue (MTT) conversion assay both for in vitro and in vivo tests, whereas dermatokinetics of CPC in LSE-high and mouse skin were evaluated using HPLC. RESULTS: The time course of cell viability in the skin after application of CPC to intact skin was distinctly different from that of stratum-corneum-stripped skin in both LSE-high and hairless mice. Biphasic behavior characterized by two first-order rates with an inflection time point was observed in intact skin, whereas cell viability monoexponentially decreased immediately after CPC application in stripped skin. The time courses of cell viability in the skin and dermatodynamics were closely related to that of dermatokinetics of CPC. CONCLUSION: The present study demonstrates that the in vitro cytotoxic profile was similar to the in vivo cytotoxicity test and that dermatodynamics was related to dermatokinetics of CPC.     

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.     

In Vivo and in Vitro Analysis of Skin Penetration Enhancement Based on a Two-Layer Diffusion Model with Polar and Nonpolar Routes in the Stratum Corneum

In vitro and in vivo skin penetration of three drugs with different lipophilicities and the enhancing effects of l-geranylazacycloheptan-2-one (GACH) were studied in rats. In vivo drug absorption profiles obtained by deconvolution of urinary excretion profiles were compared to the corresponding in vitro data obtained with a diffusion experiment. In vivo skin penetration of lipophilic butylparaben was considerably greater than that observed in vitro, while hydrophilic mannitol and acyclovir showed low penetration in both systems without GACH pretreatment. On the other hand, GACH enhanced mannitol and acyclovir penetration, especially in the in vivo system. Analysis of absorption profiles, using a two-layer skin model with polar and nonpolar routes in the stratum corneum, suggested that the diffusion length of a viable layer (viable epidermis and dermis) was shorter in vivo than in vitro and the effective area of the polar route in the stratum corneum was larger in vitro without GACH pretreatment. GACH increased the partitioning of acyclovir into the nonpolar route to the same extent in both systems. In addition, GACH increased the effective area of the polar route in vivo, probably because of enhanced water permeability; however, this effect was smaller in vitro since the stratum corneum was already hydrated even without GACH pretreatment.     

Evaluation of Bioequivalence of Highly Variable Drugs Using Monte Carlo Simulations. I. Estimation of Rate of Absorption for Single and Multiple Dose Trials Using Cmax

Purpose. A Monte Carlo simulation study was done to investigate the effects of high intrasubject variation in clearance (CL), and volume of distribution (V) on the calculation of the 90% confidence interval (CI) for Cmax for single dose and multiple dose studies. Methods. Simulations were done for both immediate release and sustained release scenarios. The simulated data were compared with clinical data from bioequivalence studies performed on indomethacin and verapamil. Results. Previous reviews and simulations have shown that the probability of failure for the Cmax for single dose studies was always greater than that for multiple dose studies. However, the results for the simulated scenarios currently investigated indicate that if intrasubject (period-to-period) variation in CL and V is high (% CV's above 25%, and 12%, respectively), multiple dose studies can exhibit a higher probability of failure for Cmax than do single dose studies. Furthermore, Cmax values from studies performed with a sustained release scenario are more sensitive to changes in Ka, CL, and V than are results of studies on immediate release products. As an example, the probability of failure for immediate release products in simulated single dose studies is about 11% and 21% when the mean difference in Ka is 10% and 20%, respectively; while, the probability of failure for multiple dose studies is about 36% regardless of the difference in Ka. The corresponding values for the probability of failure for sustained release products were 25%, 53% for single dose studies and 39% for multiple dose studies. The simulations also indicate that changes in the fraction absorbed have a greater effect on the estimation of Cmax in multiple dose regimens than in single dose studies. Conclusions. The results from these investigations indicate that multiple dose studies do not necessarily always reduce variability in Cmax.     

Analysis of Chromameter Results Obtained from Corticosteroid-Induced Skin Blanching. I: Manipulation of Data

Purpose. One of the unresolved issues in the FDA Guidance document for topical corticosteroid bioequivalence testing is the method of manipulation suggested for the chromameter data. The purpose of this study was to manipulate the instrumental data from a typical blanching study in a number of ways to investigate the appropriateness of these procedures for comparison with the subjective visually-assessed results. Methods. The human skin blanching assay methodology routinely practiced in our laboratories was utilised and the vasoconstriction produced by two corticosteroid formulations of different potency was assessed visually and instrumentally by use of a Minolta chromameter. The instrumental data were corrected for zero-time and unmedicated site readings. In addition, Euclidean distances were calculated using all data generated by the instrument. Results. Individually the a-, b- and L-scale chromameter values are imprecise and there is negligible vasoconstriction response recorded for the moderately potent formulation. Arithmetical manipulation of the data as suggested by the FDA does not appear to improve the quality of the data in any way. Euclidean distance analysis more closely resembles the visual data and appears to have better precision. Conclusions. It is clear that mathematical correction of chromameter data is unnecessary, especially since the instrumental data are extremely imprecise. Furthermore, the assessment of each individual chromameter index does not adequately characterise the blanching response profile. It is therefore suggested that Euclidean distance may be a better measure on which to base an analysis of bioequivalence than the truncated data set methodology currently suggested by the FDA.     

Pharmacokinetics of promazine: I. Disposition in patients with acute viral hepatitis B

Concentrations of promazine in plasma, plasma water, red blood cells, and urine were measured after oral administration of the drug to six patients during and after apparent recovery from the acute phase of viral hepatitis B. None of the promazine pharmacokinetic parameters were significantly different during and after the acute phase; these parameters included clearance, free drug clearance, metabolic clearance, volume of distribution, distribution and elimination half-life values, plasma protein binding, and per cent excreted in the urine. During the acute period of the illness, SGOP, SGPT, alkaline phosphatase, and total bilirubin were increased in all patients; they returned to within or near the upper limits or normal after recovery. Despite the unchanged promazine disposition, four out of six patients had more severe promazine side-effects, such as sedation, postural hypotension, and dizziness during the acute phase of the illness. This study suggests that promazine disposition was not significantly altered as a consequence of viral hepatitis. However, the pharmacodynamic effects of promazine were changed significantly. Care must be taken with patients who are taking promazine during the acute phase of viral hepatitis B.     

Use of Shed Snake Skin as a Model Membrane for in Vitro Percutaneous Penetration Studies: Comparison with Human Skin

The potential usefulness of shed snake skin as a model membrane for transdermal research was examined. There are similarities between shed snake skin and human stratum corneum in terms of structure, composition, lipid content, water permeability, etc. The permeability of various compounds and the contribution of several functional groups to the permeability were also found to be similar between shed snake skin and human skin. Moreover, the permeability of compounds through shed snake skin was increased by Azone, one of the most extensively studied transdermal penetration enhancers. Considering the similarities between shed snake skin and human skin, ease of storage and handling, and low cost, shed snake skin may offer a good model membrane for transdermal research.     

Compartmental Modeling of Transdermal Iontophoretic Transport: I. In Vitro Model Derivation and Application

PURPOSE: The objective of this study was to develop a family of compartmental models to describe in a strictly quantitative manner the transdermal iontophoretic transport of drugs in vitro. METHODS: Two structurally different compartmental models describing the in vitro transport during iontophoresis and one compartmental model describing the in vitro transport in post-iontophoretic period are proposed. These models are based on the mass transfer from the donor compartment to the acceptor compartment via the skin as an intermediate compartment. In these models, transdermal iontophoretic transport is characterized by 5 parameters: 1) kinetic lag time (tL), 2) steady-state flux during iontophoresis (Jss), 3) skin release rate constant (K(R)), 4) the first-order rate constant of the iontophoretic driving force from the skin to the acceptor compartment (I1), and 5) passive flux in the post-iontophoretic period (Jpas). The developed models were applied to data on the iontophoretic transport in human stratum corneum in vitro of R-apomorphine after pretreatment with phosphate buffered saline pH 7.4 (PBS) and after pretreatment with surfactant (SFC), as well as the iontophoretic transport of 0.5 mg ml(-1) rotigotine at pH 5 (RTG). RESULTS: All of the proposed models could be fitted to the transport data of PBS, SFC, and RTG groups both during the iontophoresis and in the post-iontophoretic period. The incorporation of parameter I1 failed to improve the fitting performance of the model. This might indicate a negligible contribution of iontophoretic driving force to the mass transfer in the direction from the skin to the acceptor compartment, although it plays an important role in loading the skin with the drug. The estimated values of Jss of PBS, SFC, and RTG were identical (p > 0.05) to the values obtained with the diffusion lag time method. Moreover, time required to achieve steady-state flux can be estimated based on the parameter tL and the reciprocal value of parameter K(R). In addition, accumulation of drug molecules in the skin is reflected in a reduction of the value of the K(R) parameter. CONCLUSIONS: The developed in vitro models demonstrated their strength and consistency to describe the drug transport during and post-iontophoresis.     

Use of a Dynamic in Vitro Lipolysis Model to Rationalize Oral Formulation Development for Poor Water Soluble Drugs: Correlation with in Vivo Data and the Relationship to Intra-Enterocyte Processes in Rats

Purpose To examine the correlation between the in vitro solubilization process of lipophilic compounds from different lipid solutions and the corresponding in vivo oral bioavailability data. In particular, to assess the influence of intra-enterocyte processes (metabolism and lymphatic absorption) on this correlation.Materials and Methods The dissolution of progesterone and vitamin D₃ in long (LCT), medium (MCT) and short (SCT) chain triglyceride solutions were tested in a dynamic in vitro lipolysis model. The absolute oral bioavailability of the drugs from the tested formulations was investigated in rats. Vitamin D₃ bioavailability was also examined following lymphatic transport blockage induced by cycloheximide (3 mg/kg).Results The dynamic in vitro lipolysis experiments indicated a rank order of MCT > LCT > SCT for both progesterone and vitamin D₃. The bioavailability of progesterone correlated with the in vitro data, despite its significant pre-systemic metabolism. For vitamin D₃, an in vivo performance rank order of LCT > MCT > SCT was obtained. However, when the lymphatic transport was blocked the bioavailability of vitamin D₃ correlated with in vitro data.Conclusions The in vitro lipolysis model is useful for optimization of oral lipid formulations even in the case of pre-systemic metabolism in the gut. However, when lymphatic transport is a significant route of absorption, the in vitro lipolysis data may not be predictive for actual in vivo absorption.     

Induction of micronuclei by phenol in the mouse bone marrow: I. Association with chemically induced hypothermia.

Doses of xenobiotics at or near LD50 may result in substantial hypothermia in mice. Hypothermia has previously been associated with an increase in micronuclei (MN) formation. The present series of investigations examined the potential for phenol to induce hypothermia in mice and its correlation to previously reported MN induction. In order to examine the potential etiology of phenol-induced MN, evaluation of kinetochore status of MN was also carried out. Phenol-induced hypothermia was assessed in CD1 mice following a single ip dose of phenol ranging from 0-500 mg/kg. Phenol at 300 mg/kg or above caused significant and prolonged hypothermia in male and female mice (up to 7 degrees C decrease). In the micronucleus test, single ip doses of phenol to CD1 mice at 0, 30, 100, or 300 mg/kg produced a significant and prolonged hypothermia and a significant increase in MN only at 300 mg/kg; no marked effect on either body temperature or MN was observed at lower doses. A statistically significant increase in kinetochore-positive MN was observed at the 300-mg/kg dose; however, the response was considerably less than that observed for a known spindle poison. Hence, the induction of MN by phenol occurred only at a dose that produced substantial and prolonged physiologic hypothermia, but interruption of the cell spindle apparatus appeared to play only a minor role in MN formation. These data are suggestive of a threshold mechanism for the induction of MN by phenol treatment in mice.     

A Novel in-Vitro Technique for Studying Percutaneous Permeation with a Membrane-Coated Fiber and Gas Chromatography/Mass Spectrometry: Part I. Performances of the Technique and Determination of the Permeation Rates and Partition Coefficients of Chemical Mixtures

Purpose. To develop a novel in-vitro technique for rapid assessment of percutaneous absorption of chemical mixtures. Methods. A silastic membrane was coated on to a fiber to be used as a permeation membrane. The membrane-coated fiber was immersed in the donor phase to partition the compounds into the membrane. At a given partition time, the membrane-coated fiber was transferred into a GC injector to evaporate the partitioned compounds for quantitative and qualitative analyses. Results. This technique was developed and demonstrated to study the percutaneous permeation of a complex mixture consisting of 30 compounds. Each compound permeated into the membrane was identified and quantified with GC/MS. The standard deviation was less than 10% in 12 repeated permeation experiments. The partition coefficients and permeation rates in static and stirred donor solution were obtained for each compound. The partition coefficients measured by this technique were well correlated (R² = 0.93) with the reported octanol/water partition coefficients. Conclusions. This technique can be used to study the percutaneous permeation of chemical mixtures. No expensive radiolabeled chemicals are required. Each compound permeated into the membrane can be identified and quantified. The initial permeation rate and equilibrium time can be obtained for each compound, which could serve as characteristic parameters regarding the skin permeability of the compound.     

A physiologically based toxicokinetic model for dietary uptake of hydrophobic organic compounds by fish: I. Feeding studies with 2,2',5,5'-tetrachlorobiphenyl.

A physiologically based toxicokinetic (PBTK) model was developed to describe dietary uptake of hydrophobic organic compounds by fish. The gastrointestinal (GI) tract was modeled using four compartments corresponding to the stomach, pyloric ceca, upper intestine, and lower intestine, and the lumenal volume of each compartment was allowed to change in time as a function of bulk flow down the GI tract and (for the pyloric ceca and upper intestine) nutrient uptake. The model was developed using data from rainbow trout that were fed a single meal of 60-day-old fathead minnows contaminated with [UL-(14)C] 2,2',5,5'-tetrachlorobiphenyl ([(14)C] PCB 52). Chemical partitioning coefficients for the gut contents and tissues were adjusted to account for changes in chemical affinity associated with uptake of dietary lipid. Permeability constants for the absorbing gut segments were then fitted by modeling to measured [(14)C] PCB 52 concentrations in gut contents and tissues. The model accurately describes observed patterns of gastric evacuation and bulk flow of digesta, the concentration time course for [(14)C] PCB 52 in contents and tissues of the GI tract, and [(14)C] PCB 52 distribution to other major tissues. Most of the [(14)C] PCB 52 was taken up in the pyloric ceca and upper intestine during the period of peak lipid absorption. It is concluded, however, that a kinetic limitation acting along the entire length of the GI tract resulted in a chemical disequilibrium between feces and tissues of the lower intestine.     

Discussion of: Statistical and Regulatory Issues with the Application of Propensity Score Analysis to Nonrandomized Medical Device Clinical Studies by Lilly Q. Yue

A nonlinear mixed-effects model suitable for characterizing repeated measurement data is described. The model allows dependence of random coefficients on covariate information and accommodates general specifications of a common intraindividual covariance structure, such as models for variance within individuals that depend on individual mean response and autocorrelation. Two classes of procedures for estimation in this model are described, which incorporate estimation of unknown parameters in the assumed intraindividual covariance structure. The procedures are straightforward to implement using standard statistical software. The techniques are illustrated by examples in growth analysis and assay development.