Predicting in vivo absorption of chloramphenicol in frogs using in vitro percutaneous absorption data.

BACKGROUND: Infectious disease, particularly the fungal disease chytridiomycosis (caused by Batrachochytrium dendrobatidis), is a primary cause of amphibian declines and extinctions worldwide. The transdermal route, although offering a simple option for drug administration in frogs, is complicated by the lack of knowledge regarding percutaneous absorption kinetics. This study builds on our previous studies in frogs, to formulate and predict the percutaneous absorption of a drug for the treatment of infectious disease in frogs. Chloramphenicol, a drug with reported efficacy in the treatment of infectious disease including Batrachochytrium dendrobatidis, was formulated with 20% v/v propylene glycol and applied to the ventral pelvis of Rhinella marina for up to 6 h. Serum samples were taken during and up to 18 h following exposure, quantified for chloramphenicol content, and pharmacokinetic parameters were estimated using non-compartmental analysis. RESULTS: Serum levels of chloramphenicol reached the minimum inhibitory concentration (MIC; 12.5 µg.mL- 1) for Batrachochytrium dendrobatidis within 90-120 min of exposure commencing, and remained above the MIC for the remaining exposure time. Cmax (17.09 ± 2.81 µg.mL- 1) was reached at 2 h, while elimination was long (t1/2 = 18.68 h). CONCLUSIONS: The model, based on in vitro data and adjusted for formulation components and in vivo data, was effective in predicting chloramphenicol flux to ensure the MIC for Batrachochytrium dendrobatidis was reached, with serum levels being well above the MICs for other common bacterial pathogens in frogs. Chloramphenicol's extended elimination means that a 6-h bath may be adequate to maintain serum levels for up to 24 h. We suggest trialling a reduction of the currently-recommended continuous (23 h/day for 21-35 days) chloramphenicol bathing for chytrid infection with this formulation.

Can models of percutaneous absorption based on in vitro data in frogs predict in vivo absorption?

The primary aim of in vitro testing of chemicals delivered via the percutaneous route is to predict the absorption that would ensue if exposure occurred in live animals. While there is mounting evidence that in vitro diffusion studies in mammalian skin can provide valid information regarding likely in vivo absorption, little is known whether such a correlation exists between in vitro diffusion testing and in vivo blood levels in amphibians. The current study used previously-reported in vitro absorption data for caffeine, benzoic acid, and ibuprofen across isolated skin from the cane toad (Rhinella marina) to produce a series of linear mixed-effect models of the absorption parameters flux and permeability coefficient (Kp). Models investigated the relative impacts of animal weight, physicochemical characteristics of the applied chemical (logP or molecular weight), and site of application. The top models were then used to predict the flux, Kp and serum concentrations of the same three model chemicals. Finally, the absorption of these chemicals was determined in live cane toads, and results compared to the model predictions. LogP and site of application were included in all top models. In vivo absorption rates were lower than predicted for all chemicals, however, the models provided reasonable predictions of serum concentration, with factors of difference (FOD) ranging from 2.5-10.5. Ibuprofen, the chemical with the highest relative lipophilicity, had the poorest predictive performance, consistently having the highest FOD for all predictions. This report presents the first models of percutaneous absorption in an amphibian. These models provide a basic method to establish the approximate in vivo absorption of hydrophilic and moderately-lipophilic chemicals through frog skin, and could therefore be used to predict absorption when formulating such chemicals for treatment of disease in frogs, or for risk-assessments regarding chemical pollutants in frog habitats.

Percutaneous absorption between frog species: Variability in skin may influence delivery of therapeutics.

Frogs have permeable skin, so transdermal delivery provides a practical alternative to traditional dosing routes. However, little is known about how frog skin permeability differs interspecifically, and there are different reported clinical outcomes following topical application of the same chemical in different frog species. This study collated in vitro absorption kinetic data previously reported for two frog species: the green tree frog (Litoria caerulea) and the cane toad (Rhinella marina), and used linear mixed-effects modelling to produce a model of absorption. Histology of skin samples from each species was performed to observe morphological differences that may affect absorption. Absorption kinetics differed significantly between species, with the logP of the applied chemical a better predictor of permeability than molecular weight. Application site also influenced permeability, with dorsal permeability consistently higher in cane toads. Ventral permeability was more consistent between species. Skin thickness differed between species and skin regions, and this may explain the differences in absorption kinetics. Guidelines for selecting chemicals and dosing site when treating frogs are presented. The permeability differences identified may explain the poor reproducibility reported in the treatment of disease across frog species, and reinforces the importance of considering interspecies differences when designing therapeutic treatments for frogs.

Permeability of frog skin to chemicals: effect of penetration enhancers.

Rarely do commercial chemical products contain solely the active chemical/ingredient. It is therefore important to consider whether ingredients other than the active may: 1) alter absorption of the active chemical, or 2) be absorbed themselves, resulting in systemic effects. Frogs have highly permeable skin and are routinely exposed to commercial chemical products in the environment or therapeutically. Ethanol and propylene glycol (PG), which have known penetration-enhancing effects, are commonly included in such products. The current study has therefore investigated the in vitro absorption kinetics through Rh. marina skin of three model chemicals - caffeine, benzoic acid, and ibuprofen - formulated individually as solutions containing: 1%, 10% or 30% v/v ethanol, or 20% v/v PG. Differential scanning calorimetry and histology were used to characterise fresh frog skin, investigate the mechanism of these enhancers in frog skin, and to determine whether these enhancers significantly affected skin structure. Results showed that the extent of absorption enhancement was influenced by chemical, enhancer and skin region, and that enhancement was generally not consistent for individual enhancers or skin regions. The exception was 1% v/v ethanol, which did not significantly alter flux across the skin for any of the chemicals evaluated. Caffeine absorption was not enhanced by any of the investigated penetration enhancers, and was in fact significantly reduced by 30% v/v ethanol and PG. Ethanol caused concentration-dependant changes in skin morphology and should be avoided in concentrations ≥10% v/v. PG, however, caused minimal changes to the skin and consistently improved absorption of benzoic acid and ibuprofen through all skin regions. Owing to the significant changes in skin structure following ≥10% v/v ethanol exposure, it is recommended to avoid its use in frogs. For enhancement of penetration of moderately-to-highly lipophilic chemicals, this study has identified 20% v/v PG should to be the enhancer of choice.

Effects of skin region and relative lipophilicity on percutaneous absorption in the toad Rhinella marina.

Owing to the dynamic interaction between frog skin and the environment, xenobiotics in frog habitats are of particular concern, and knowledge of percutaneous absorption in frog skin is necessary for risk-mitigation purposes. Baseline transdermal kinetics in adult aquatic and arboreal frog species have recently been reported; however, there is little information regarding absorption kinetics in adult terrestrial species. The present study investigated the in vitro absorption kinetics of 3 model chemicals-caffeine, benzoic acid, and ibuprofen-through different skin regions in the terrestrial toad Rhinella marina. Caffeine flux was consistently higher than that of the other 2 chemicals (p < 0.001), whereas the fluxes of the moderately and highly lipophilic chemicals (benzoic acid and ibuprofen) were similar, regardless of skin region. When considering individual chemicals, caffeine demonstrated increased flux through the ventral pelvic skin compared with the ventral thoracic or dorsal skin regions. Flux did not differ between skin regions for either benzoic acid or ibuprofen. These findings have implications for management of environmental contamination in frog habitats, as many environmental xenobiotics are of moderate to high lipophilicity and would be expected to be equally absorbed from all skin surfaces in terrestrial toads. Environ Toxicol Chem 2019;38:361-367. © 2018 SETAC.

Regional variation in percutaneous absorption in the tree frog Litoria caerulea.

Frog skin structure and physiology differs between skin regions, however little is known about how these differences affect transdermal absorption of chemicals. Further, no information is available regarding how the relative lipophilicity of a chemical influences its transdermal pharmacokinetics in frog skin. This study investigated the in vitro percutaneous absorption of three model chemicals - benzoic acid, caffeine, and ibuprofen - through dorsal and ventral skin of the tree frog Litoria caerulea. Flux was significantly higher through the ventral skin for all chemicals. Relative lipophilicity affected flux differently in different skin regions. These differences are likely due to significantly thicker dorsal skin increasing absorption path length, and also possibly owing to lipoid secretions on the dorsum providing an additional diffusional barrier. This knowledge can advise risk mitigation of xenobiotics in agricultural and industrial settings, and also guide selection of chemicals and doses when considering transdermal drug therapy in captive frogs.

Repackaged sodium valproate tablets--Meeting quality and adherence to ensure seizure control.

PURPOSE: Sodium valproate, which is commonly repacked to assist with adherence to ensure seizure control, is hygroscopic and therefore sensitive to moisture. The aim of this study was thus to determine the stability implications of removing the enteric coated tablets from their original packaging and repackaging into a Dose Administration Aid (DAA) with storage under various environmental conditions. METHODS: Physicochemical stability of enteric coated sodium valproate tablets repackaged into a DAA and stored at controlled room temperature, accelerated and refrigerated conditions was evaluated for 28 days. A validated high performance liquid chromatography method was used for the quantitation of the drug content. RESULTS: Although the chemical stability (sodium valproate between 95 and 105% of labelled content) was maintained for 28 days for all storage conditions, for those tablets stored under accelerated conditions the integrity of the enteric coat was compromised after only 8 days. CONCLUSIONS: Repackaging of enteric coated sodium valproate should be undertaken with caution and be informed by storage climate. This is particularly relevant for those patients living in hot, humid environments where they should be advised to store their DAA in a refrigerator.

Stability of sodium valproate tablets repackaged into dose administration aids.

OBJECTIVES: Since sodium valproate, a commonly used antiepileptic drug, has been reported to be unstable in the presence of moisture, our objective was to investigate the effect of repackaging into dose administration aids. METHODS: Sodium valproate 100 mg immediate-release tablets were repackaged and stored for 56 days at accelerated conditions (40 degrees C/75% relative humidity), room temperature (25 degrees C) and under refrigeration (2-8 degrees C). Samples were analysed at 3, 7, 10, 14, 21, 35, 49 and 56 days to determine chemical stability using high-performance liquid chromatography, while physical testing included assessment of weight changes and dissolution behaviour. KEY FINDINGS: The results revealed that the sodium valproate content in the tablets remained within the acceptable range of 90-110% under all storage conditions for 56 days. Physical stability, however, was not maintained, with a total weight gain of 12.36% under accelerated conditions over the 56 days. Samples stored under all conditions showed variable dissolution compared to the controls, with the amount of sodium valproate in solution following 45 min of dissolution testing below 75% for half of all the intervals determined. CONCLUSIONS: Repackaging sodium valproate tablets into dose administration aids results in unacceptable weight variation and changes in the dissolution profiles.

Compliance aids and medicine stability: new evidence of quality assurance.

Although increasing use of compliance aids is resulting in improved clinical outcomes for patients, the stability of some drugs being repackaged into these aids is being questioned. This is due to the fact that despite their widespread use, there is limited availability of relevant stability data. This review presents clinical evidence for repackaging into Dose Administration Aids (DAAs), the Australian Pharmaceutical Advisory Committee and other guidelines on general stability issues related to repackaging and a summary of evidence for stability studies conducted in the practice. For frusemide and prochlorperazine chosen as candidates for study because of their light sensitivity, while discoloration on light exposure rendered them unacceptable for patient use, precautions in repackaging and patient counselling can easily overcome this problem. In the case of sodium valproate however, hygroscopicity results in these tablets being unusable after exposure to accelerated storage conditions. In the absence of specific data on the stability of drug products repackaged into compliance aids, the guidelines, practical recommendations for repackaging and the management of compliance aids put forward in this article provide the pharmacist with the tools to make an informed decision on this process.