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.     

Regional variations in percutaneous absorption of methimazole: an in vitro study on cat skin

The use of transdermal gel medications in cats has become popular in veterinary medicine due to the ease of administration compared to oral medication. The research to support systemic absorption of drugs after transdermal gel administration and the preferred skin region to apply these drugs in cats is limited. The aim of this study was to characterize the effect of different skin regions on the percutaneous absorption pharmacokinetics of a commercially available transdermal methimazole after a finite dose was applied to feline skin in vitro. A commercial formulation of methimazole (10 mg) was applied to four skin regions (the inner stratum corneum of the ear, groin, neck, and thorax regions) from six cats. The receptor medium was sampled up to 36 h postapplication, and methimazole concentrations were measured using high‐performance liquid chromatography. Methimazole was absorbed more completely across the pinnal skin, compared to the groin, neck, and thorax (P < 0.001), which justifies application to the pinna to maximize efficacy and also to minimize the effects of grooming.     

中国林蛙皮肤抗菌肽的分离纯化及部分特性研究

从长白山林蛙养殖场购买林蛙 ,用冷水清洗林蛙 ,断头后迅速剥离皮肤 ,清洗血液后剪碎 ,经 80 %的甲醇抽提 3次 ;再经组织捣碎机捣碎 ,用 80 %的甲醇抽提 2次 ;再经葡聚糖凝胶过滤。实验结果表明 ,林蛙皮肤中小分子活性多肽经葡聚糖凝胶过滤得到纯度较高的抗菌活性肽 ;林蛙皮肤中小分子活性多肽对革兰氏阳性细菌、革兰氏阴性细菌都具有一定的抗菌作用 ;经醋酸纤维薄膜电泳 ,表明其具有较强的阳离子特征 ;SDS - -PAGE电泳分析表明为单一区带 ,林蛙皮肤中小分子抗菌活性多肽的相对分子量为 6 .2 8ku。     

Wild‐Caught and Farm‐Reared Amphibians are Important Reservoirs of Salmonella,A Study in North‐East Thailand

The role of amphibians as Salmonella reservoirs has not been as well studied as in reptiles, where the literature is abundant. Recent outbreaks of salmonellosis associated with exotic pet frogs have occurred in United States. Frog farming and wild frog harvesting have increased the international trade in these species. This necessitates a better understanding of the risk of salmonellosis transmission from amphibians to humans. We explored the presence of Salmonella in amphibians (frogs and toads) in Thailand, where farmed and wild frogs as well as toads are present. These live animals are easily found in the local markets and are used as food. Exportation of frog meat from Thailand is common. During March–June 2014, ninety‐seven frogs were collected from several habitats, including frog farms, urban areas and protected natural areas. The collected amphibians were tested for the presence of Salmonella. The overall prevalence of Salmonella was 69.07% (90.00% in farm animals, 0% in urban area animals and 44.83% in protected area animals). Eight serovars of Salmonella were isolated: subsp. diarizonae ser. 50:k:z, Hvittingfoss, Muenchen, Newport, Stanley, Thompson, Panama and Wandsworth. Six of the identified serovars, Hvittingfoss, Newport, Panama, Stanley, Thompson and Wandsworth, have been detected in humans in Thailand. According to our results, amphibians are reservoirs of Salmonella and can be a public health concern when used as a source of protein for humans.     

First isolation and identification of Elizabethkingia meningoseptica from cultured tiger frog, Rana tigerina rugulosa

Elizabethkingia meningoseptica has been recognised as an occasional but serious opportunistic bacterial pathogen to human beings. Recently, it was frequently isolated from tiger frog, Rana tigerina rugulosa, with cataract disease, which is the most common disease of unknown aetiology of frogs in Hainan, China. The purpose of this study was to identify and characterise the bacterial strains isolated from the recent outbreaks of cataract disease in farmed tiger frog in Hainan, China, and to evaluate their pathogenicity to the frog and their sensitivity to 20 chemotherapeutic agents.The 16S rRNA gene sequences of strains W0701 (1478 bp), W0702 (1477 bp) and W0703 (1478 bp) showed 98.6–98.7% similarity with the sequence of E. meningoseptica type strain (ATCC 13253) and 99.9–100% similarity with that of E. meningoseptica NTU 870424-IL. Six strains (W0701–W0706) were selected to represent 24 isolates retrieved from six moribund frogs. The morphological, physiological and biochemical characteristics of the six representative isolates were consistent with those of E. meningoseptica strains. The organisms were only susceptible to vancomycin and moderately susceptible to cefoperazone among the 20 investigated chemotherapeutic agents. Virulence test with strain W0702 was conducted and pathogenicity (by intramuscular injection) was demonstrated in the tiger frog. In conclusion, 24 isolates obtained from frogs with cataract disease were the E. meningoseptica strains highly pathogenic to tiger frog, and this is the first report of E. meningoseptica as a pathogen for tiger frog.     

Prediction of formulation effects on dermal absorption of topically applied ectoparasiticides dosed in vitro on canine and porcine skin using a mixture‐adjusted quantitative structure permeability relationship

Topical application of ectoparasiticides for flea and tick control is a major focus for product development in animal health. The objective of this work was to develop a quantitative structure permeability relationship (QSPeR) model sensitive to formulation effects for predicting absorption and skin deposition of five topically applied drugs administered in six vehicle combinations to porcine and canine skin in vitro. Saturated solutions (20 μL) of ¹⁴C‐labeled demiditraz, fipronil, permethrin, imidacloprid, or sisapronil were administered in single or binary (50:50 v/v) combinations of water, ethanol, and transcutol (6 formulations, n = 4–5 replicates per treatment) nonoccluded to 0.64 cm² disks of dermatomed pig or dog skin mounted in flow‐through diffusion cells. Perfusate flux over 24 h and skin deposition at termination were determined. Permeability (logKp), absorption, and penetration endpoints were modeled using a four‐term Abrahams and Martin (hydrogen‐bond donor acidity and basicity, dipolarity/polarizability, and excess molar refractivity) linear free energy QSPeR equation with a mixture factor added to compensate for formulation ingredient interactions. Goodness of fit was judged by r², cross‐validation coefficient, coefficients (q²s), and Williams Plot to visualize the applicability domain. Formulation composition was the primary determinant of permeation. Compounds generally penetrated dog skin better than porcine skin. The vast majority of permeated penetrant was deposited within the dosed skin relative to transdermal flux, an attribute for ectoparasiticides. The best QSPeR logKp model for pig skin permeation (r² = 0.86, q²s = 0.85) included log octanol/water partition coefficient as the mixture factor, while for dogs (r² = 0.91, q²s = 0.90), it was log water solubility. These studies clearly showed that the permeation of topical ectoparasiticides could be well predicted using QSPeR models that account for both the physical–chemical properties of the penetrant and formulation components.     

Percutaneous absorption of methimazole: an in vitro study of the absorption pharmacokinetics for two different vehicles

The use of transdermal medications in cats has become popular in veterinary medicine due to the ease of administration compared to oral medication. However, the research to support systemic absorption of drugs applied to the pinna after transdermal administration in cats is limited. The aim of this study was to characterize the percutaneous absorption pharmacokinetics of methimazole in a lipophilic vehicle compared to methimazole in Pluronic^® lecithin organogel (PLO) using a finite dose applied to feline ear skin in an in vitro Franz cell model. The two formulations of methimazole (10 mg) were applied to the inner stratum corneum of six pairs of feline ears. The receptor medium was sampled up to 30 h post–administration, and methimazole concentrations were measured using high‐performance liquid chromatography (HPLC). Histological examination of all ears was undertaken as small differences in the thickness of ear skin may have contributed to inter‐individual differences in methimazole absorption between six cats. Methimazole was absorbed more completely across the pinnal skin when administered in the lipophilic vehicle compared to administration in the PLO gel (P < 0.001).     

Regional differences in the in vitro penetration of methylsalicylate through equine skin

Commercial formulations of non-steroidal anti-inflammatory drugs (NSAIDs) are developed for human use but the extent to which they will pass through equine skin is unknown. Skin was harvested from five Thoroughbred geldings from the thorax, groin and leg (dorsal metacarpal) regions and frozen (-20 degrees C) until required. Two grams of methylsalicylate (MeSa) gel was applied to defrosted full-thickness samples in diffusion cells and the penetration of MeSa and its active metabolite, salicylate (Sa), through skin samples were measured over 24 h. Significantly higher (P < or = 0.02) total salicylate (AUC; MeSa + Sa) penetrated through skin from the leg region (5491.3 h mg/L), compared to thorax (3710.7 h mg/L) and groin (3571.5 h mg/L). In addition, there was a significantly higher (P0.01) rate of penetration of total Sa through leg skin in the first 6h after application. It was concluded that the commercial formulation of MeSa would achieve therapeutic levels of total salicylate beneath sites of topical application, with a faster and more pronounced response through the leg region, compared to the upper body.     

Once in a blue moon: Lipid keratopathy and intrastromal hemorrhage in a Mission golden‐eyed tree frog (Trachycephalus resinifictrix)

Mimicking natural anuran biology is a major challenge faced in the husbandry management of frogs, and the quandary of lipid keratopathy in frogs under human care has plagued keepers and practitioners for decades. Unlike corneal lipid dystrophy or lipidosis secondary to degeneration, where there is limited or no vascular in‐growth or inflammatory response, lipid keratopathies are associated with vascularization, most often following the appearance of lipid. Hemorrhage from stromal neovascularization has not been described in a frog; however, the presence of vessels in lipid keratopathy certainly heralds the possibility. We report a rather unique case of lipid keratopathy in a 6‐year‐old female Mission golden‐eyed tree frog (Trachycephalus resinifictrix) that not only had concurrent intrastromal hemorrhage, but blue plasmoid staining of the corneal stroma. Current views on both the function of blue plasma in several species and lipid keratopathy are briefly discussed. Overall, evidence suggests that the cause of lipid keratopathy is probably multifactorial and will not successfully be rectified until anuran biology and husbandry are better understood.     

Bacterial flora on Cascades frogs in the Klamath mountains of California

Amphibians are experiencing global declines due in part to the infectious disease chytridiomycosis. Some symbiotic bacteria residents on frog skin have been shown to inhibit the growth of Batrachochytrium dendrobatitis (Bd) but few studies have attempted to fully describe the resident bacterial flora of frog skin. We cultured and sequenced 130 bacterial isolates from frogs collected from the California Klamath Range, recovering predominantly Gram-negative bacteria from 20 higher order taxa and 31 genera. There were also a large number of unclassifiable isolates. Forty-three isolates were assessed for their ability to inhibit the growth of Bd in vitro; of these, two had strong and three had slight anti-Bd activity. We suggest that many bacterial species may play a secondary role in Bd resistance, acting synergistically with inhibitory species. Future research is required in order to characterize these interactions. Understanding the relationships between bacterial strains may be important in predicting and managing the effects of future anti-Bd treatments such as antimicrobial compounds or probiotic bacteria.     

Trombiculid mites (Hannemania sp.) in canyon tree frogs (Hyla arenicolor).

Six wild-caught, captive canyon tree frogs (Hyla arenicolor) purchased as a group and housed at the North Carolina Zoological Park were diagnosed with intradermal Hannemania mites, the first described infestation of this frog species by this mite. Typical gross lesions were orange-colored skin vesicles approximately 1 mm in diameter, predominantly located on the ventrum and ventral hind limbs. The larval mites had ovoid bodies approximately 44 microm in length and 240 microm in width. Three of the frogs died, and three became free of mites and pathologic changes after approximately 28 wk of treatment with transcutaneous ivermectin and manual mite removal. The frogs remained free of mites at a 1-yr recheck.     

人工养殖东北林蛙生长与发育规律的初步研究

2008年4—10月,在黑龙江省佳木斯市某东北林蛙养殖场,对1龄、2龄和3龄人工养殖东北林蛙的体重（Y）、体长（1）、前臂及手长（f）、后肢长（X）和头宽（z）体尺指标进行测量与分析。结果表明：1龄、2龄东北林蛙在7月的绝对生长率最大,3龄东北林蛙在5月和8月的绝对生长率高于其他月;1龄、3龄东北林蛙5月的相对生长率最大,2龄东北林蛙5月和7月的相对生长率大于其他月;不同种源的1龄东北林蛙体重差别显著（P〈0．05）;东北林蛙各体尺指标之间的相关性均显著（P〈0．01）;利用多元线性回归建立了东北林蛙体重与其他体尺指标的最优回归方程为Y=-18．374＋4．417x＋1．246z（R=0．923、R^2=0．853、校正R^2=0．851）。     

Quantification of percutaneous absorption of metronidazole and levamisole in the fire-bellied toad (Bombina orientalis)

Spot-on application has proved to be an effective way to reach therapeutic doses of metronidazole and levamisole in fire-bellied toads. The percutaneous absorption of metronidazole and levamisole was quantified, using an aqueous solution of 1.008 mg/ml of metronidazole and an aqueous solution of 3.767 mg/ml of levamisole. High Performance Liquid Chromatography (HPLC) analysis of the percutaneous absorption revealed that of the amount applied, 75% of metronidazole and 90% of levamisole was absorbed. This resulted during 3 days of application in dosages of 23 mg/kg BW of metronidazole and 94 mg/kg BW of levamisole. Of the absorbed substances, 48% of metronidazole and 9% of levamisole were excreted in urine and faeces as unmetabolised substances.     

Comparative in vitro characterization of moxidectin and doramectin percutaneous absorption through bovine skin

Topical formulations have achieved worldwide acceptance in veterinary medicine because their administration is an easy, less labor-intensive and nonstressing form. Any chemical compound that comes in contact with the skin has the potential to be locally and/or systemically absorbed. However, many factors related to the features of animal skin, composition of the topical formulation and to the drug itself can determine marked differences in the percutaneous absorption process. The aim of the current work was to characterize the pattern of in vitro percutaneous absorption for moxidectin (MXD) and doramectin (DRM), two of the most worldwide used topical macrocyclic lactone antiparasitic compounds in cattle. The work included the development of a simple and inexpensive in vitro assay useful to predict in vivo drug percutaneous absorption in cattle. Both drugs were administered as the commercial formulations intended for their topical administration to cattle. The in vitro studies were carried out using modified Franz-type vertical diffusion cells. Cattle skin slices of 500 μm thickness were prepared using a dermatome to separate the stratum corneum and upper epidermis from dermis and subcutaneous tissue. The receptor medium was sampled up to 72 h postadministration and drug concentrations were measured by HPLC. The parameters used to estimate the comparative in vitro skin permeation showed marked differences between DRM and MXD. A 5.29-fold longer lag time (T(lag)) was observed for DRM. Similarly, the flux (J) (2.93-fold) and the permeation coefficients (K(p) ) (2.95-fold) in cattle skin were significantly higher (P < 0.05) for DRM compared to those obtained for MXD. Additionally, the data obtained from the in vitro permeation studies was correlated with the plasma concentrations of both compounds achieved in vivo in cattle treated with the same topical formulations. Correlation coefficients between percentage of drug permeated in vitro vs. percentage of drug absorbed in vivo (up to 48 h post-treatment) were 0.856-0.887 (MXD) and 0.976-0.990 (DRM). However, the highest in vitro-in vivo correlations for both molecules were observed up to 24 h post-treatment A rapid screening method for testing different topical formulations can be achieved with the simple in vitro cattle skin permeation technique described here, which has been successfully adapted to test the comparative percutaneous absorption of MXD and DRM.     

Effect of rain on absorption after transdermal application of flunixin in calves

Flunixin is a nonsteroidal anti-inflammatory drug (NSAID) that has anti-inflammatory, anti-pyretic, and analgesic effects. Recently, a novel transdermal formulation was developed (Finadyne^® Transdermal, MSD Animal Health) and is now the first NSAID registered to be administered as a pour-on product in cattle. According to the manufacturer's instructions, the pour-on product should be applied only to dry skin and exposure to rain should be avoided for at least 6 hr after application. The objective of the study was to evaluate the effect of simulated exposure to light or heavy rain on flunixin absorption and bioavailability within the first 4 hr after administration. Therefore, an isocratic HPLC method was developed to quantify flunixin concentrations in bovine serum by UV detection. Light rain decreased flunixin absorption only when rain started immediately after flunixin administration, while light rain starting more than 30 min after administration of flunixin had no effect on absorption. Absorption and bioavailability of flunixin was impacted under simulated heavy rain conditions, when exposure to rain occurred within one hour after the application of the pour-on formulation, but not later.     

Evaluation of Plasma Detectable Concentrations of Two Lidocaine Transdermal Formulations and Their Analgesic Effect in the Horse

Transdermal absorption of four lidocaine (L) patches (5%) was compared with the transdermal absorption of L cream (5%) to evaluate the pharmacokinetics of the two formulations applied on the same anatomic region under dressing in eight horses. The animals were also assessed for antinociceptive effect after removal of the patches and cream, using a psychophysical method visual analog scale, by pricking the patient's skin to test the response to pain. Horses were randomly assigned to four treatment groups: in groups I and II, four L patches were applied for a period of 24 hours with and without alcohol precleaning, respectively; in group III, 5% L cream was applied every 2 hours over a 24-hour period on the same anatomic site. Group IV was the control. No clinical side effects were noted with either formulation. L was detectable in plasma 6 to 24 hours after application of the patches, and the highest plasma concentrations were reached between 12 and 18 hours. The use of alcohol to preclean the skin appeared to reduce the transdermal drug absorption over time. After L cream application, the peak plasma drug concentration occurred at 24 hours. Nociception assessment after L patch or L cream application showed a decreased response when the L cream was used. The results of this study showed that there was minimal absorption from the L patches compared with the L cream in horses. Also, the L cream treatment reduced significantly the intensity of nociception quality as measured by the visual analog scale.     

Isoflurane Potency in the Northern Leopard Frog <Emphasis Type="Italic">Rana pipiens</Emphasis> is Similar to That in Mammalian Species and is Unaffected by Decerebration

Amphibians are commonly used in biomedical research, including studies of mechanisms of anaesthetic action. There is, however, little published work describing the kinetics of inhaled anaesthetic agents or the potency of isoflurane in amphibians. Ten Northern leopard frogs were exposed to a constant isoflurane concentration of 1.0%, 1.2% or 1.5% atm for 4 h, and their response to a noxious stimulus was tested every 20 min. Each frog was anaesthetized with each concentration in random order and allowed at least 16 h to recover between anaesthetic exposures. Frogs were then pithed and the protocol was repeated. Frogs first displayed immobility during stimulus application at 80 min, and the proportion of animals becoming immobile steadily increased to reach a stable level at 4 h. The 50% effective dose for isoflurane in intact and pithed frogs did not differ, and was 1.15 and 1.25% atm, respectively. The potency of isoflurane in leopard frogs was similar to that reported in mammalian species. Cutaneous uptake of anaesthetic is effective given sufficient time, approximately 4 h in this study. Forebrain structures appear to be unimportant for the immobilizing action of isoflurane in the frog.     

Flavobacterium indologenes infection in leopard frogs.

An investigation of an epidemic of infectious disease in a frog (Rana pipiens) colony was conducted. Six of 40 frogs in a continuous (once through) water flow housing system had weight loss, swollen abdomen, corneal edema, uveitis, subcutaneous edema, petechial hemorrhage, incoordination, and respiratory distress. The frogs had lesions consistent with bacterial septicemia. A gram-negative, nonfermenting bacillus, Flavobacterium indologenes (Flavobacterium sp biovar IIb), was isolated in pure culture from tissues and blood. The clinical isolate was used to inoculate healthy frogs sc. An isolate identical to the one isolated from the sick frogs was recovered from tissues and blood of the inoculated frogs. Inoculation of the housing water in a nonflow-through system did not result in disease, despite proliferation of the Flavobacterium spp in the water; therefore, it is likely that establishment of infection requires the presence of the organism in sufficient numbers and a portal of entry into the body.