J. Edmond Riviere

The isolated perfused porcine skin flap (IPPSF). I. A novel in vitro model for percutaneous absorption and cutaneous toxicology studies.

This article describes the development of a novel in vitro alternative animal model for dermatology and cutaneous toxicology. A single-pedicle, axial-pattern, island-tubed skin flap was created in crossbred Yorkshire weanling pigs in one surgical procedure, then transferred 2 or 6 days later to a computer-controlled temperature-regulated perfusion chamber for 10-to 12-hr studies. Perfusate consisted of Krebs-Ringer bicarbonate buffer (pH 7.4) containing albumin and glucose. Viability was assessed by glucose utilization, lactate production, an absence of significant concentrations of the intracellular enzyme lactate dehydrogenase in the perfusate, and light and electron microscopy. A mean lactate to glucose ratio of 1.6 for flaps harvested 2 days after surgery and 1.8 for flaps taken 6 days after surgery suggested primarily anaerobic glycolysis. This preparation would be a humane alternative animal model for studies in cutaneous toxicology, physiology, oncology, and percutaneous drug absorption and metabolism.

Determination of Lidocaine Concentrations in Skin After Transdermal Iontophoresis: Effects of Vasoactive Drugs

The purpose of this study was to investigate the effect of vasoactive drugs on transdermal lidocaine iontophoresis by measuring the concentrations of radiolabeled lidocaine which has penetrated the skin. Previous studies had demonstrated that coiontophoresis of vasoactive drugs could modulate the transcutaneous flux of lidocaine and suggested that a dermal depot of lidocaine was involved. To address this, lidocaine hydrochloride (14C) was iontophoresed in vivo in anesthetized weanling pigs either alone or with the vasodilator tolazoline or the vasoconstrictor norepinephrine. Tissue cores under the active electrode were then collected, quick-frozen, and sectioned on a cryostat, and then the radioactivity was determined in each 40-µm section. Coiontophoresis with norepinephrine resulted in increased concentrations of lidocaine in skin up to a depth of 3 mm. These concentrations decreased to lidocaine-alone levels after a 4-hr washout. Tolazoline decreased tissue concentrations of lidocaine. Concentrations were intermediate when lidocaine alone was administered. These studies support the hypothesis that coiontophoresis of vasoactive drugs modulates the transdermal delivery of lidocaine, in part by altering the cutaneous “depot.”

The isolated perfused porcine skin flap: III. Percutaneous absorption pharmacokinetics of organophosphates, steroids, benzoic acid, and caffeine

The isolated perfused porcine skin flap (IPPSF) has been developed as an alternative in vitro tool for examining the pharmacokinetics and mechanisms of percutaneous absorption. In this study, dosing solutions of seven 14C-radiolabeled compounds representing three chemical classes--organic acid/base [benzoic acid (B), caffeine (C)], organophosphate (OP) pesticides (diisopropylfluorophosphidate, malathion, parathion), and steroid hormones (progesterone, testosterone)--were prepared in ethanol and applied topically at a surface concentration of 40 micrograms cm-2 to the IPPSF. A three-compartment pharmacokinetic model used to stimulate mass transfer from the surface (C1), diffusion through epidermis and dermis (C2), and transfer into the capillary perfusate (C3), was developed based on flux through the IPPSF from 0 to 8 hr. This basic model accurately stimulated measured IPPSF fluxes for five of seven compounds, including the OPs and steroids. The model was modified to simulate the shunting of drug to fast and slow release pathways, which occurred for B 3-4 hr postapplication, and to account for flow-dependent flux increases seen for C at 6 hr postapplication. The latter may be due to a direct pharmacologic effect, since C is a known vasodilator. Extrapolated (to 6 days) areas under the curve from the model simulations were compared with in vivo percutaneous absorption estimates, obtained from 6-day excretion studies in pigs. The in vivo-in vitro correlation, based on simple linear regression across compounds, was excellent (R2 = 0.88, R = 0.94, p less than 0.002). These results suggest that xenobiotic penetration in the 8-hr IPPSF experiments is highly predictive of in vivo absorption totals (6-day studies). In addition, since pig and human skin are similar physiologically and pharmacologically, the IPPSF may eventually have applications in formulating human dermal risk assessment models.

Pharmacokinetics and toxicity of oral and intravenous lonidamine in dogs

Abstract Plasma lonidamine concentration and toxicity were investigated in dogs receiving 100, 200, 400, 800, 1200 mg/m2 orally twice daily for 30 days and in dogs receiving single intravenous doses of 200, 400, 800, 1200 mg/m2. Physical or laboratory signs of toxicity were not observed in dogs receiving oral lonidamine, but severe vomiting and signs of acute hepatic and pancreatic toxicity were observed in dogs receiving intravenous doses that exceeded 400 mg/m2. The area under the lonidamine concentration versus time curve (AUC) in dogs receiving 200, 400, and 800 mg/m2 of lonidamine intravenously was a 1.8-, 3.3-, and 8.7-fold higher than in dogs receiving oral lonidamine. This suggests that the bioavailability of oral lonidamine may be limited. However, centrilobular hepatocellular swelling and vacuolation were observed in dogs receiving oral lonidamine. Serum alanine aminotransferase (ALT) activity was increased in dogs receiving intra-venous lonidamine. These findings suggest that lonidamine is hepatotoxic in dogs. However, serum ALT was increased in only 1/4 dogs receiving 400 mg/m2 of lonidamine intravenously and plasma concentration were within the range capable of sensitizing hyperthermia and chemotherapy. Therefore, this dose and route appears to be a viable and controllable method for prospective quantification of lonidamine interaction with systemic chemotherapy and/or hyperthermia.

Percutaneous Absorption and Excretion of Xenobiotics after Topical and Intravenous Administration to Pigs

Interspecies comparisons suggest that the weaning pig is a suitable surrogate for man in percutaneous absorption studies. Despite known anatomical and physiological similarities between porcine and human skin, very few investigations of percutaneous absorption phenomena have been conducted in pigs. This study examined radiolabel excretion patterns after intravenous (iv) and topical administration of six 14C-radiolabeled compounds in weanling Yorkshire sows. Radiolabel recovery from excrement collected over 6 days following iv doses in physiological saline (200 micrograms, 10 muCi) showed that malathion (M), parathion (P), caffeine (C), and benzoic acid (B) were primarily excreted into urine (greater than 80%), while greater fractions of testosterone (T, 72%) and progesterone (R, 35%) were fecally eliminated. Percutaneous absorption was determined from total urine and fecal excretion of radiolabel after topical application, corrected for incomplete excretion following iv administration. Topical doses in ethanol (200 micrograms, 10 muCi) were applied at a surface concentration of 40 micrograms cm-2 and penetrated in the following rank order (percentage dose): B (25.7%) greater than R (16.2%) greater than C (11.8%) greater than T (8.8%) greater than P (6.7%) greater than M (5.2%). Fecal clearances of radiolabel, expressed as a percentage of total excretion, were greater after topical administration for four of the six compounds (B, C, P, and T, p less than 0.05). Calculations based on urinary excretion alone underestimated percutaneous absorption determined from total excretion by 5-30%, although the difference between the two estimates was statistically significant only for C (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

PARATHION METABOLISM DURING PERCUTANEOUS ABSORPTION IN PERFUSED PORCINE SKIN

The metabolism and disposition of topical parathion was examined in the isolated perfused porcine skin flap (IPPSF), a novel organ perfusion method developed for in vitro studies of the pharmacology and toxicology of skin. Ethanol solutions of 14C-radiolabeled parathion (1.0 mg/ml, 0.05 mCi/ml) were applied to the surface at a dose of 40 μg/cm2 on skin flaps representing three treatment groups: control (N = 5), occluded (N = 4), and 1-aminobenzotriazole (ABT)-pretreated (N = 2). Radiolabel uptake in the perfusion medium from 0–8 hr postapplication indicated that total chemical flux and peak rates of absorption in occluded preparations were 59% and 47% lower than in the controls (P 75% lower in the ABT-pretreated IPPSFs. Thin-layer chromatographs of ethyl acetate extracts demonstrated that most of the absorbed radiolabel recovered in the perfusion medium of the controls was paraoxon (67.8 ± 2.3%, mean ± SE), with a lesser amount of p-nitrophenol (14.5 ± 5.2%) and unmetabolized parathion (16.9 ± 4.1%). Occlusion of the application site increased the fraction of p-nitrophenol in the perfusion medium (43.0 ± 7.8%, P < 0.05), at the apparent expense of paraoxon (39.6 ± 17.8%, P < 0.05), without altering the mean percentage of parent compound recovered (17.4 + 10.1%). Pretreatment of the IPPSF by addition of 50 μg ABT/g wet tissue weight into the perfusion medium blocked most of the paraoxon formation (6.6%), but not that of p-nitrophenol (11.9%), while allowing 78.5% of the parathion absorbed to penetrate intact. Total flux assessments for parathion plus its metabolites (total radiolabel absorption) in vitro varied in conjunction with its fraction metabolized within the skin. Net permeation of unchanged parathion was similar in the control (580 ng/cm2) and ABT-pretreated skin flaps (660 ng/cm2), suggesting that the diffusivity of parathion molecules through skin was not altered by this metabolic inhibitor. In contrast, occlusion reduced the net flux of intact parathion molecules to approximately 240 ng/cm2. These findings show that parathion undergoes significant biotransformation following topical application to porcine skin and that the resultant cutaneous metabolite profiles can be altered by both physical (occlusion) and chemical (ABT) means.

Transdermal Lidocaine Iontophoresis in Isolated Perfused Porcine Skin

AbstractThe isolated perfused porcine skin flap (IPPSF) is an alternative animal model for quantitatively assessing percutaneous drug absorption. The ability to place drugs or chemicals on the surface of viable skin with a functional microcirculation maintained in a controlled ambient environment, coupled with the measurement of arterial and venous drug concentrations, makes this model well suited for modeling percutaneous drug flux after transder-mal delivery. Lidocaine hydrochloride was used as the model drug to investigate the dynamics of transdermal delivery using iontophoresis. Total lidocaine flux (bioavailability) was a function of lidocaine concentration, current density, and duration of application. Rate of transdermal flux was a function of concentration, current density, and the presence or absence of coadministered epinephrine (reduced) or tolazoline (increased). The presence of vasodilators significantly affected the magnitude of peak venous

Pharmacokinetics and comparative nephrotoxicity of fixed-dose versus fixed-interval reduction of gentamicin dosage in subtotal nephrectomized dogs

There is presently no consensus as to the relative safety of fixed-interval/reduced dose (FI) vs fixed-dose/increased interval (FD) dosage adjustment regimens for use in renal insufficiency. This study compared their nephrotoxic potential using gentamicin in beagle dogs with renal insufficiency secondary to subtotal surgical nephrectomy. Pharmacokinetic analysis in six dogs showed that this surgical procedure resulted in a decreased total body clearance of drug and a marginally contracted volume of the central compartment. An allometric analysis of gentamicin disposition in different species was used to derive a human-equivalent maximum canine nontoxic dose of 9 mg kg-1 day-1. Nephrotoxicity was detected by histopathologic analysis and changes in the pre- and post-drug treatment, creatinine clearance, and daily drug elimination rate constants. This allometric dose did not produce clinical toxicity in a control group of six dogs with intact kidneys given drug for 14 days. Dosage adjustments within the FI and FD groups were based on serum creatinine concentrations 10 days after surgery. Statistical analysis of morphological and functional parameters indicated that the FD method was significantly less toxic than the FI regimen.

Cutaneous toxicity and absorption of paraquat in porcine skin

Paraquat, a commonly used herbicide, has been shown to be toxic in exposed field workers. The objectives of this study were to (a) assess the cutaneous toxicity of paraquat in vivo in pig skin and in vitro in the isolated perfused porcine skin flap (IPPSF) and (b) quantitate its absorption in the IPPSF. The amounts of 3, 24, and 200 mg of paraquat were topically applied (5 cm2 surface area) on the ventral abdomen of pigs and biopsied after 6-8 hr for light microscopy (LM) and transmission electron microscopy (TEM). IPPSFs were topically dosed with the same concentrations and perfused for 8 hr (n = 4/treatment). The dosed area of the skin was sampled for LM, TEM, and enzyme histochemistry. IPPSFs were also treated topically with [14C]paraquat dichloride at the aforementioned concentrations (n = 4/dose) and hourly perfusate samples were collected for radiolabel determination and assessment of biochemical and physiological parameters. The epidermal changes were similar both in vivo and in vitro. The changes included epidermal intercellular edema which increased with dose and epidermal-dermal separation at the 200-mg dose. Acid phosphatase and nonspecific esterase activities were increased in the upper layers of the epidermis, while alkaline phosphatase showed a greater activity in the stratum basale layer. Glucose utilization of all treated IPPSFs was lower than that of the controls and a variation in the vascular resistance profiles was seen in all the treated flaps. Radiotracer studies indicated that a majority of the compound remained on top of the application site and minimal absorption or penetration into skin was observed. Thus, at high concentrations and prolonged exposure, paraquat may have deleterious effects on epidermal morphology in the absence of significant percutaneous absorption.

Dose-response studies of gentamicin nephrotoxicity in rats with experimental renal dysfunction: II. Polyvinyl alcohol glomerulopathy

Animal studies involving concurrent pathophysiologic states, including experimental renal dysfunction, are useful for a proper understanding of the mechanisms of gentamicin nephrotoxicity and acute renal failure. This study examined gentamicin nephrotoxicity in a model of glomerular dysfunction in rats. Administration of medium molecular weight polyvinyl alcohol (PVA) produced a glomerulopathy, with characteristic accumulation of macromolecular PVA in the glomerular mesangium without altering glomerular filtration or causing proteinuria. Subsequent daily doses of gentamicin ranging from 0 to 120 mg/kg elicited a dose-response nephrotoxicity in both control and PVA-pretreated rats after 6 or 12 days of drug. Based on statistical analysis of renal clearances of creatinine, urea, sodium, and potassium; serum creatinine and urea nitrogen; urinary N-acetyl-beta-D-glucosaminidase excretion (6 days only); in vitro renal cortical transport of tetraethylammonium (TEA) (6 days); and quantified light-microscopic data (12 days), PVA induced an early (6 days) sensitivity to gentamicin nephrotoxicity. By 12 days, there were no differences in the responses of control and PVA rats to gentamicin. Single-dose gentamicin clearance, volume of distribution, and half-life were not altered by PVA and renal concentrations at 6 days were generally lower in these rats. Results of TEA transport studies tend to rule out PVA-induced metabolic lesions in the proximal tubular epithelium as the mechanism for the early sensitivity. This investigation demonstrates altered gentamicin nephrotoxicity in rats with an otherwise benign glomerulopathy and, combined with similar conclusions from a related study in subtotally nephrectomized rats, presents further evidence that the underlying pathophysiologic state of the kidney is an important factor in the renal response to nephrotoxins.