Toxicokinetics and Metabolism of N-[c]n-methyl-2-pyrrolidone in Male Sprague-dawley Rats: in Vivo and in Vitro Percutaneous Absorption

Neat N-methyl-2-pyrrolidone (NMP) rapidly penetrated into the skin of male Sprague-Dawley rats after in vivo and in vitro topical application. At the two topical doses tested in vivo, no steady state was observed. The maximal absorption fluxes were 10 and 20 mg/cm/h for 20 l/cm and 40 l/cm, respectively. Similar results were observed after in vitro topical application of neat [C]NMP (25–400 l/cm) in fresh full-thickness skin. Whatever the dose tested, the percutaneous absorption fluxes increased with exposure time to reach a maximum value (Fmax) and then decreased. Fmax and the time to reach it (Tmax) increased as the dose increased. At the highest dose, which may be considered as an “infinite dose,” the maximal flux (7.7 1.1 mg/cm/h, n 12) occurred 6 h after the topical application of NMP. The decrease on percutaneous absorption flux was correlated with the dilution of neat NMP with water from the receptor fluid. A semi-quantitative mathematical model was developed to describe the absorption flux of NMP taking into account the transfer of water through the skin. The Kp values determined from the different aqueous solutions of NMP (1:1 to 1:32, v/v) were not significantly different. The mean value was 6.4 (10 3 cm/h) (range, 4.7 to 7.6). Occlusion did not affect the percutaneous absorption flux of neat NMP. Desquamation increased the percutaneous absorption of NMP slightly. The skin did not metabolize NMP. The flux was dependent on the thickness of the skin and was proportional to the concentration of NMP. These findings suggest a passive diffusion of NMP through the skin. N-Methyl-2-pyrrolidone (NMP) is a limpid liquid, which is soluble in water and a wide range of organic solvents. Its volatility is low (vapor pressure 32 Pa at 25°C). The use of NMP as a solvent is increasing, in particular as a substitute for methylene chloride in paint strippers. One of the major uses of NMP is the extraction of aromatics from lubricating oils. NMP is also used as a vehicle for drugs or to facilitate their percutaneous absorption. Acute toxicity is low. LD50 in rabbits was 4 to 8 g/kg and 1.5 to 7 g/kg in rats after topical application. The LD50 in mice after intravenous (i.v.), intraperitoneal, and oral administration was 3.5, 4.3, and 7.5 ml/kg, respectively. In rats, the LD50 was 2.2, 2.4, and 3.8 ml/kg, respectively (Bartsh et al., 1976). Studies on reproductive toxicity have shown that NMP causes developmental toxicity at doses causing no or mild maternal toxicity (Hass et al., 1994; Solomon et al., 1995). Cases of stillbirth after occupational exposure to NMP have been reported (Solomon et al., 1996; Bower, 1997). NMP is well absorbed through gastrointestinal, pulmonary tract, and skin (Midgley et al., 1992; Akesson and Paulsson, 1997; Ursin et al., 1999). Metabolites of NMP are intensively excreted in urine mainly as 5-hydroxypyrrolidone (5-HNMP) (Wells et al., 1992; Akesson and Jonsson, 1997; Payan et al., 2002). A percutaneous absorption rate of 25.3 g/cm/h for NMP has been calculated from an in vivo experiment in rats with a co-exposition of NMP and 2-vinylpyrrolidone (Midgley et al., 1992). This value is about 3 orders of magnitudes lower than the flux determined (17 mg/cm/h) in human skin (Ursin et al., 1999). The low percutaneous flux determined in rats is incompatible with the use of NMP as a percutaneous absorption enhancer for drugs (Barry and Bennett, 1987; Sugibayashi et al., 1989). Thus, this work was carried out to determine in vivo and in vitro the percutaneous absorption of neat [C]NMP in rats. Additional experiments were also conducted in vitro with aqueous solutions of NMP (1:2–1:32, v/v). Materials and Methods Chemicals. Radiolabeled N-[C]methyl-2-pyrrolidone ([C]NMP) was supplied by Amersham International Plc (Buckinghamshire, England). Radiochemical purity exceeding 99% was determined by HPLC before each experiment. The specific activity was 1.04 GBq/mmol (28 mCi/mmol). Animals. Male haired Sprague-Dawley rats (Iffa Credo, Saint-Germain-surl’Arbresle, France) weighing 250 to 300 g were used for all studies. The animals were acclimatized to laboratory conditions for at least 4 days prior to initiating the studies in rooms with a 12-h light/dark cycle, that were designed to control relative humidity at 50 5% and temperature at 22 1°C. Commercial food pellets (UAR Alimentation-Villemoison, Epinay-sur-Orge, France) and tap water were available ad libitum. This study was sponsored by Public Health Service Grant NCCAM R21 AT00511-01. 1 Abbreviations used are: NMP, N-methyl-2-pyrrolidone; LD50; 5-HNMP, 5-hydroxy-NMP; HPLC, high performance liquid chromatography; ANOVA, analysis of variance; Fmax, maximal percutaneous absorption flux; Fmax Nor, Fmax for a skin thickness of 1.3 mm; AUC, area under the plasma curves. Address correspondence to: Dr. Jean Paul Payan, Institut National de Recherche et de Sécurité, Avenue de Bourgogne, B.P. No. 27, 54501 Vandoeuvre Cedex, France. E-mail: [email protected] 0090-9556/03/3105-659–669$7.00 DRUG METABOLISM AND DISPOSITION Vol. 31, No. 5 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 994/1062080 DMD 31:659–669, 2003 Printed in U.S.A. 659 at A PE T Jornals on A ril 8, 2017 dm d.aspurnals.org D ow nladed from In Vivo Percutaneous Penetration and Absorption of [C]NMP by Sacrifice. One day before dosing, the middle of the back of the rats was clipped with electric clippers, and a circular ring (10 cm) was glued. After topical application of neat [C]NMP, 20 or 40 l/cm, the skin was covered by a perforated circular plastic cap to allow aeration. Batches of three to five hairy male rats were sacrificed at different times (0.5, 0.75, 1, and 2 h) after dosing by bleeding the abdominal aorta under mild ether anesthesia. The blood was collected on heparin. After sacrifice, the skin area of the application site was washed five times with 200 l of water to remove the unabsorbed fraction of NMP. A preliminary study showed that 5 min after topical application of [C]NMP, more than 95% of the radioactivity was removed by this washing process (n 3). The radioactivity of the application skin area and the skin area around the ring (about 30 cm) was measured after digestion in KOH solution. Radioactivity in the carcass and excreta (urine and feces) was also analyzed. In Vivo Percutaneous Penetration and Absorption of [C]NMP by Catheterism. For the sequential collection of urine and blood, a catheter was introduced into the carotid artery and bladder, respectively, 1 week before topical administration of [C]NMP. The catheters (internal diameter, 0.58 mm; external diameter, 0.96 mm) were introduced subcutaneously, exteriorized through the back of the neck, and inserted into a protective stainless tubing (about 2 g in weight) ligatured firmly to the skin. Urine was excreted by injecting saline solution (2 ml) into the bladder. Blood was collected on heparin. The rats were clipped 1 day before dosing. Neat [C]NMP (20 l/cm) was applied on the skin (10 cm). The application area was washed with water 24 h after dosing. Blood and urine were collected at different times until the animal was sacrificed (72 h). In Vitro Percutaneous Absorption of NMP. In vitro percutaneous absorption was assessed with static diffusion cells using fresh full-thickness skin of male rats (0.9–1.5 mm, 1.3 0.0, n 73). The rats were sacrificed with Pentobarbital. The whole dorsal region was shaved, and the excess of subcutaneous tissue carefully removed. The skin section was cut into circular sections (four per rat, 1.76 cm) and placed, stratum corneum side up, in diffusion cells. The diffusion cells were maintained at a temperature of 36°C with a circulating water bath, yielding a skin surface temperature of 32 1°C. The dermis side was kept in contact with the RPMI receptor fluid (Life Technologies, Paisley, Scotland) containing 2% bovine albumin and 1% penicillin-streptomycin. The fluid receptor was previously filtered through a sterile Millex (Millipore, Bedford, MA) 0.22m pore size filter and degassed with a vacuum pump. Preliminary experiments had shown that absorption flux was not significantly different when the receptor fluid was NaCl 0.9%. The integrity of the skin samples was assessed by determining the trans-epidermal water loss (Tewameter, TM210, Courage Khazaka) after an equilibrium time of 1 h. Neat or aqueous solutions of [C]NMP were applied on a skin surface area of 1.76 cm. The cells were non-occluded. An aliquot (200 l) of receptor fluid (5.15 ml) was collected at regular intervals from 24 to 52 h after sacrifice with an automatic fraction collector (Gilson FC 204, Middleton, WI). The same volume of fresh receptor fluid was automatically introduced into the cell to maintain the volume of the receptor fluid constant. At the end of the experiment, the unabsorbed dose of [C]NMP was removed with water (1 500 l) and cotton swabs. The skin was digested in KOH (25%, w/v). The radioactivity contained in the receptor fluid samples, the washing water, and the skin homogenates was measured by adding 10 ml of liquid scintillation solution (Pico Fluor 30; Packard, St. Louis, MO). Counting efficiency was determined by quenching correction curves for the various addition and scintillation fluids with a liquid scintillation spectrophotometer (CA 1900; Packard). Metabolism of NMP. Neat NMP was applied (200 l/cm) on fresh skin samples of one rat. After 2 h of exposure, the radioactivity contained in the receptor fluid, and skin homogenates in water were analyzed by the HPLC method described below. Reproducibility. Maximal percutaneous absorption rate (Fmax) and the time to reach it (Tmax) were determined in three independent experiments (three rats, two skin samples per rat) with neat NMP (400 l/cm) for 24 h of exposure. Fmax was normalized for a skin thickness of 1.3 mm according to eq. 1. Fmax Nor Fmax thickness of the skin sample/1.3 Effect of Neat NMP Doses. Fmax Nor and Tmax were determined after topical application of neat NMP (25–400 l/cm) for a 24-h exposure period. Effect of Hydration on NMP Absorption Flux. Different doses of neat [C]NMP (100, 200, and 400 l/cm) or H2O (400 l/cm ) were applied on skin samples, and aliquots of the receptor fluid were collected for 4 h. At the end of the experiment, the volumic radioactive concentration of unabsorbed NMP was determined. In a second experiment, neat [C]NMP was applied on the skin of male rats (400 l/cm) aliquot of unabsorbed dose (10 l) and of the receptor fluid (160 l) were collected at different times for 30 h to determine the evolution of the volumic radioactive concentration of the unabsorbed dose and percutaneous absorption fluxes, respectively. Additionally, neat NMP (400 l/cm) was introduced on a glass watch, and aliquots of NMP were collected to measure the radioactive concentration. Effect of NMP on the Transfer of H2O from the Receptor Fluid. Four groups of excised skin (n 3) were treated as follows. Group 1: 400 l of water were deposited on skin sample and H2O was introduced into the receptor fluid to measure the transfer of water from receptor fluid to the deposited water on skin. Group 2: 400 l of H2O were deposited on skin to measure the transfer of water from the deposited area to the receptor fluid. Group 3: 400 L of NMP were deposited on skin and H2O was introduced into receptor fluid to measure the transfer of water from the receptor fluid to deposited skin NMP. Group 4: 400 l of [C]NMP were deposited on skin to measure the absorption flux of NMP. At the end of the exposure (4 h), unabsorbed water or NMP was collected to measure H2O content for groups 1 and 3. For groups 2 and 4, radioactivity present in the receptor fluid was analyzed. Effect of Aqueous Dilution of NMP. An identical volume of aqueous dilutions of NMP (400 l/cm) at different concentrations (1:2 to 1:32, v/v) was applied on the skin. Percutaneous absorption was determined for 24 or 52 h. Effect of Occlusion or Desquamation. Percutaneous absorption fluxes of neat NMP (400 l/cm) were compared between skin samples of haired rats, with or without occlusion. Percutaneous absorption fluxes of neat NMP (100 l/cm) were compared between skin samples of hairless rats after desquamation (20 stripping, 100 g/cm) or without desquamation. HPLC Analysis. An HPLC method to analyze unchanged NMP and its main metabolites has been previously described (Payan et al., 2002). Briefly, proteins from plasma were precipitated in methanol. Unchanged NMP and its main metabolites (5-HNMP, 2-hydroxy-N-methylsuccinimide, and N-methylsuccinimide) contained in methanolic phase from plasma or aliquot of urine are analyzed by HPLC with a mixture of H2SO4 0.001 N and acetonitrile (85:15, v/v). The radioactivity contained in the HPLC eluates was measured with a liquid scintillation spectrophotometer. Analysis of Radioactivity. Samples of urine (500–1000 l) and plasma (500 l) were accurately weighed and added directly to liquid scintillation vials containing 10 ml of liquid scintillation solution (Pico Fluor 30; Packard). Samples of fresh feces were weighed and homogenized in water (1:5, w/v) in glass vials. Tissues (liver or kidney) were homogenized in water (1:5, w/v). Aliquots of feces or tissue homogenates (250–500 mg) were mixed with 10 ml of Pico Fluor 30. The radioactivity of all the samples was measured in a Packard liquid scintillation spectrophotometer model 1900. Counting efficiency was determined by quenching the correction curves of the various additions and scintillation fluids. Expression of Data and Statistical Analysis. Values were expressed as the percentage of [C]NMP dose per organ (%Qo/organ) or as the percentage of [C]NMP dose per gram (%Qo/g) of fresh tissue. The one-way ANOVA test was used to determine the significance of the means. The level of significance was set at p 0.05. The terminal elimination rate ( ) of NMP and its main metabolites in plasma were obtained by log-linear concentration time data. The area under the plasma curves of NMP and its metabolites from time 0 to the end of the experiment (AUC0-t) were calculated by the linear trapezoidal rule. The AUC from infinity was estimated by the calculated concentration at t divided by . The sum of both areas was AUC0-inf. The percentage of the absorbed dose was calculated from: a) the radioactivity content in the excreta and carcass % absorbed dose % applied dose in urine feces carcass (1) 660 PAYAN ET AL. at A PE T Jornals on A ril 8, 2017 dm d.aspurnals.org D ow nladed from b) the ratio of the AUC of NMP or 5-HNMP after topical application versus intravenous administration (500 mg/kg) % absorbed dose AUC0-inf topical application % dose/ml h 100 AUC0-inf % dose/ml h (2) c) the ratio of the total radioactivity, NMP, or 5-HNMP excreted in the urine after topical application versus intravenous administration (500 mg/kg) % absorbed dose % topical dose in urine 100 % intravenous dose in urine (3) Intravenous values are from Payan et al. (2002).