Short Communication PHARMACOKINETIC STUDIES OF 2-AMINO-9-(3-ACETOXYMETHYL-4- ISOPROPOXYCARBONYL-OXYBUT-1-YL)PURINE, AN ORAL PRODRUG FOR THE ANTIVIRAL AGENT PENCICLOVIR

2-Amino-9-(3-acetoxymethyl-4-isopropoxycarbonyloxybut-1-yl)purine (SK1899) was tested as an oral prodrug for penciclovir. SK1899 was administered orally to rats and dogs at doses up to 2 and 0.68 mmol/kg, respectively. SK1899 was well absorbed, and the major metabolites detected in plasma and urine were penciclovir, the active antiviral compound, and 6-deoxypenciclovir (M4) in both species. In rats, SK1899 was rapidly and extensively metabolized to penciclovir, which reached the peak plasma concentration (Cmax) of 39.5 mM at 0.5 h after 0.2-mmol/kg dosing. The area under the plasma concentration-time curve (AUC) for penciclovir was 57.5 mM z h. After an oral dose of 0.034 mmol/kg to dogs, extensive conversion of SK1899 to penciclovir also occurred with slower rate of formation of penciclovir from M4 than in rats. The mean Cmax and AUC for penciclovir were 4.5 mM at 2.7 h and 28.2 mM z h, respectively. The 0to 24-h urinary recovery of penciclovir represented 36.1 and 36.3% of dose to rats and dogs, respectively. Radioactivity was found in fetuses following an oral administration of [C]SK1899 to pregnant rats, but no significant accumulation was observed. Although substantial milk transfer of [C]SK1899 occurred in rats, the radioactivity in milk was rapidly cleared. The values of Cmax, AUC, and urinary recovery of penciclovir after dosing with SK1899 to rats and dogs were similar or slightly higher than those from famciclovir. These data indicate that introduction of an isopropoxy carbonate group into one of the two hydroxyl groups of M4 did not significantly alter the oral bioavailability of penciclovir compared with famciclovir. Penciclovir is a potent and highly selective inhibitor of herpes viruses such as herpes simplex virus types 1 and 2, varicella-zoster virus, Epstein-Barr virus, and also of hepatitis B virus (Boyd et al., 1987, 1988a; Harnden et al., 1987; Sutton and Boyd, 1993; Korba and Boyd, 1996). However, penciclovir showed poor bioavailability when administered orally to humans (Boyd et al., 1988b). To improve this poor oral bioavailability, the diacetate ester of the 6-deoxy derivative of penciclovir, famciclovir, was developed as a prodrug of penciclovir (Harnden et al., 1989; Vere Hodge et al., 1989; Pue and Benet, 1993). After an oral administration of famciclovir to rats (0.125 mmol/kg) and dogs (0.078 mmol/kg), the urinary recoveries of penciclovir represented 36.0 and 36.4% of the dose, respectively (Filer et al., 1995). In humans, approximately 60% of dose was excreted as penciclovir in urine following an oral dose of 500 mg (1.56 mmol) of famciclovir (Pue et al., 1994). To further improve the oral bioavailability of famciclovir, SK1899 was developed in our laboratory as an oral prodrug of penciclovir. SK1899 showed higher urinary recovery of penciclovir than famciclovir after oral administration to mice, and effective in vivo antiviral activities against herpes simplex virus type 1 and duck hepatitis B virus (Kim et al., 1999). These results led us to undertake pharmacokinetic studies of SK1899 to compare plasma concentration-time profiles and urinary excretions of its active metabolite penciclovir and a major intermediate metabolite M4 in rats and dogs with those of famciclovir. Transfer into the fetus and milk of [C]SK1899 was also investigated. Experimental Procedures Materials and Animals. SK1899 and [C]SK1899 (specific activity, 16.4 mCi/mg) were synthesized by SK Chemicals (Suwon-Si, Korea). Chemical purity of SK1899 and radiochemical purity of [C]SK1899 detected by reverse phase HPLC were 99.5 and 99.1%, respectively. Trichloroacetic acid, sodium bicarbonate, sodium azide, and oxytocin were purchased from Sigma Chemical Co. (St. Louis, MO). Sep-Pak cartridges were obtained from Waters Co. (Milford, MA). Methanol and water were of HPLC grade. Male and female rats were purchased from Charles River Japan, Inc. (Yokohama, Japan) and housed in temperatureand humidity-controlled facilities on a 12-h light cycle with free access to food and water. Beagle dogs were supplied by Marshall Farms, Inc. (North Rose, NY). Pharmacokinetic Study. Male Sprague-Dawley rats (250–300 g) housed separately in metabolic cages were each given a single oral dose of SK1899 (0.2 or 2 mmol/kg) or famciclovir (0.2 mmol/kg) with an intragastric needle. Blood sample was collected from the rat’s tail into heparinized Pasteur pipette. One-hundred fifty microliters of each sample was immediately mixed with the same volume of 16% trichloroacetic acid in a separate tube and centrifuged, and 200 ml of the supernatant was neutralized with 40 ml of saturated solution of sodium bicarbonate. The urine sample was collected in a metabolic cage, which efficiently separated urine and feces. A 5% solution of sodium azide 1 Abbreviations used are: SK1899, 2-amino-9-(3-acetoxymethyl-4-isopropoxycarbonyloxybut-1-yl)purine; Cmax, peak plasma concentration; Tmax, time of peak plasma concentration; AUC0-`, area under the plasma concentration-time curve extrapolated to infinity; t1/2, elimination half-life; HPLC, high-performance liquid chromatography. Address correspondence to: Guang-Jin Im, Life Science Research Center, SK Chemicals, 600 Jungja-1-Dong, Changan-Ku, Suwon-Si, Kyungki-Do 440745, Korea. E-mail: [email protected] 0090-9556/01/2907-945–949$3.00 DRUG METABOLISM AND DISPOSITION Vol. 29, No. 7 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 257/911081 DMD 29:945–949, 2001 Printed in U.S.A. 945 at A PE T Jornals on Jne 5, 2017 dm d.aspurnals.org D ow nladed from (0.4 ml per estimated 100 ml of urine) was added to each urine receptacle before collection to prevent bacterial growth. The samples collected from the animals were stored in 270°C until analysis. Three male beagle dogs (approximately 10 months old; 9–13 kg) were housed individually in labeled metabolic cages. The dogs were dosed once orally by capsule at 0.034 and 0.68 mmol/kg of SK1899 and at 0.034 mmol/kg of famciclovir. Samples of blood (approximately 3 ml) were withdrawn from each animal via a cephalic vein up to 24 h after dosing. After collection, blood samples were processed as described above. Urine sample of each dog was separated from feces and collected in a metabolic cage. The urine and plasma samples from rats and dogs dosed with SK1899 or famciclovir were analyzed by reverse phase HPLC (Waters 2690, Waters Co.) as described previously (Kim et al., 1999). Placental Transfer. A single oral dose of [C]SK1899 (0.2 mmol/kg, 30 mCi/kg) was given to three pregnant Sprague-Dawley rats (17th day of gestation, 275–320 g) for each sampling time point. Blood sample was collected from tail into heparinized tube and centrifuged to obtain plasma. After each group of animals was anesthetized with diethyl ether, fetus, placenta, and amniotic fluid were taken from pregnant rats. Excretion in Milk. Before oral administration of [C]SK1899 to lactating rats, the pups were removed from their mothers for 5 min from day 4 until day 11 after parturition, and each litter was reduced to six pups at day 6 after parturition. A single oral dose of [C]SK1899 (0.2 mmol/kg, 30 mCi/kg) was given to three Sprague-Dawley rats (12th day after parturition, 285–350 g) for each sampling time point. The maternal rats were separated from their pups at 1 h before sampling and injected with 20 IU/kg oxytocin intraperitoneally at 15 min before sampling to stimulate milk secretion. Radioactivity Measurements. The aliquots of urine, amniotic fluid, and milk were mixed with Lumagel Safe (Lumac*LSC, Groningen, The Netherlands) and counted directly for radioactivity. Plasma was solubilized with Soluene-350 (Packard Instrument Co., Meriden, CT) and assayed for radioactivity following the addition of Lumagel Safe and glacial acetic acid to minimize chemiluminescence. Fetus and placenta were air-dried and combusted with a sample oxidizer (Tri-Carb model 307, Packard) without further processing. Feces were homogenized with water and then combusted using aliquots. The resulting CO2 was adsorbed on Carbo-sorb E (Packard) and then mixed with Permafluor E scintillation fluid (Packard). The radioactivity of sample was measured using a liquid scintillation analyzer (Tri-Carb 1500, Packard) and converted to equivalents of SK1899 based on the specific radioactivity of the administered [C]SK1899. Pharmacokinetic and Statistical Analyses. Pharmacokinetic parameters were analyzed by noncompartmental model using the WinNonlin program (Scientific Consulting Inc., Cary, NC). The experimental results were evaluated by analysis of variance for statistical significance.