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ABSTRACT: Development, validation and application of an HPLC assay for new antiviral nucleoside analogues AM365 and AM188 in isolated perfused rat liver perfusate and bile were performed. An analytical column (Phenosphere-NEXT, 250 x 4.6 mm, C(18), 4 microm, Phenomenex) was used in tandem with a guard column (4 x 3 mm, C(18), Phenomenex) and operated at 25 degrees C. The mobile phase [methanol:10 mmol/L sodium orthophosphate buffer (pH 7.0), 15:85, v/v] was pumped at 1 mL/min. The signal from a diode array detector was collected from 190 to 300 nm. The chromatogram was processed at 220 and 252 nm for AM365 and AM188, respectively. The HPLC method was validated by six intraday and seven interday runs. Standard curves were linear in the range 0.125-8.00 microg/mL for AM365 and AM188, and the lower limit of quantification for AM365 and AM188 was 0.125 microg/mL. Mean interday precision and accuracy of IPL perfusate quality control samples were within 8.8%, and mean intraday precision and accuracy were within 13.1%. The assay has been successfully used in the study of metabolism and disposition of AM365 in the isolated perfused rat liver.
Biomedical Chromatography 04/2006; 20(3):244-50. · 1.97 Impact Factor
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ABSTRACT: The present study was designed to investigate the hepatic disposition of the prodrug AM365 and the generated antiviral guanosine analogue, AM188 in the isolated perfused rat liver (IPL). The livers of rats (n=12) were isolated and perfused with Krebs-Henseleit pH 7.4 buffer to which AM365 was added as a bolus to achieve an initial perfusate concentration of 22.4 micromol/L. During the 120-min period after administration of AM365, bile was collected in 10-min intervals and perfusate was collected at the mid-point of these intervals. Concentrations of AM365 and AM188 in perfusate and bile were quantified by HPLC. Following administration of AM365, its concentration in perfusate declined and the concentration of AM188 increased; the sum of the molar concentrations remained constant. The clearance and hepatic extraction ratio of AM365 were 3.3+/-2.4 mL/min and 0.110+/-0.079, respectively. The cumulative amount of AM365 excreted in bile during the 120-min perfusion period was approximately 0.21% of the bolus dose, and 0.36% of the total amount of AM365 cleared by the liver during the period. The cumulative amount of AM188 excreted in bile was about 0.48% of the total amount of AM188 formed during the perfusion period. In conclusion, AM365 was metabolised to AM188, which appeared to be the only metabolite and was not further biotransformed. The biliary excretion of AM365 and AM188 was negligible.
Current Drug Metabolism 11/2005; 6(5):487-93. · 5.11 Impact Factor
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ABSTRACT: To investigate the effects of potential inhibitors of membrane transport on the tubular secretion of AM188, an antiviral guanosine analog, in the isolated perfused rat kidney (IPK).
AM188 was administered to the IPK perfusate as a bolus/infusion regimen. In inhibitor groups, probenecid, p-aminohippuric acid (PAH), cimetidine, or nitrobenzylthioinosine was added to the perfusing medium.
In control IPKs, the ratio of renal clearance of AM188 (CLR) to GFR was 7.7 +/- 0.51 (mean +/- SD). The CL(R)/GFR ratio for AM188 was 6.20 +/- 0.41*, 2.85 +/- 0.20*, 1.45 +/- 0.07*, and 0.80 +/- 0.01* when the concentration of probenecid in perfusate was 10, 50, 100, and 1000 microM, respectively (*p < 0.05 compared to control group); the ratio was 7.71 +/- 0.38, 6.02 +/- 0.42*, 1.71 +/- 0.15*, and 0.91 +/- 0.07* for the PAH group and 6.42 +/- 1.70*, 5.33 +/- 1.53*, 3.16 +/- 0.81*, and 1.21 +/- 0.20* for the cimetidine group when the concentrations were 10, 100, 1000 and 10,000 microM, respectively; and the ratio was 5.33 +/- 0.21* when the concentration of nitrobenzylthioinosine was 5 microM.
These results suggest that renal tubular secretion of AM188 involves organic anion and cation transport systems.
Pharmaceutical Research 06/2004; 21(6):982-8. · 4.09 Impact Factor
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ABSTRACT: The rat isolated perfused kidney (IPK) preparation is a very useful model for pharmacokinetic and pharmacologic studies. Bovine serum albumin (BSA) is the oncotic agent used most commonly in IPK models, but the protein is expensive and varies significantly in quality. The present study evaluated the use of dextran to replace a large proportion of BSA as the oncotic agent, to establish a more reliable and economic IPK model for pharmacokinetic studies.
The right kidneys of male Sprague-Dawley rats were isolated and perfused in recirculating mode with Krebs-Henseleit pH 7.4 buffer containing amino acids, glucose and 65 g/l BSA (BSA group, n=11) or 6.5 g/l BSA plus 36 g/l dextran (dextran/BSA group, n=6). (14)C-Inulin was added to the perfusate to permit estimation of glomerular filtration rate (GFR). An antiviral guanosine analogue, AM188, was administered to the IPK perfusate to investigate its renal disposition. During the 130-min experimental period, urine was collected in 10-min intervals and perfusate was collected at the midpoint of these intervals.
The kidney functional parameters were generally better and more stable in the dextran/BSA IPKs when compared to the BSA group. At a similar perfusate flow rate, the IPKs in the dextran/BSA group exhibited lower renal artery perfusion pressure, a higher GFR, and more extensive tubular reabsorption of water, glucose, and sodium. These functional parameters were acceptable and stable throughout the whole experimental period in the dextran/BSA group. The renal clearance of AM188 was higher in the dextran/BSA group compared with that in the BSA group.
Using a large proportion of dextran and a small proportion of BSA as oncotic agent in perfusate provides an improved IPK preparation. This offers a reliable and economic rat IPK model for pharmacokinetic studies.
Journal of Pharmacological and Toxicological Methods 49(2):105-13. · 2.32 Impact Factor
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ABSTRACT: 1. AM 188 is an antiviral guanosine analogue that undergoes extensive renal excretion in humans. The present study was designed to investigate the disposition of AM 188 over a range of concentrations in the rat isolated perfused kidney (IPK) to explore the mechanisms involved in its renal handling. 2. Right kidneys of male Sprague-Dawley rats (n = 23) were isolated and perfused in recirculating mode with Krebs'-Henseleit (pH 7.4) buffer containing 0.65% bovine serum albumin, 3.6% dextran, amino acids and glucose. [14C]-Inulin was added to the perfusate reservoir to permit estimation of glomerular filtration rate (GFR). [3H]-AM 188 and unlabelled AM 188 were added to the perfusate as a bolus initially, followed by a constant rate of infusion at 5, 25, 125, 500 or 1000 microg/min to achieve initial target perfusate concentrations of 1, 5, 25, 100 or 200 microg/mL, respectively. During the 130 min over which AM 188 was infused, urine was collected in 10 min intervals (commencing 10 min after the bolus dose) and perfusate was collected at the mid-point of these intervals to permit calculation of the renal clearance (CLR) of AM 188. Binding of AM 188 in perfusate, measured using ultrafiltration, was negligible. 3. The bolus dose and infusion regimen produced relatively stable AM 188 concentrations in perfusate in the 5, 25 and 125 micro g/min groups and progressively increasing concentrations in the 500 and 1000 microg/min groups. High-pressure liquid chromatography analysis of IPK perfusate and urine suggested that there was no or negligible metabolism of AM 188 in the kidney. The CLR/GFR ratio for AM 188 (mean+/-SD) was 5.76 +/- 1.57, 5.99 +/- 0.52, 6.02 +/- 1.47, 3.38 +/- 0.26 and 1.08 +/- 0.42 in the 5, 25, 125, 500 and 1000 microg/min groups, respectively, showing significant reductions at the two highest infusion rates (P < 0.05). Although there was no difference between the five groups in the distribution of AM 188 between kidney tissue and perfusate (KT/P), at the end of perfusion the corresponding urine-to-tissue concentration ratio declined significantly in the 1000 microg/min group. 4. AM 188 undergoes substantial net renal secretion over a wide range of perfusate concentrations. A reduction in renal clearance at perfusate concentrations above 25 microg/mL could be due to saturation of carrier-mediated transport at the brush border membrane and/or a solubility limitation leading to precipitation of AM 188 in tubular cells and/or tubular urine.
Clinical and Experimental Pharmacology and Physiology 31(1-2):29-34. · 1.85 Impact Factor
