Application of the isolated perfused rat kidney model to assess gender effects on drug excretion.

PURPOSE: To study the effect of gender on the renal disposition of two organic anions, p-aminohippuric acid (PAH) and furosemide (FSM) in the isolated perfused rat kidney (IPK). METHODS: IPK experiments (3-4 per treatment group) were conducted using kidneys from male and female Sprague Dawley rats. PAH was administered as a continuous infusion (with loading dose, targeted steady-state concentration 10 ug/mL). FSM was added as a bolus dose (2.65 mg, targeted concentration 33 ug/mL). Urine was collected in 10-min. intervals and perfusate was sampled at the midpoint of each collection period. Control (drug naïve) perfusions were performed for both genders. PAH and FSM were measured by HPLC. Kidney viability (GFR [estimated using inulin clearance], sodium reabsorption, glucose reabsorption) was monitored continuously during each perfusion experiment (2-h duration). RESULTS: Good kidney function was maintained across all study groups, and lower GFR estimates in female kidneys were due to differences in kidney weight. For PAH, kidney weight corrected renal clearance (0.88 +/- 0.37 mL/min/g vs. 0.59 +/- 0.19 mL/min/g) and excretion ratio (3.8 +/- 1.7 vs. 2.2 +/- 0.72) were significantly higher in male kidneys. For FSM, renal clearance was significantly lower in female (0.10 +/- 0.05 mL/min/g) compared to male kidneys (0.15 +/- 0.07 mL/min/g). Mass balance analysis showed that FSM cumulative urinary excretion was significantly higher and kidney accumulation was significantly lower in experiments with male kidneys. CONCLUSIONS: The study demonstrates that the IPK is a useful model to assess gender effects on renal drug disposition. The renal excretion of organic anions is reduced in female rats, possibly due to gender differences in expression and/or activity of membrane transporters (both basolateral and luminal) in the kidney.

Alternative high-performance liquid chromatographic assay for p-aminohippuric acid (PAH): effect of aging on PAH excretion in the isolated perfused rat kidney.

Para-aminohippuric acid (PAH), an indicator of renal plasma flow, is a commonly used marker of organic anion transport by the kidney. An analytical method for PAH using HPLC was developed. The method is simple, fast and requires a minimum amount of organic solvent. Sample preparation involved protein precipitation with zinc sulfate. Para-amino benzoic acid was utilized as an internal standard (IS). Chromatography was performed using a reversed-phase phenyl column with UV detection at a wavelength of 254 nm. Mobile phase consisted of 0.1 M acetic acid and acetonitrile (99:1) at a flow rate of 1 ml/min. The assay was validated over a standard concentration range from 1 to 25 microg/ml. Accuracy, precision, reproducibility and specificity of the method was established with coefficients of variation <10%. The method was sensitive and showed linear response in peak height ratio (analyte:IS) over the concentration range studied (r(2)>0.99). The assay was used to study the effect of aging on PAH excretion in the isolated perfused rat kidney model. Experiments were conducted in kidneys from young (2-3 months, n=6), adult (6-9 months, n=5) and aged (12-16 months, n=3) male Sprague-Dawley rats at an initial drug concentration of 20 microg/ml. Significant differences in kidney function (e.g. glomerular filtration rate and glucose reabsorption) were observed in aged kidneys. Despite a 5-fold reduction in glomerular filtration rate, PAH renal clearance (kidney weight-corrected) decreased by only 2-fold in aged (2.2+/-0.42 ml/min per gram) compared to young (4.6+/-0.70 ml/min per gram, P<0.05) rats. Furthermore, renal excretion ratio was significantly higher in aged rats (27+/-8.0 vs. 15+/-5.0, P<0.05). These preliminary findings challenge the 'Whole Nephron Hypothesis' that assumes parallel reductions in renal filtration and secretory capacity secondary to disease or aging.

In vitro characterization of the erythrocyte distribution of methazolamide: a model of erythrocyte transport and binding kinetics.

The rate and extent of binding of methazolamide to human erythrocytes was studied in vitro. All experiments were carried out at physiological temperature (37 C) and pH (7.4). Methazolamide (MTZ) buffer concentrations were analyzed by HPLC. Distributional equilibrium between buffer and washed red blood cells was achieved after 1 hr. Results of equilibrium studies were consistent with two classes of binding sites for MTZ within the erythrocyte: a low affinity, high capacity site (CA-I) and a high affinity, low capacity site (CA-II). A two-binding site model was fitted to experimental data generating estimates for binding parameters Ka1 (0.0017 +/- 0.00022 microM-1) nM1 (636 +/- 5.23 microM), Ka2(0.46 +/- 0.0083 microM-1), and nM2(80.9 +/- 0.389 microM). Based upon these findings, kinetic studies were performed in order to characterize the rate of drug distribution. The rate of erythrocyte uptake of MTZ was mathematically modeled using a series of differential equations describing drug diffusion across the red blood cell membrane and subsequent complexation with intracellular binding sites. The model assumed that penetration of MTZ into the red blood cells was passive but drug binding to the carbonic anhydrase isozymes was not instantaneous. Using a novel curve fitting technique, parameter estimates of RBC membrane permeability (0.0102 +/- 0.000618 cm/min), and binding rate constants k-1(0.254 +/- 0.0213 min-1), k1 (0.0022 +/- 0.00020 ml/microgram-min), k-2(1.59 +/- 0.0358 min-1), and k2(3.1 +/- 0.035 ml/microgram-min) were obtained. The model characterized the observed biphasic decline of MTZ buffer concentrations over time and may help explain the prolonged residence of MTZ in vivo.

Blood disposition and urinary excretion kinetics of methazolamide following oral administration to human subjects.

The pharmacokinetic disposition of methazolamide (MTZ) was studied in five healthy volunteers following administration of a single oral dose. Drug concentrations in blood, plasma, and urine were measured by HPLC. Over the range of plasma concentrations observed in vivo, MTZ free fraction (measured by ultrafiltration) was 0.28. Being a carbonic anhydrase inhibitor, MTZ would be expected to distribute into, and be sequestered by, red blood cells. For this reason, MTZ disposition was characterized utilizing blood concentrations as the reference. Using a two-compartment model, a series of differential equations were simultaneously fitted to blood concentrations and urinary excretion data generating estimates for k10 (0.035 +/- 0.019 h(-1)), k12 (0.200 +/- 0.036 h(-1)), k21 (0.077 +/- 0.046 h(-1)), k(a) (0.304 +/- 0.064 h(-1)), Vc (1.1 +/- 0.18 L) and f(r) (fraction excreted renally, 0.61 +/- 0.14). Total blood clearance was 0.037 +/- 0.020 L h(-1). The model estimate of elimination half-life (126 +/- 61 h) was consistent with drug binding to a high affinity carbonic anhydrase isozyme in the erythocyte. Estimates of MTZ renal clearance and renal excretion ratio were 0.021 +/- 0.010 L h(-1) and 0.16 +/- 0.06, respectively. Overall, the prolonged elimination of MTZ from the blood is the result of extensive erythrocyte distribution and tubular reabsorption by the kidney.

Determination of methazolamide concentrations in human biological fluids using high performance liquid chromatography.

Methazolamide is a carbonic anhydrase inhibitor used to treat glaucoma. In vivo, methazolamide readily distributes into red blood cells. Therefore, both blood and plasma concentration data are needed in order to characterize the pharmacokinetics of methazolamide. In the present study, an analytical method using high performance liquid chromatography was validated for determination of methazolamide concentrations in several biological fluids. Through slight modification of a previously reported method for acetazolamide, another carbonic anhydrase inhibitor, methazolamide was readily quantitated in whole blood, plasma and urine. Sample preparation involved liquid-liquid extraction with ethyl acetate followed by a washing step using phosphate buffer (pH 8.0). After back extraction into glycine buffer (pH 10.0), samples were then washed with ether and injected onto the chromatograph. Chromatography was performed using a C-18, 5 microns reverse-phase column with UV detection at a wavelength of 285 nm. Mobile phase consisted of 0.05 M sodium acetate (pH 4.0) and acetonitrile (20%). The assay was validated over two standard concentration ranges from 1 to 100 micrograms ml-1, concentrations reflective of those expected in vivo, Calibration curves were linear for all biological fluids and coefficients of variation for interday and intraday reproducibility studies were less than 8% (range 3.1-7.9%). The method was used to measure methazolamide concentrations in blood, plasma and urine following oral administration to five human subjects.

Application of a first-pass effect model to characterize the pharmacokinetic disposition of venlafaxine after oral administration to human subjects.

Venlafaxine (VEN), a drug used in the treatment of depression, undergoes significant first-pass metabolism after oral dosing to O-desmethylvenlafaxine (ODV), a metabolite with comparable therapeutic activity to that of parent drug. The pharmacokinetic disposition of VEN was characterized using a "first-pass" model that incorporates a presystemic compartment (liver) to account for the first-pass metabolism of VEN to ODV. A series of differential equations were simultaneously fitted to plasma concentrations of parent and metabolite. A good fit of the model to observed data was demonstrated, generating estimates for the following parameters: ka (1.31 +/- 0.009 hr-1), VVEN (252 +/- 87.6 liters), CLint (65.8 +/- 39.7 liters/hr), RL (liver:plasma partition coefficient, 29.6 +/- 18. 3), VODV (181 +/- 84.1 liters), and CLODV (23.5 +/- 12.5 liters/hr). Parameter estimates correlated closely with those obtained through noncompartmental methods. These results indicate that the time-course disposition of a compound undergoing first-pass hepatic metabolism after oral dosing can be successfully modeled.

Evidence for reversible sequestration of morphine in rat liver.

The residence of morphine in the systemic circulation is prolonged despite a high systemic clearance, suggestive of significant extravascular sequestration. The present study was conducted to test the hypothesis that morphine binds significantly in tissues, and that the liver plays an important role in morphine binding. [14C]Morphine was administered to male Sprague-Dawley rats 55 min before unlabeled morphine or saline. Blood 14C increased immediately after injection of unlabeled morphine; the area under the blood concentration-time curve (AUC) for 14C increased approximately 2-fold after morphine compared with saline injection. Residual radioactivity in the liver was lower in morphine-treated rats than in controls, suggesting that unlabeled drug displaced [14C]morphine (or a metabolite) from binding sites. To examine this phenomenon more directly, a recirculating isolated perfused liver system was employed. [14C]Morphine was added to the perfusate reservoir 15 min before unlabeled morphine or saline; perfusate and bile samples were collected for 120 min. Upon termination of perfusion, the liver was fractionated to identify the hepatic subcellular fraction(s) in which morphine was sequestered. The perfusate AUC for [14C]morphine was increased approximately 2-fold in response to unlabeled drug, consistent with the in vivo experiment. Morphine was associated preferentially with the cytosolic fraction, and [14C]morphine in all relevant fractions was reduced after administration of unlabeled morphine. In contrast, unlabeled drug had no influence on derived [14C]morphine-3-beta,D-glucuronide. These data are consistent with significant, reversible binding of morphine in hepatic tissue.

Concentration-dependent tubular secretion of acetazolamide and its inhibition by salicylic acid in the isolated perfused rat kidney.

An isolated perfused rat kidney (IPK) technique was used to study the effect of salicylic acid (SA) on the excretion of acetazolamide (AZ). Initial experiments were conducted in the absence of interactants at three nominal AZ concentrations (50, 100, and 250 micrograms/ml). Over the concentration range studied, AZ demonstrated net tubular secretion in the IPK. Significant decreases in excretion ratio (4.97 +/- 0.79-2.66 +/- 1.1) and secretory clearance (0.809 +/- 0.23-0.541 +/- 0.28) were observed with increasing AZ concentration, consistent with saturation of tubular secretion. Using a facilitated model for renal secretion, values of tubular transport parameters were obtained from a plot of excretion ratio vs. unbound AZ concentration: tmax = 118 +/- 29.4 micrograms/min, KM = 53.4 +/- 22.4 micrograms/ml, and tmax(A) = 6.31 +/- 2.82 micrograms/min. In the presence of SA (200 micrograms/ml), renal secretion of AZ was inhibited, as demonstrated by significant decreases in renal clearance (0.731 +/- 0.21-0.147 +/- 0.03) and excretion ratio (3.77 +/- 0.82-0.378 +/- 0.07). Although these findings were indicative of a reabsorption component to AZ excretion in the IPK that had not been previously proposed, the results were consistent with a previous investigation of concomitant administration of AZ and SA in humans (Br. J. Clin. Pharmacol. 27, 866, 1989), thereby endorsing utilization of the IPK as a screening tool for renal clearance mechanisms in humans.

Renal disposition and drug interaction screening of (-)-2'-deoxy-3'-thiacytidine (3TC) in the isolated perfused rat kidney.

PURPOSE: Dideoxynucleoside bases are used for the treatment of acquired immune deficiency syndrome (AIDS), acting by inhibiting reverse transcriptase and preventing human immunodeficiency virus (HIV) replication. Currently, AZT (zidovudine), ddC (zalcitibine), and ddI (didanosine) are available to the medical community to prevent the onset of AIDS in HIV-infected individuals. 3TC (-)-2'-deoxy-3'-thiacytidine, lamivudine), a new dideoxynucleoside base, is currently undergoing Phase II/III trials, and has exhibited anti-HIV replication activity, a favorable adverse event safety profile, and is eliminated via renal mechanisms. Concomitantly administered drugs could potentiate the effects of 3TC due to interaction in the kidney. METHODS: An isolated perfused rat kidney (IPK) technique was used to screen several clinically relevant drugs for potential interaction with 3TC. The following perfusions were performed: baseline 3TC; and 500 ng/mL 3TC with clinically relevant concentrations of AZT, ddC, ddI, probenecid, trimethoprim, sulfamethoxazole, ranitidine, and cimetidine. RESULTS: Renal clearance of 3TC was nonlinear between 500 and 5000 ng/mL, decreasing from 3.06 to 1.74 mL/min. Excretion ratio also decreased, from 3.67 (500 ng/mL) to 2.49 (5000 ng/mL), consistent with a decrease in 3TC secretion. AZT, ddI, and ddC elicited no or minimal effects on 3TC elimination at the concentrations studied. However, trimethoprim caused significant reductions in 3TC elimination parameters: clearance and excretion ratio decreased to 1.25 mL/min and 1.43, respectively. CONCLUSIONS: These results indicate that caution should be exercised when the combination of 3TC and trimethoprim are administered to AIDS patients.

The influence of protein binding on the elimination of acetazolamide by the isolated perfused rat kidney: evidence of albumin-mediated tubular secretion.

The impact of albumin on the renal elimination of acetazolamide, a low extraction ratio compound, was investigated in the isolated perfused rat kidney. Perfusion studies were conducted over a wide range of protein concentrations (0.25, 0.5, 0.75, 1.0, 4.0 and 6.0 g/100 ml) and an initial drug concentration of 100 micrograms/ml. Kidney viability was within normal limits among all treatment groups. Over the range of albumin levels studied, an approximate 3.4-fold increase in drug-free fraction effected a 2.8-fold increase in renal clearance. Although this finding contradicted conventional wisdom regarding extraction ratio and renal elimination, the results were consistent with a proposed ancillary role of albumin in renal tubular transport processes. An alternative clearance model was developed, analogous to earlier models of hepatic elimination. The facilitated renal clearance model utilized and validated in this investigation represents a composite of previously proposed theories, modified to account for albumin-mediated tubular secretion.