Renal Disposition of Recombinant Human Interleukin-11 in the Isolated Perfused Rat Kidney

Purpose. To clarify the mechanism of the renal clearance of recombinant human interleukin-11 (rhIL-11), we investigated the renal disposition characteristics of rhIL-11 in the perfused rat kidney. Methods. The disposition characteristics of ¹¹¹In-labeled rhIL-11 were analyzed using a single-pass indicator dilution technique and statistical moment analysis in the perfused rat kidney under filtering and nonfiltering conditions. Results. Steady-state distribution volume (V _d ) calculated from the venous outflow patterns of rhIL-11 at the doses of 0.3 to 10 g/kidney was between 0.35 and 0.40 ml/g kidney. However, V _d at the highest dose decreased to a value almost identical to that of bovine serum albumin, suggesting that there is a reversible and saturable interaction between the capillary wall and rhIL-11 molecule. In filtering kidney, a remarkable accumulation of rhIL-11 was observed while its urinary excretion was highly restricted at all doses. In nonfiltering kidney, rhIL-11 showed a decreased but still significant renal uptake. Taken together, the marked renal uptake of rhIL-11 may be explained by both efficient tubular reabsorption and significant uptake from the capillary side. These processes were not saturable within the tested dose range. These characteristics of rhIL-11 are likely based on non-specific electrostatic interaction with the tissues due to its cationic charge in the cytokine. Conclusions. The renal disposition processes of rhIL-11 were clarified at organ level in a quantitative manner. These findings agree well with previous observations in an in vivo disposition study in mice.     

Stereoselective Disposition of Ibuprofen Enantiomers in the Isolated Perfused Rat Kidney

The renal clearance of ibuprofen enantiomer was studied separately in the isolated perfused rat kidney at initial perfusate concentrations of 10 µg/ml (n = 4) and 100 µg/ml (n = 4). Perfusate and urine samples were measured for R(–) and S( + )-ibuprofen using a stereospecific HPLC assay; urine samples were also analyzed after alkaline hydrolysis. Functional viability of the kidney was assured by determining the fractional excretion of glucose and glomerular filtration rate (GFR) at similar perfusion pressures. The clearance of ibuprofen was equivalent to the apparent formation clearance of conjugated enantiomer since unchanged ibuprofen could not be detected in the urine. At 10 and 100 µg/ml, the clearance (±SD) of R(–)-ibuprofen was 2.50 ± 1.28 and 2.19 ± 1.42 µl/min, respectively. At 100 µg/ml, the clearance of S( + )-ibuprofen was 0.805 ± 0.290 µl/min. The protein binding of ibuprofen was found to be concentration dependent and favored the R(–)-enantiomer. The excretion ratio (clearance corrected for free fraction and GFR) of R(–)-ibuprofen was 0.398 ± 0.209 and 0.295 ± 0.209 for perfusate concentrations of 10 and 100 µg/ml, respectively. The excretion ratio of S( + )-ibuprofen was 0.0886 ± 0.0335 for perfusate concentrations of 100 µg/ml. These results demonstrate that the sum of renal mechanisms involved for the clearance of R(–)- and S( + )-ibuprofen was net reabsorption. Ibuprofen was recovered in the urine solely as conjugated material and no evidence of R(–) to S( + ) conversion was observed. In addition, the data suggest that R(–)-ibuprofen is cleared through the kidney faster than its S( + )-enantiomer.     

Transepithelial Transport of Levofloxacin in the Isolated Perfused Rat Kidney

Purpose. The transepithelial transport of levofloxacin was evaluatedin the isolated perfused kidney to investigate its renal secretorymechanisms. Methods. Levofloxacin was instantaneously administered into the renalartery together with inulin and Evans blue-labeled albumin, and thesingle-pass dilution curves of the renal venous and urinary outflowwere determined in the absence or presence of various compounds.Kinetic parameters were computed based on non-compartmentmoment analysis. Results. The ratio of fractional excretion to filtration fraction (FE/FF)for levofloxacin was 2.99 ± 0.18, indicating the involvement of tubularsecretion. In the presence of cimetidine and quinolones, the FE/FF oflevofloxacin was significantly decreased and the transepithelial meantransit time (¯T_cell) of levofloxacin was prolonged. The ¯T_cell showed anegative correlation with renal secretion of levofloxacin, while thevolume of distribution of levofloxacin showed no correlation. Conclusions. Transport on the brush-border membrane plays adetermining step in the renal secretion of levofloxacin, and cimetidineand quinolones interact with levofloxacin transport on thebrush-border membrane.     

Assessment of Drug Disposition in the Perfused Rat Brain by Statistical Moment Analysis

Drug disposition in the brain was investigated by statistical moment analysis using an improved in situ brain perfusion technique. The right cerebral hemisphere of the rat was perfused in situ. The drug and inulin were injected into the right internal carotid artery as a rapid bolus and the venous outflow curve at the posterior facial vein was obtained. The infusion rate was adjusted to minimize the flow of perfusion fluid into the left hemisphere. The obtained disposition parameters were characteristics and considered to reflect the physicochemical properties of each drug. Antipyrine showed a small degree of initial uptake. Therefore, its apparent distribution volume (V _i) and apparent intrinsic clearance (CL_int,i) were small. Diazepam showed large degrees of both influx and efflux and, thus, a large V_i. Water showed parameters intermediate between those of antipyrine and those of diazepam. Imipramine, desipramine, and propranolol showed a large CL_int,i compared with those of the other drugs. The extraction ratio of propranolol significantly decreased with increasing concentrations of unlabeled propranolol in the perfusion fluid. These findings may be explained partly by the tissue binding of these drugs. In conclusion, the present method is useful for studying drug disposition in the brain.     

Statistical Moment Analysis of Hepatobiliary Transport of Phenol Red in the Perfused Rat Liver

A new experimental system was applied to study hepatobiliary transport of drugs. Rat livers were perfused using a single-pass technique, and phenol red was momentarily introduced to this system from the portal side. Outflow dilution patterns of phenol red were analyzed using statistical moment theory, and kinetic parameters of hepatic distribution and elimination of phenol red were calculated from moments, namely, the hepatic extraction ratio (E _i) and elimination rate constant (k _el,i). A larger distribution volume (V _i) was obtained for phenol red than for ¹³¹I-human serum albumin (HSA) and ⁵¹Cr-red blood cells (RBC), indicating its extravascular diffusivity. The biliary excretion of conjugated phenol red was delayed relative to that of the free agent. The larger biliary mean transit time (t _bile,conj.) represents the processes of biliary transport and intrahepatic metabolism. Further, the effects of dose and perfusion temperature on the hepatobiliary transport of phenol red were determined. With high doses or low perfusion temperatures (20 and 27°C), E _i, k _el,i, and intrinsic clearance (CL_int,i) of phenol red and biliary recovery of free and conjugated phenol red (F _bile,free, F _bile,conj) significantly decreased. The temperature-dependent and saturable processes in hepatic uptake, metabolism, and biliary excretion of phenol red were assessable to moment analysis.     

Physiological Disposition of Ibuterol and Terbutaline in the Isolated Perfused Rat Lung

Abstract The absorption from the airways, uptake from the vascular compartment and biotransformation of ³H–ibuterol (diisobutyrate ester of terbutaline) and ³H–terbutaline have been studied using isolated perfused and ventilated rat lungs. After endotracheal instillation, ibuterol was more rapidly absorbed than terbutaline (P < 0.01 at 5 and 10 min. after administration) at doses of 55 and 550 nmol/lung. No difference was noted at 5.5 nmol/lung. When the drugs were added to the perfusion medium the calculated lung uptake for ibuterol was about 30 times that for terbutaline. Ibuterol was shown to be rapidly hydrolyzed to terbutaline. No or minor biotransformation of terbutaline was noted.     

Metabolism of I4C-Codeine in the Isolated,Perfused Rat Liver

Abstract: The metabolism of ¹⁴C-codeine in the isolated rat liver was studied in single-pass and recirculation perfusion experiments. The perfusate, a semi-synthetic medium with bovine erythrocytes, was delivered at a constant rate (12 ml/min.) and contained codeine in the range of 3–57 nmol/ml. Samples of perfusate were collected and analyzed for codeine and its metabolites after extraction and thin-layer chromatographic separation. In single-pass perfusion, steady state was reached within 20 min. The codeine concentration in the effluent perfusate varied from 17 to 48% of that in the affluent corresponding to extraction ratios of 0.83 to 0.52. There was a significant negative correlation between codeine dose and extraction ratio (r = 0.86, P<0.05, N = 6). The steady state concentration of free and conjugated morphine made a total of 21 to 49% of the molar concentration of codeine at the inflow side. The recovery of radioactivity at the end of the perfusions was on an average 89%. In recirculation perfusion experiments the codeine extraction ratios varied from 0.65 to 0.35. The amount of free morphine in the reservoir increased to a maximum within 25–45 min. Our results suggest a relatively high hepatic first-pass metabolism of codeine in the rat which is apparently dose-dependent. The quantitatively most significant metabolites of codeine are morphine and conjugated morphine. The rate and extent of morphine formation is compatible with the hypothesis that metabolically produced morphine may be responsible for the analgesic effect of codeine.     

Localization of Epidermal Growth Factor (EGF) Binding Sites on Antiluminal Plasma Membrane of Rat Kidney: Autoradiographic Study Using Nonfiltering Perfused Rat Kidney

We previously demonstrated that the specific binding of EGF to the antiluminal plasma membrane was a prerequisite step for the renal uptake of EGF. In the present study, the localization of ¹²⁵I-EGF binding sites on the antiluminal plasma membrane was investigated by tissue sampling and X-ray autoradiography in the nonfiltering kidney. The binding of ¹²⁵I-EGF was recognized over the whole kidney and was highest in the inner medulla followed by the cortex and outer medulla. The binding of ¹²⁵I-EGF in the nonfiltering kidney was completely inhibited in the presence of 20 nM unlabeled EGF, suggesting specific binding of ¹²⁵I-EGF to its receptor. Further, we used a histologic tissue staining method to confirm the location of the ¹²⁵I-EGF binding sites. Binding of ¹²⁵I-EGF was demonstrated on the proximal straight tubules (PST), cortical collecting ducts (CCD), inner medullary collecting ducts (IMCD), and thin limb of Henle in the inner medulla (IMTLH). We found that the binding of ¹²⁵I-EGF was high in the IMTLH. In addition, we determined the grain density both on the cell surface membrane and in the intracellular space of the proximal straight tubules, where the grain density on the antiluminal plasma membrane was approximately 50% that in the intracellular space at 20 min after the start of ¹²⁵I-EGF perfusion, suggesting the internalization of ¹²⁵I-EGF from the antiluminal plasma membrane to the intracellular compartment. In conclusion, the binding sites of ¹²⁵ I-EGF, which were accessible from the antiluminal side, were broadly distributed over the whole kidney and were most dense around the IMTLH.     

Characterization of LLC-PK1 Kidney Epithelial Cells as an in Vitro Model for Studying Renal Tubular Reabsorption of Protein Drugs

Purpose. The purpose of this study was to assess whether LLC-PK₁ renal epithelial cells could serve as an in vitro model for studying the renal tubular reabsorption of protein drugs. Methods. The association of ¹¹¹In-labeled model protein drugs, bovine serum albumin (BSA), superoxide dismutase (SOD), soybean trypsin inhibitor (STI), and [Asu^1,7]-eel calcitonin (Asu-ECT), with the monolayers of LLC-PK₁ renal epithelial cells was characterized under various conditions. Results. The cellular association of these proteins was temperature-dependent and varied according to the protein. Saturation kinetics were observed for STI association, with the apparent Km and Vmax values determined to be 66.3 µg/ml and 250 ng/mg protein/min, respectively. The association of STI decreased with increases in medium pH from 5.4 to 8.4 and was inhibited significantly by 2,4-dinitrophenol, sodium azide, cytochalasin B, and colchicine, suggesting that the cellular association involved endocytosis. Mutual inhibition was observed in competitive binding experiments with the four protein drugs, suggesting that they shared a common binding site on the luminal membrane of LLC-PK₁ cells. Taken together, these findings show that a variety of protein drugs bind to LLC-PK₁ cells in a non-specific manner and possibly undergo endocytosis, a phenomenon that is similar to in vivo proximal tubular reabsorption. Conclusions. LLC-PK₁ renal epithelial cells would be a suitable model system for the study of the renal proximal tubular reabsorption of protein drugs.     

Effect of 3,4-Dihydroxyphenylpyruvic Acid on the Metabolism of L-DOPA in Isolated Perfused Rat Liver

Abstract: The effect of 3,4-dihydroxyphenylpyruvic acid (DHPPA) on the hepatic metabolism of L-DOPA was investigated in isolated perfused rat liver. DHPPA decreased the initial hepatic extraction and prolonged the elimination half-life of L-DOPA when they were added simultaneously at the ratio 1:4 (L-DOPA: DHPPA) to the perfusate. At the ratio 1:1 DHPPA had no effect on the elimination half-life, but decreased the initial hepatic extraction of L-DOPA. When L-DOPA was added alone the initial loss of L-DOPA was 30% at 5 min. At that time the perfusion medium had passed the liver 2.5 times. Between 5 to 60 min. about 5% of the dose was extracted in a single pass through the liver. DHPPA added alone or together with L-DOPA was rapidly converted to L-DOPA, only about half of the DHPPA dose remained unmetabolized in the “plasma” at 2.5 min. The main metabolites in bile of all the compounds tested were conjugates of homovanillinic acid and 3,4-dihydroxyphenylacetic acid.     

Effects of Low and High Density Lipoproteins on Renal Cyclosporine A and Cyclosporine G Disposition in the Isolated Perfused Rat Kidney

Purpose. This study investigated the effects of low (LDL) and high density lipoproteins (HDL) on renal cyclosporine A (CsA) and cyclosporine G (CsG) disposition in the isolated perfused rat kidney model. Methods. Kidneys were perfused with CsA or CsG in perfusion medium containing 6% protein, bovine serum albumin only (BSA) (Control), LDL (200 mg/dl) and BSA, or HDL (200 mg/dl) and BSA. In vitro protein binding studies were conducted with CsA and CsG in the same media. Results. The unbound fractions (f_u) of CsA and CsG were significantly reduced with LDL and HDL in the perfusion media. In the presence of LDL, f_u for CsA and CsG was 3.9% and 5.9%, respectively. With HDL, f_u was 2.1% for CsA and 1.8% for CsG. f_u for the controls was 14.7% for CsA and 11.9% for CsG. Renal clearance (CL_R) of CsA and CsG was significantly reduced when perfused with perfusion medium containing LDL and HDL. LDL and HDL had similar effects on reducing CsA and CsG CL_R, and were four-fold lower when compared to controls (0.006 Vs. 0.023 ml/min). Renal CsA and CsG tissue (whole organ, cortex and medulla) concentrations were lower than corresponding controls when perfused with LDL or HDL. Conclusions. The interaction of CsA and CsG with LDL and HDL significantly reduced the CL_R and extent of renal tissue distribution of both compounds.     

农药叶枯灵在大鼠原位灌流肝中的代谢研究

采用反相HPLC、TLC、UV、IR和MS对农药叶枯灵经大鼠原位灌流肝代谢后所形成的代谢产物进行分离和鉴定。结果表明,叶枯灵在大鼠肝脏中进行了广泛的代谢,包括S-氧化作用、水解作用、丙酮酸缩合作用和乙酰化作用,共分离鉴定出5种代谢产物。     

Reduced Inactivation of Tyrosine Aminotransferase in the Perfused Rat Liver in the Presence of Ethanol

Abstract Livers from fasted male rats (250–300 g body weight) were isolated and perfused. The effect of ethanol in the perfusate (approximate concentration 0.2 (w/v)) on the activity of tyrosine aminotransferase was investigated in the presence and absence of dexamethasone (final concentration 20 (μ/ml) and/or cycloheximide (final concentration 25 μg/ml). The following observations were made: (1) Ethanol increased tyrosine aminotransferase activity both in the presence and absence of dexamethasone. (2) The addition of ethanol or its main metabolite acetaldehyde to the enzyme assay mixture did not increase enzyme activity. Neither did ethanol reduce the leakage of enzyme from liver to perfusate or bile. (3) Tyrosine aminotransferase activity declined more slowly in ethanol-treated livers than in control livers when protein synthesis was inhibited by cycloheximide. (4) Ethanol did not influence either the activity of another liver enzyme, tryptophan oxygenase, or the radioactivity remaining in labelled proteins 1 hr 30 min. after administration of cycloheximide. It is concluded that ethanol increased tyrosine aminotransferase activity in perfused livers by specific inhibition of enzyme inactivation.     

Disposition Characteristics of Plasmid DNA in the Single-pass Rat Liver Perfusion System

Purpose. To define the hepatic uptake mechanism of a plasmid DNA, we quantitated the uptake of pCAT (plasmid DNA encoding chloramphenicol acetyltransferase reporter gene fused to simian virus 40 promoter), a model plasmid, after a single pass through the perfused rat liver using albumin- and erythrocyte-free Krebs-Ringer bicarbonate buffer (pH 7.4). Methods. [³²P]pCAT was introduced momentarily into this system from the portal vein as a bolus input or constant infusion mode, and the outflow patterns and hepatic uptake were evaluated using statistical moment analysis. Results. The venous outflow samples had electrophoretic bands similar to that of the standard pCAT, suggesting that the plasmid is fairly stable in the perfusate during liver perfusion. In bolus experiments, pCAT was largely taken up by the liver and the uptake was decreased with increase in injected dose. Statistical moment analysis against outflow patterns demonstrated that the apparent volume of distribution of pCAT was greater than that of human serum albumin, indicating a significant reversible interaction with the tissues. The results of collagenase perfusion experiments suggest that the hepatic accumulation of pCAT occurred preferentially in the nonparenchymal cells (NPC). The amount of total recovery in the liver decreased substantially by preceding administration of polyinosinic acid, dextran sulfate, succinylated bovine serum albumin, but not by polycytidylic acid. This suggests that pC AT is taken up by the liver via scavenger receptors for polyanions on the NPC. In constant infusion experiments, the presence of 2,4-dinitrophenol and NH₄C1 caused a significant increase in the outflow concentration of [³²P]pCAT and decrease by half in the total hepatic recovery than that of plasmid DNA administered alone, suggesting that plasmid DNA may undergo internalization by the NPC. Conclusions. The liver plays an important role in the elimination of plasmid DNA and a successful delivery system will be required to avoid its recognition by the scavenger receptors on the liver NPC.     

Disposition of Glycosidase Inhibitors in the Isolated Perfused Rat Liver: Hepatobiliary and Subcellular Concentration Gradients of 1-Deoxymannojirimycin and N-Methyl-1-Deoxynojirimycin

The hepatic disposition of two glycosidase inhibitors was studied in the isolated perfused rat liver and after subcellular fractionation. The mannosidase inhibitor 1-deoxymannojirimycin (dMM) and the glucosidase inhibitor N-methyl-1-deoxynojirimycin (MedNM) exhibited minimal binding to albumin and reached liver concentrations that approximately equaled their medium concentrations, after 30 min (MedNM) or 90 min (dMM). Within 2 hr 0.5% of the dose of MedNM and 2.9% of dMM were excreted in bile. No metabolites were found for MedNM, whereas minor (bio)degradation was inferred for dMM. After subcellular fractionation, dMM and MedNM were found predominantly in the cytosolic fraction. Compared to the other paniculate fractions, MedNM was elevated in the microsomal fraction, and both compounds were slightly enriched in the lysosomal fraction. We conclude that dMM and MedNM will likely inhibit liver enzymes when sufficiently high plasma levels are reached.     

Hepatic Disposition Characteristics of Electrically Charged Macromolecules in Rat in Vivo and in the Perfused Liver

The effect of electric charge on the hepatic disposition of macromolecules was studied in the rat. Charged derivatives of dextran (T-70) and bovine serum albumin (BSA), mitomycin C–dextran conjugates (MMC-D), and lactosaminated BSA (Lac-BSA) were employed as model macromolecules. After intravenous injection, cationic macromolecules were rapidly eliminated from plasma because of their extensive hepatic uptake, while anionic and neutral macromolecules were slowly eliminated. Cationic macromolecules were recovered from parenchymal and nonparenchymal hepatic cells at a cellular uptake (per unit cell number) ratio of 1.4–3.2, while that of Lac-BSA was 14. During liver perfusion using a single-pass constant infusion mode, cationic macromolecules were continuously extracted by the liver, with extraction ratios at steady-state (E _ss) ranging between 0.03 and 0.54, whereas anionic and neutral macromolecules were almost completely recovered in the outflow at steady state. The E _ss for cationized BSA (Cat-BSA) and cationic MMC-Dcat were concentration dependent and decreased at low temperatures and in the presence of colchicine and cytochalasin B. The possible participation of the internalization process in the uptake of cationic macromolecules by hepatocytes was suggested.     

Comparison of the Hepatic Uptake Clearances of Fifteen Drugs with a Wide Range of Membrane Permeabilities in Isolated Rat Hepatocytes and Perfused Rat Livers

The hepatic uptake clearances of 15 ligands with a wide range of permeabilities were determined in rats using two techniques: centrifugal filtration with isolated hepatocytes and the multiple indicator dilution (MID) method with isolated perfused livers. Some of the uptake clearance values were taken from the literature. Uptake clearance values obtained from isolated hepatocytes were extrapolated to that per gram liver (PS _{inf, cell}), assuming that 1 g of liver has 1.3 × 10⁸ cells. The values of PS _{inf, cell} varied from approximately 0.1 to 72 (mL/min/g liver). The values of PS _{inf, cell} were similar to those (PS _inf,MID) determined by the MID method for ligands with uptake clearances below approximately 1 mL/min/g liver. However, for the ligands with larger uptake clearances, the PS _{inf, MID} values were lower than the PS _inf,cell values and appeared to reach an upper limit (approx. 15–20 mL/min/g liver). The PS_inf,cell values of 1-propranolol, tetraphenylphosphonium (TPP⁺), and diazepam were 72, 43, and 22 mL/min/g liver, respectively, whereas their uptake clearances (PS _inf,MID) determined by the MID method were 4 to 10 times lower. One of the possible mechanisms for this discrepancy is that an unstirred water layer, which may exist in Disse's space in isolated perfused livers (and probably under in vivo condition), limits the hepatic uptake rate of ligands with extremely high membrane permeabilities.     

Renal Secretion of the Antiviral Nucleoside Analog AM188 Is Inhibited by Probenecid,p-Aminohippuric Acid,and Cimetidine in the Isolated Perfused Rat Kidney

Purpose. 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). Methods. 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. Results. In control IPKs, the ratio of renal clearance of AM188 (CL_R) 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 M, 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 M, respectively; and the ratio was 5.33 ± 0.21* when the concentration of nitrobenzylthioinosine was 5 M. Conclusions. These results suggest that renal tubular secretion of AM188 involves organic anion and cation transport systems.     

蛋白多肽类药物长效化技术研究进展

近年来,蛋白多肽类药物因在治疗疾病方面具有显著的优点,普遍受到研究者的青睐;另一方面,蛋白多肽类药物半衰期一般较短,影响患者用药顺应性。结合蛋白多肽类药物药动学特点,总结蛋白多肽类药物长效化方法的研究进展,主要包括氨基酸替代、定点修饰突变、通过基因手段与其他蛋白融合、糖基化、环化、与聚合物缀合、钉合肽,以及新剂型开发等方法,希望能对药物研发工作者有所帮助。