Influence of pH on the Uptake and Pharmacodynamics of Quinidine in the Isolated Perfused Rat Heart

Using the single-pass isolated perfused rat heart preparation we examined the effect of perfusate pH (pH 7.05, 7.46, 7.71, 7.92) on quinidine output concentration (C_out) and ?QT. Eight hearts were perfused at 2.5 ml/min. with quinidine (20 μM) for 35 min. followed by a 35-40 min. washout period with drug-free perfusate. This procedure was repeated four times in each preparation with the pH sequence varied and the same pH used in the first and last phases. Increasing pH slowed the rate of equilibration of C_out, the equilibration rate constant (k) decreasing from 0.273 min.^-1 at pH 7.05 to 0.095 min.^-1 at pH 7.92. A modified Kety-Renkin-Crone equation was fitted to the C_out versus time data for each pH. The estimated volume of distribution (V) increased significantly with pH from 11.5 × 1.1 to 32.5 × 2.9 ml/g, but the permeability surface product did not change with pH (mean 17.7 ml/min./g). There was a linear relationship between V and calculated un-ionised quinidine C_out, with an intercept of 5.70 ml/g corresponding to the V of ionised drug. This indicates that ionised and un-ionised drug readily enter the heart and that the slower equilibration with pH is due to the increased V which results from increased partitioning of un-ionised quinidine into myocardial tissue. Perfusion pH did not directly affect baseline QT interval, but the rate of attainment of maximum ?QT decreased with increasing perfusate pH. Plots of ?QT versus calculated coronary output quinidine concentration did not change with pH, showing that this drug effect was due to both ionised and un-ionised moieties. This study shows that myocardial permeability and pharmacodynamic effect (?QT) of quinidine are not influenced by perfusion pH over the range 7.0 to 7.9, although rate of equilibration of both C_out and effect vary with pH.     

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.     

Hepatic Distribution and Clearance of Antisense Oligonucleotides in the Isolated Perfused Rat Liver

Purpose. This study was conducted to investigate the impact of backbone modifications on the hepatobiliary disposition of oligonucleotides. Methods. The disposition of backbone-modified antisense oligonucleotides [phosphorothioate (PS) and methylphosphonate (MP)] of the same base-length and sequence (5-TAC-GCC-AAC-AGC-TCC-3), complementary to the codon 12 activating mutation of Ki-ras, was investigated in the isolated perfused rat liver. Livers were perfused for 2 hr; perfusate and bile concentrations were analyzed by HPLC. Hepatocellular distribution was examined by measuring the amount of radiolabeled PS oligonucleotide associated with hepatocytes and Kupffer cells. Protein binding of the PS and MP oligonucleotides was determined in rat serum by ultrafiltration. Results. MP oligonucleotide perfusate concentrations remained constant during the 2-hour perfusion. In contrast, PS oligonucleotide was eliminated slowly by the isolated perfused liver [Cl = 1.05 ± 0.21 mL/min; extraction ratio = 0.06 ± 0.01]. Uptake of PS oligonucleotide by Kupffer cells appeared to exceed uptake by hepatocytes, based on standard cell separation techniques as well as confocal microscopy. The degree of protein binding in rat serum was greater for the PS oligonucleotide (79.9 ± 2.2%) than for the MP oligonucleotide (53.0 ± 4.7%). Conclusions. Backbone modifications significantly influence the hepatic clearance of oligonucleotides. Uncharged MP oligonucleotides are not extracted by the isolated perfused rat liver, whereas the charged PS oligonucleotide is processed more readily.     

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.     

Effect of Plasma Protein Binding on Kinetics of Capillary Uptake and Efflux

We examined the effect of plasma protein binding on the kinetics of organ accumulation and washout of drugs using the single-pass Kety–Renkin–Crone capillary model. An equation relating the accumulation and washout rate constant (k) with the plasma unbound fraction (f _u) was derived. Simulations showed that k was highly dependent on f _u if capillary permeability was high but was independent of f _u if permeability was low. The effect of plasma protein binding was to increase the rate of tissue accumulation and washout of drug but to decrease the equilibrium amount of drug taken up by the tissue, both effects mediated via a decrease in the volume of distribution. This model was used to analyze published data on the effect of plasma protein binding on the kinetics of accumulation and washout of isradipine and propafenone in the isolated perfused heart preparation. The relationship between k and f _u and the directly measured volume of distribution were in accordance with the model. Although more complex models relating k and f _u could be proposed, taking into account unequal flows in capillaries, slow dissociation of ligand from protein, and unstirred layer constraints, this simple model appears adequate for describing the effect of f _u on myocardial accumulation and washout of isradipine and propafenone.     

Enantioselective Kinetics of Verapamil and Norverapamil in Isolated Perfused Rat Livers

The kinetics of the individual enantiomers of verapamil (VER) and its metabolite, norverapamil (NOR), were studied in isolated perfused rat livers (IPRLs) after administration of racemic drug or the preformed metabolite. After constant infusion of 20 µg/min of racemic VER to single-pass IPRLs, the hepatic availabilities (F) of the enantiomers were low (S-VER, 0.069 ± 0.030; R-VER: 0.046 ± 0.025) and stereoselective (S:R ratio, 1.6 ± 0.2). After administration of similar doses, the F values of the preformed NOR enantiomers (S-NOR: 0.24 ± 0.04; R-NOR, 0.10 ± 0.02) were higher than those of the VER enantiomers. However, the stereoselectivity in F of NOR (S:R ratio, 2.2 ± 0.1), was in the same direction of that of VER. Further, the fractions of R enantiomers unbound to bovine serum albumin in the perfusate were higher than those of their antipodes for both VER (R:S ratio, 1.9 ± 0.1) and NOR (R:S ratio, 2.6 ± 0.2). Therefore, for unbound moieties, modest stereoselectivity in the metabolism of VER in favor of the S-isomer and no stereoselectivity in the metabolism of NOR were observed. Overall, our data suggest that the stereoselective protein binding is a primary determinant of stereoselectivity in the hepatic availability of VER and NOR in IPRLs.     

Influence of Unbound Fraction and Perfusate Flow Rate on Taurocholate Elimination by Perfused Rat Liver: Applicability of Three Distributed Models

The applicability of three different distributed models to the kinetics of elimination of taurocholate by isolated perfused rat liver was examined by fitting each model to literature data. Each of the models was able to predict the effect of changing hepatic blood flow on elimination, but only the model which incorporates separate density functions describing the degree of sinusoidal heterogeneity of blood flow and intrinsic clearance was able to predict the effect of changing unbound fraction on elimination.     

Pharmacokinetic Analysis and Cellular Distribution of the Anti-HIV Compound Succinylated Human Serum Albumin (Suc-HSA) in Vivo and in the Isolated Perfused Rat Liver

After intravenous injection of a low dose (25 µg/kg) in rats, the anti HIV-1 compound succinylated human serum albumin (Suc-HSA) is taken up mainly in the liver and spleen and is proteolytically degraded. Ten minutes after injection of ¹²⁵I-Suc-HSA, 72 and 14% of the dose were found in the liver and spleen, respectively. With immunohistochemistry we demonstrated that in both organs, Suc-HSA was specifically endocytosed in endothelial cells. In the isolated perfused rat liver preparation, liver uptake was shown to be saturable, with a K _m of 2.9 10^–8 M and a V _max of 2.4 µg/inin/100 g body weight. The apparent K _m and V _max in vivo were 2.2 10^–7 M and 10.3 µg/min/100 g, respectively. Uptake in liver and spleen was inhibited by preadministration of an excess of formaldehyde-treated albumin and with polyinosinic acid, indicating the involvement of the scavenger receptor, as anticipated for such polyanionic compounds. Suc-HSA is not absorbed intact from the colon and the ileum. After injecting (i.v.) rats with a high dose of Suc-HSA (10 mg/kg), the elimination t _1/2 was 3 hr, and therefore, sustained plasma levels above the concentration needed for in vitro anti-HIV-1 activity can be achieved.     

The Effect of Nifedipine on the Isolated Rat Heart

Abstract The mode of action of a calcium-antagonistic drug, nifedipine, on the spontaneously beating, isolated, perfused rat heart was studied at two different calcium concentrations of the Ringer solution, [Ca⁺⁺]_o, (2.0 and 5.0 meq./l). The sinus node discharge, the aortic pressure amplitude and hence the work index were greater at 5.0 than at 2.0 meq./l. Nifedipine 100 μg/1 caused a reduction in these parameters, the effects being most pronounced at the lowest [Ca⁺⁺]_o studied. ECG tracings demonstrated that the PR-interval was longer at 2.0 than at 5.0 meq./l of calcium. The AV conduction time further increased after the addition of nifedipine. This effect was enhanced at the lowest [Ca⁺⁺]_o used, where varying degrees of AV block occurred in several experiments. The drug also reduced the R-wave voltage and the RT-interval. Calcium-induced ventricular tachyarrhythmias were produced, which were suppressed by nifedipine. Nifedipine 100 μg/l produced a small and transient increase in the coronary flow, but a tremendous increase in the ratio coronary flow/work index was observed. This indicates improvement of the cardiac energy balance caused by an increase in the coronary oxygen supply in relation to the myocardial oxygen demand.     

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.     

Contribution of Monoamine Oxidase(MAO) to the Binding of Tertiary Basic Drugs in Isolated Perfused Rat Lung

The effect of tertiary basic drugs on mitochondrial MAO activity and the effect of MAO inhibitors (MAOIs) on basic drug accumulation in the isolated perfused rat lung were studied to clarify the role of MAO in drug binding to lung tissue. In the perfused lung preparation, the inhibition of MAO by basic drugs correlated well with their lipid solubilities and followed competitive kinetics. The inhibitory rank order (imipramine diphenhydramine > quinine > metoclopramide > procainamide) also correlated with their accumulation in the perfused lung. Moreover, MAOI treatment decreased the accumulation of basic drugs in the lung, and the potency of MAOIs to inhibit drug accumulation in the lung correlated with their MAO inhibitory activity. These results indicate that lung MAO has specific binding sites for basic drugs and may function as a drug reservoir.     

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.     

Hepatic Extraction of Hexarelin,a New Peptidic GH Secretagogue,in the Isolated Perfused Rat Liver

Purpose. To assess the hepatic extraction of hexarelin (HEX), a novel peptidyl GH secretagogue, in the isolated perfused rat model and document the in vitro binding of HEX to plasma proteins using plasma from rats, dogs, pigs, and humans. Methods. Rat liver was perfused in situ using a recirculating system. The recirculating perfusate consisted of a Krebs Henseleit buffer containing 20% (v/v) prewashed bovine red blood cells, 1 % albumin, and 1g/L dextrose. Three HEX concentrations of 5, 50, and 500 ng/ml were examined. In vitro plasma binding was determined by the ultrafiltration method. Results. The disappearance rate constant (K), half-life (t_1/2), clearance (Cl), and hepatic extraction ratio (E) were: K = 0.013–0.014 min^–1, t_1/2 = 45–55 min, Cl = 0.345–0.401 ml/min/g liver, and E = 19-21% for the different concentrations of HEX. A linear increase in AUC (270-24334 min pmol/ml) was observed with increasing concentrations. Binding of HEX to plasma proteins of rats, dogs, pigs, and humans was 68.7 ± 0.8%, 78.7 ± 0.6%, 67.3 ± 0.7%, and 65.2 ± 0.6% respectively. Plasma binding was concentration-independent in the range between 0.003–3 M for the four species examined. Conclusions. These results show that 1) the hepatic extraction of HEX is low, 2) the hepatic clearance is concentration independent up to 500 ng HEX/ml of perfusate, and 3) the plasma protein binding of HEX is significant over the dose range studied. HEX exhibits a low hepatic extraction ratio, allowing us to predict that its hepatic clearance may be limited upon HEX protein binding.     

Effects of Quinuronium Sulfate Pre-exposure on Hepatic Excretion of Sulfobromophtalein (BSP) as Measured by Isolated Perfused Rat Liver

Abstract: Effects of quinuronium sulphate pre-exposure on hepatic excretion of sulfobromophthalein (BSP) as measured by isolated perfused rat liver was investigated. Seventeen rats were used in the study. Rats were divided into controls (Group 1, n = 5), low quinuronium treated animals (Group 2, n = 6), and high quinuronium treated animals (Group 3, n = 6) receiving 0, 1.5 or 2.3 mg quinuronium sulphate/kg intraperitoneally, respectively. Each animal was dosed 30 min. before sacrifice. After 30 min. of equilibrium perfusion, BSP was introduced and the experiment continued for 2 hours. Perfusate and bile were collected at designated time intervals and the volume of bile was noted. BSP in perfusate and bile was assayed. Half-life in minutes (t_1/2$pLS.E.) of BSP disappearance from perfusate in groups 1, 2 and 3 were 12.5$pL1.8, 13.7$pL1.0 and 16.8$pL3.3, respectively. Group 2 and 3 rats had a significant decrease of BSP excretion in bile (P<0.01) compared to group 1 rats. Bile volumes decreased in both quinuronium pre-exposed groups of rats compared to control rats. These observations indicate that quinuronium affects liver function.     

Combined Effects of Ethanol and pH-Change on Protein Synthesis in Isolated Rat Hepatocytes

Abstract: Rat liver parenchymal cells were isolated and incubated for 80 min. in buffered salt solutions. ¹⁴C-valine incorporation in the presence of a high concentration of unlabelled valine (4.2 mM) was taken as a measure of protein synthesis. The pH dependence of the synthesis of cell and medium proteins was studied at pH values varying from 6.6 to 7.9. The results showed that maximum protein synthesis occurred close to the physiological pH value. Protein synthesis was reduced at both lower and higher pH-values. Protein synthesis was inhibited by the addition of ethanol (30 mM). The relative inhibition caused by ethanol got more pronounced as the pH of the hepatocyte suspensions was lowered. The metabolism of ethanol resulted in lowering of the pH in cell suspensions. It is suggested that the depression of protein synthesis by ethanol could be mediated by the fall in pH due to ethanol metabolism.     

Effect of Neutral Endopeptidase Inhibition on the Natriuresis and Renal Clearance of Atrial Natriuretic Peptide in Perfused Rat Kidney

Pharmaceutical Research -     

pH值对右旋布洛芬体外透皮性能的影响研究

目的:研究pH值对右旋布洛芬(DI)体外透皮性能的影响,为其透皮给药制剂的开发提供实验依据。方法:建立DI的高效液相色谱分析方法,测定其在水和不同pH的磷酸盐缓冲液(PBS)中的平衡溶解度;用摇瓶法测定DI的正辛醇溶液在正辛醇饱和蒸馏水与不同pH的PBS中的表观油水分配系数;以稳态渗透速率(Js)和渗透系数(Ps)为评价指标,检测不同pH的PBS对DI体外透皮性能的影响。结果:32℃时,DI在水中难溶;在pH7.4的PBS中平衡溶解度、Js、Ps均最大,表观油水分配系数极小,分别为5421.6μg·mL-1、339.97μg·cm-2·h-1、0.063cm·h-1、0.17。结论:pH值对DI体外透皮性能有很大影响,pH7.4时DI有较高的溶解度、脂溶性及较理想的皮肤通透性。     

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.