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In our previous study [Lin, Sugiyama, Awazu, and Hanano: J. Pharmacokin. Biopharm. 10,649 (1982)], we successfully applied a physiological pharmacokinetic model to quantitative prediction of the elimination and distribution kinetics of ethoxybenzamide in rats and rabbits. The predictions of the time course of ethoxybenzamide concentrations in plasma were good at lower doses (10 and 20 mg/kg), whereas those at high dose (80 mg/kg) were poor. In the present study, therefore, product inhibition was suspected and examined. Product inhibition of ethoxybenzamide deethylation by its metabolite, salicylamide, was demonstrated both in vivo and in vitro studies. The plasma disappearances of ethoxybenzamide after a 20 mg/kg iv injection were determined both in the control and the salicylamide-treated rabbits. In the salicylamide-treated rabbits, the plasma disappearance of ethoxybenzamide was significantly delayed compared to that in control rabbits. This delay was quantitatively explained by the physiological pharmacokinetic model taking the competitive-type of product inhibition into consideration. The apparent dissociation constant for the salicylamide-enzyme complex in vivo was estimated as 0.14 mM. The inhibition of ethoxybenzamide de-ethylation by salicylamide was observed also in in vitro study using liver microsome of rabbits.

The interaction between tolbutamide (TB) and sulfonamide (SA) in rabbits was quantitatively investigated by both in vivo and in vitro experiments, and the mechanisms of species difference between rabbits and rats were analyzed by comparing the two total body clearances (CLtot) obtained from in vivo and in vitro studies. The sulfonamides used were sulfaphenazole (SP) and sulfadimethoxine (SDM). In vivo CLtot of TB was changed little by SA in rabbits, which was contrary to the phenomenon seen in rats, i.e. CLtot was markedly decreased by SA in rats (Sugita et al., Biochem. Pharmacol., 30, 3347, 1981). The total body clearance defined as CL in vitro tot.pred was predicted for TB in the presence and absence of SA by the equation: CL in vitro tot.pred congruent to fB CL in vitro int, where fB is the blood-free fraction and CL in vitro int is the hepatic intrinsic clearance of unbound drug obtained from in vitro experiments using liver microsomal fraction. A comparatively good agreement was observed between the two CLtot obtained from in vivo and in vitro data. The analysis in rabbits based on these in vitro experiments showed that the small gross changes of in vivo CLtot induced by SA were due to the compensative changes of two factors, i.e. fB and CL in vitro int which in the presence of SA were approximately 200 and 50% of the control, respectively. However, in rats, the marked decrease of in vivo CLtot induced by SA was explained by the greater degree of the decrease in CL in vitro int than that of the increase in fB.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to simulate the distribution and elimination of radioiodinated human beta-endorphin (125I-beta-EP) after iv bolus injection in rats, we proposed a physiologically based pharmacokinetic model incorporating diffusional transport of 125I-beta-EP across the capillary membrane. This model assumes that the distribution of 125I-beta-EP is restricted only within the blood and the tissue interstitial fluid, and that a diffusional barrier across the capillary membrane exists in each tissue except the liver. The tissue-to-blood partition coefficients were estimated from the ratios of the concentration in tissues to that in arterial plasma at the terminal (pseudoequilibrium) phase. The total body plasma clearance (9.0 ml/min/kg) was appropriately assigned to the liver and kidney. The transcapillary diffusion clearances of 125I-beta-EP were also estimated and shown to correlate linearly with that of inulin in several tissues. Numerically solving the mass-balance differential equations as to plasma and each tissue simultaneously, simulated concentration curves of 125I-beta-EP corresponded well with the observed data. It was suggested by the simulation that the initial rapid disappearance of 125I-beta-EP from plasma after iv injection could be attributed in part to the transcapillary diffusion of the peptide.

We investigated the effects of various phenothiazines on the Na+-H+ exchanger in brush-border membrane vesicles isolated from the proximal small intestine of the rabbit. The Na+-H+ exchanger activity was assayed by measuring the dimethylamiloride-sensitive Na+ uptake into these vesicles in the presence of an outward-directed H+ gradient ([pH]o greater than [pH]i). All phenothiazines that were tested inhibited the Na+-H+ exchanger and chlorpromazine sulfoxide (CPZ-SO) was the most potent inhibitor among them. CPZ-SO inhibited the dimethylamiloride-sensitive Na+ uptake in a dose-dependent manner and IC50 for this process was 90 microM. Even though CPZ-SO drastically reduced the initial rates of Na+ uptake, the equilibrium uptake of Na+ into the vesicles was not affected, indicating that the integrity of membrane vesicles was not altered by the drug. Furthermore, the inhibition of the exchanger activity by phenothiazines was not due to nonspecific dissipation of the H+ gradient, because CPZ-SO inhibited the dimethylamiloride-sensitive Na+ uptake even in the absence of a H+ gradient. The inhibition of the exchanger by CPZ-SO was freely and completely reversible. Kinetic analyses of the Na+-H+ exchanger carried out in the presence and absence of CPZ-SO revealed that the nature of the inhibition depended on the composition of the buffers used in the assay of the exchanger because the inhibition was of a mixed type in the mannitol medium, whereas it was of a competitive type in the KCl medium.

Transport characteristics of benzylpenicillin in the central nervous system was examined using ATP-depleted rat choroid plexus. In the presence of an outwardly directed Cl- gradient, accumulation of benzylpenicillin was stimulated markedly compared with the accumulation in the absence of Cl- gradient. Under Cl- gradient conditions, benzylpenicillin was accumulated transiently at a concentration approximately 1.7-fold higher than at equilibrium ("overshoot") implying the uphill transport. The Cl- gradient-stimulated accumulation of benzylpenicillin was not solely due to the changes in the membrane potential or in the intracellular pH. Accumulation of benzylpenicillin in choroid plexuses preloaded with HCO3-, SCN- or benzylpenicillin also was stimulated compared with the accumulation in unpreloaded choroid plexuses. The Cl- gradient-stimulated benzylpenicillin accumulation was saturable (Km = 13.6 microM and Vmax = 3.76 nmol/ml of tissue per min), and was reduced by sulfhydryl reagents (p-hydroxymercuribenzoic acid, p-chloromercuribenzoic acid, p-chloromercuribenzenesulfonic acid and N-ethylmaleimide). Probenecid and anionic exchange inhibitors (furosemide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) inhibited the Cl- gradient-stimulated benzylpenicillin accumulation in a dose-dependent fashion. An inwardly directed Na+ gradient did not stimulate the accumulation of benzylpenicillin. These findings suggest that the principal mechanism for the uphill transport of benzylpenicillin in the rat choroid plexus is via an anionic exchanger.

To characterize the transport system by which cephalosporin antibiotics are accumulated by the choroid plexus, kinetic analysis of cefodizime transport was performed. Accumulation of cefodizime was against an electrochemical potential gradient via a saturable process (Km = 470 microM, Vmax = 174 nmol/ml of tissue per min) that was inhibited by metabolic inhibitors (KCN and 2,4-dinitrophenol), hypothermia, a sulfhydryl reagent (p-hydroxymer-curibenzoic acid) and anion transport inhibitors (probenecid and 4,4'-diisothiocyanostilbene -2,2'-disulfonic acid). Accumulation of cefodizime was inhibited competitively by benzylpenicillin with an inhibition constant of aproximately 100 microM. Cefodizime inhibited competitively the accumulation of benzylpenicillin with an inhibition constant of approximately 500 microM. Kinetic analysis using 16 kinds of beta-lactam antibiotics also supported the view 1) that the transport system of cefodizime is shared by benzylpenicillin and 2) that these beta-lactam antibiotics are transported via a common transport system. These findings indicate that the major transport system of cephalosporin antibiotics in the rat choroid plexus is via a carrier-mediated active anion transport process. The affinity of beta-lactam antibiotics for this transport system in the choroid plexus may be a major factor in determining their pharmacokinetics in the cerebrospinal fluid.

To evaluate the relationship between the pharmacological effect of benzodiazepine (BZP) and BZP receptor (BZP-R) binding in the conscious mouse brain, a response of the local cerebral metabolic rate of glucose utilization (GU) to clonazepam (CNZ) was measured as an index for the pharmacological effect. Two glucose analogs (3-O-[3H]methylglucose and 2-[14C]deoxyglucose) method, originally presented by A. Gjedde was used for determination of GU. In the cerebral cortex, GU decreased to 70 to 80% at 60 min after i.v. administration of CNZ (0.005-1.0 mg/kg), but CNZ did not change the lumped constant, and this effect was diminished completely by the administration of a BZP antagonist, Ro-15-1788 (5 mg/kg). The maximum effect of CNZ on GU (about 30% decrease) was found at 0.1 mg/kg of CNZ, but increasing the dose to 1 mg/kg had very little additional effect. In vivo BZP-R occupancy was measured using [3H]-Ro-15-1788. Receptor occupancy increased from less than 10% at a dose of 0.005 mg/kg up to essentially 100% at doses of 1 mg/kg or greater. ID50 in dose-response curve of the receptor occupancy for CNZ and ED50 in that of decrease in GU were 0.3 and 0.007 mg/kg, respectively . A nonlinear and hyperbolic relationship was observed between the receptor occupancy and the response for the glucose metabolic rate, indicating that BZP exert the maximum glucose metabolic change at a low fractional receptor occupancy (30-40%).

The transport of imipenem, a novel carbapenem antibiotic, in the rat central nervous system (CNS) was studied using in vivo, in situ and in vitro experimental techniques. After i.v. bolus administration, the imipenem concentration in the cerebrospinal fluid (CSF) rose to a peak within 30 min and declined with time. The CSF/serum unbound concentration ratio of imipenem was 0.22 at 2 hr after i.v. administration, substantially higher than that reported for benzylpenicillin. By using an in situ brain perfusion technique, we found that imipenem was transported through the blood-brain barrier principally via passive diffusion with a permeability-surface area product comparable to that of mannitol. In vitro, imipenem was accumulated by the isolated choroid plexus via an active organic anion transport system, although much less rapidly than benzylpenicillin. In vivo, after i.c.v. administration, imipenem was cleared from the CNS in a manner comparable to that of mannitol with only a small probenecid-sensitive process. Imipenem thus has minimal affinity for the organic anion transport system in the choroid plexus, resulting in the slow elimination of this drug from the CNS. These results suggest that the difference between imipenem and benzylpenicillin in the ratio of CSF to unbound serum drug concentration is determined principally by the efflux process in the choroid plexus rather than the influx process through the blood-brain barrier.

Multiplicity for the transport of organic anions across the bile canalicular membrane was studied in vivo and in vitro using dibromosulfophthalein (DBSP), [14C]cefodizime, [3H]leukotriene C4 (LTC4) and indocyanine green (ICG) as model compounds in rats. A high concentration of DBSP in plasma reduced the biliary excretion of cefodizime and leukotriene radioactivity to about 15 and 35% of their control values, respectively, but did not affect the excretion of ICG. A high plasma concentration of ICG reduced the excretion of cefodizime to about 60% of the control value, but exerted minimal effect on the excretion of leukotriene radio-activity and DBSP. In vitro, ATP-dependent uptake of LTC4 into the canalicular membrane vesicles was reduced by DBSP, cefodizime and ICG in a dose-dependent manner, with approximate IC50 values of 0.1 microM, 10 microM, and 1 microM, respectively. The hepatic unbound concentration of DBSP sufficient to reduce the excretion of cefodizime, leukotriene radioactivity and DBSP itself in vivo was calculated to be approximately 10 microM, a concentration which was also sufficient to reduce the transport of LTC4 in vitro. In contrast, the hepatic unbound concentration of ICG that saturated the excretion of ICG in vivo was calculated to be less than 0.1 microM, which was more than ten times smaller than the IC50 of ICG for the transport of LTC4 in vitro. These results suggest the presence of multiple systems for the transport of organic anions across the bile canalicular membrane: one is the predominant transport system for DBSP and cefodizime, which also accepts LTC4 as a substrate; the other is the primary transport system for ICG, which is distinct from that for DBSP, cefodizime and LTC4, although some overlap in the substrate specificity may be observed between the two systems.

Liquiritigenin (LG), 2, 3-dihydro-7-hydroxy-2-(4-hydroxyphenyl)-(S)-4H-1-benzopyran-4-1, is metabolized to five kinds of conjugates (glucuronides and sulfates) that are excreted predominantly into the bile (Shimamura et al., 1993). Using LG as a model compound, we studied the multiplicity for the biliary excretion of conjugates in vivo. LG was administered i.v. as a bolus to Sprague-Dawley rats (SD rats) that received i.v. infusions of the inhibitors [glycyrrhizin (GR), dibromosulfophthalein (DBSP) and indocyanine green (ICG)] at their maximal transport rates. The infusion of GR and that of DBSP reduced significantly the biliary excretion of all the conjugates except LG 4',7-O-disulfate (M3), whereas infusion of ICG did not affect the excretion of conjugates. To minimize the effect of the other tissues on the disposition of the conjugates, the apparent biliary excretion clearance (CLbile,app) was calculated. The CLbile,app values of LG 4'-O-glucuronide (M1), LG 7-O-glucuronide (M2), LG 4'-O-glucuronide 7-O-sulfate (M4) and LG 7-O-glucuronide 4'-O-sulfate (M5) were significantly reduced by GR and DBSP infusion, whereas the CLbile, app value of M3 was not affected by these inhibitors. The CLbile, app values of all the conjugates except M2 were not reduced by ICG infusion. In Eisai hyperbilirubinemic rats, which have a hereditary defect in the canalicular transport system for several organic anions, the biliary excretion clearance of M1, M2, M4 and M5 was markedly reduced, whereas that of M3 was comparable with that in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

To characterize the transport system by which benzylpenicillin, an organic anion, is accumulated by the isolated rat choroid plexus, kinetic analysis of benzylpenicillin transport was performed. Accumulation of benzylpenicillin was against an electrochemical potential gradient via a saturable process (Km = 58 microM and Vmax = 84 nmol/ml of tissue per min) that was inhibited by sulfhydryl reagents (p-hydroxymercuribenzoate and N-ethylmaleimide), metabolic inhibitors (KCN and 2,4-dinitrophenol) and anion exchange inhibitors (4-acetamide-4'-isothiocyanatostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), but is major transport system did not require the inward Na+ gradient. Organic anions, such as 5-hydroxyindoleacetic acid, homovanillic acid, p-aminohippuric acid and probenecid inhibited the accumulation of benzylpenicillin, whereas dipeptides did not affect it. Kinetic analysis of the accumulation of benzylpenicillin suggests that both phenoxymethylpenicillin and cefpiramide are also transported via the benzylpenicillin transport system. Other penicillin and cephalosporin derivatives inhibited the accumulation of benzylpenicillin with different affinities. Penicillin derivatives without dissociating groups in the side chain had the higher affinity for the benzylpenicillin transport system than other beta-lactam antibiotics did. Among penicillin derivatives examined, a good correlation (r = 0.92) was observed between the lipophilicity and the affinity for the benzylpenicillin transport system, whereas no correlation was observed among the cephalosporin derivatives. These findings suggest that the major transport system of benzylpenicillin in the rat choroid plexus is via a carrier-mediated active anion transport process which is distinct from that of dipeptides, and does not require the inward Na+ gradient.(ABSTRACT TRUNCATED AT 250 WORDS)

To characterize the transport system of cimetidine, an organic cation, in the blood-cerebrospinal fluid barrier, the accumulation of cimetidine by the isolated rat choroid plexus was examined. Accumulation of cimetidine was against a concentration gradient via a saturable process (Km = 53 microM, Vmax = 12 nmol/ml/min) that was inhibited by sulfhydryl reagents (p-hydroxymercuribenzoate), metabolic inhibitors (KCN and 2,4-dinitrophenol) and hypothermia (Q10 = 4.5), but did not require inward Na+ gradient. Organic cations such as 1N-methylnicotinamide, tetraethylammonium, choline, histamine and creatinine did not affect the accumulation of cimetidine at the concentration of 1 mM. Cimetidine did not affect the accumulation of tetraethylammonium. More lipophilic cations such as quinidine and quinine inhibited not only the accumulation of cimetidine but also that of an organic anion, benzylpenicillin, although the inhibitory mechanisms are not known. One millimolar of organic anions, such as 5-hydroxyindoleacetic acid, p-aminohippuric acid, homovanillic acid, salicylic acid and benzylpenicillin, inhibited the accumulation of cimetidine. Furthermore, the accumulation of organic anions (benzylpenicillin and salicylic acid) showed saturability and was inhibited by cimetidine. Cimetidine and the organic anions thus showed a mutual inhibition. Oligopeptides also inhibited the accumulation of cimetidine. These findings suggested that cimetidine transport in the choroid plexus is via carrier-mediated active transport process, but does not require inward Na+ gradient. This transport is inhibited by several compounds with different properties like oligopeptides, lipophilic cations and organic anions, although the inhibitory mechanism is not known.

The hepatobiliary transport of nonmetabolizable organic anions [cefodizime, dibromosulfophthalein, and indocyanine green (ICG)] was kinetically analyzed in Eisai hyperbilirubinemic rats (EHBR; Sprague-Dawley-derived mutant rats with conjugated hyperbilirubinemia). In EHBR, the biliary excretion of these anions was remarkably impaired, whereas the alteration in initial plasma disappearance was minimal. The kinetic analysis of the disposition of these ligands revealed 1) that the transport rate via bile canalicular membrane was severely impaired in EHBR for cefodizime and dibromosulfophthalein and 2) that the intracellular transport rate of ICG was decreased in EHBR, which contributed more than the decrease in the canalicular membrane transport to the net reduction of the excretion rate of ICG in EHBR. The latter finding was also supported by the in vitro results; the binding of ICG to ligandin(s) in EHBR was less extensive compared with that in normal rats, resulting in the higher distribution of ICG to organelle. Because the ligand molecules bound to organelle diffuse within the cells much more slowly than the molecules in the cytosol, the higher distribution of ICG to organelle in EHBR results in the reduction in the intracellular transport rate. These results indicate that the differential impairment mechanisms can be proposed for the excretion of organic anions: One is the impairment in the transport rate across the canalicular membrane, the other is the impairment in the intracellular transport rate.

Effect of benzylpenicillin (PCG) on the disposition of [14C]cefodizime (CDZM), a nonmetabolizable analog of cefotaxime, was studied in rats. Rats were divided into two groups to receive i.v. injection of [14C]CDZM (15 mg/kg) alone and during the infusion of PCG. Although the total concentration of [14C]CDZM in plasma vs. time data were almost the same between the groups, the unbound concentration of [14C]CDZM was increased 2-fold by the treatment with PCG. Hepatobiliary excretion of [14C]CDZM was reduced by the treatment with PCG, indicating that PCG inhibits the transport of CDZM across the sinusoidal and/or bile canalicular membrane(s). Further support for this hypothesis is the finding that the uptake of [14C]CDZM by the isolated hepatocytes in vitro was via an active transport process that was reduced by PCG. Furthermore, pharmacokinetic analysis of the data obtained from in vivo and in vitro experiments indicated that PCG reduced the permeability of [14C]CDZM across the sinusoidal and bile canalicular membranes to 32% and 40% of the control value, respectively. The urinary excretion of [14C]CDZM was principally via the glomerular filtration process and was not affected by the treatment with PCG. Because of the 2-fold rise in the unbound fraction of [14C]CDZM in the treated rats, the renal clearance for total [14C]CDZM was increased 2-fold by the treatment with PCG. The increase in the renal clearance compensated for the decrease in the hepatic clearance. The net effect of the alterations in these pharmacokinetic parameters resulted in no change in the time profiles of [14C]CDZM concentration in plasma between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The disposition of [14C]cefodizime, a nonmetabolizable analog of cefotaxime, in the central nervous system was studied in control and benzylpenicillin-infused rats using in vivo experimental techniques. After i.v. bolus administration of [14C]cefodizime, a concentration gradient was established from the brain extracellular fluid (ECF) to the cerebrospinal fluid (CSF) (i.e., drug penetration into the CSF after i.v. administration can be accounted for by permeation across the cerebral capillaries and diffusion through the brain ECF and across the ependymal surface into the CSF). Kinetic analysis indicated that the cerebrovascular transfer coefficient for plasma unbound [14C]cefodizime in control rats equaled 5.2 x 10(-5) ml/sec/g brain, a value comparable to that for mannitol. In rats which received an i.v. infusion of benzylpenicillin, the cerebrovascular transfer coefficient for [14C]cefodizime was reduced to approximately 1/15th of the control value, resulting in a reduced central nervous system (brain ECF and CSF)/plasma unbound concentration ratio for this drug. After i.c.v. administration, the efflux of [14C]cefodizime from CSF was comparable to that of [3H]mannitol with a small probenecid-sensitive component and was not affected by treatment with benzylpenicillin. These results indicate that the unidirectional influx of [14C]cefodizime into brain ECF and then into the CSF is facilitated by a mechanism shared with other beta-lactam antibiotics.

The effect of phenylarsine oxide (PAO) on the internalization rate of epidermal growth factor (EGF) was investigated using perfused rat liver and isolated rat hepatocytes. In perfused liver, a tracer concentration of 125I-EGF alone or with excess unlabeled EGF (20 nM) was perfused and the internalization rate constants (kint) were measured. In the absence of PAO, kint values did not differ significantly for either dose condition. However, with the addition of PAO to the perfusate, the kint value dropped to 4% of that of the control at the low concentration of EGF, while dropping to only 40% of that of the control at the high concentration of EGF. These results suggest the existence of a PAO-insensitive internalization pathway having a kint value comparable with that of the other pathway. Similar EGF concentration-dependent inhibition of 125I-EGF internalization caused by PAO was ascertained using isolated rat hepatocytes. PAO also decreased the cellular ATP content in isolated hepatocytes. However, when we lowered the cellular ATP content with rotenone, the cell-surface binding and internalization of EGF were comparable with the control levels. We concluded that there exist dual pathways for the internalization of EGF and that excess doses of EGF lead to EGF internalization not only through a PAO-sensitive pathway but also through a PAO-insensitive pathway, whereas at a tracer dose of EGF, the internalization occurs mainly via the PAO-sensitive pathway.