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To characterize pentazocine (PTZ) transport across the blood‐brain barrier (BBB), the cerebrovascular permeability‐surface area product (PSinf) of PTZ was determined by a well‐established in siturat brain perfusion technique. The uptake kinetics of PTZ by the rat brain exhibited saturability, which indicates the simultaneous mechanisms of carrier‐mediated transport and passive diffusion. The kinetic parameters were estimated as follows: maximal influx rate (Vmax), 27.2 ± 5.2 nmol/s/g brain; apparent Michaelis constant (Km) for the saturable component of PTZ uptake, 2.9 ± 0.5 mM; nonsaturable uptake rate constant (Kd), 1.5 ± 0.3 μL/s/g brain. BBB transport of PTZ was significantly inhibited by cationic drugs such as diphenhydramine, propranolol, and eptazocine (a narcotic‐antagonist analgesic), but not by choline, suggesting that the PTZ transport system is shared by cationic drugs. Furthermore, co‐perfusion of verapamil caused a significant (two‐fold) increase in the BBB permeability to PTZ. This finding indicates that PTZ may be a substrate of the endogenous BBB efflux transport system, P‐glycoprotein. These findings demonstrate that the primary mechanism governing the uptake of PTZ by the brain is carrier‐mediated transport, not passive diffusion. © 2002 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2346–2353, 2002

To characterize pentazocine (PTZ) transport across the blood-brain barrier (BBB), the cerebrovascular permeability-surface area product (PSinf) of PTZ was determined by a well-established in situ rat brain perfusion technique. The uptake kinetics of PTZ by the rat brain exhibited saturability, which indicates the simultaneous mechanisms of carrier-mediated transport and passive diffusion. The kinetic parameters were estimated as follows: maximal influx rate (Vmax), 27.2 ± 5.2 nmol/s/g brain; apparent Michaelis constant (Km) for the saturable component of PTZ uptake, 2.9 ± 0.5 mM; nonsaturable uptake rate constant (Kd), 1.5 ± 0.3 μL/s/g brain. BBB transport of PTZ was significantly inhibited by cationic drugs such as diphenhydramine, propranolol, and eptazocine (a narcotic-antagonist analgesic), but not by choline, suggesting that the PTZ transport system is shared by cationic drugs. Furthermore, co-perfusion of verapamil caused a significant (two-fold) increase in the BBB permeability to PTZ. This finding indicates that PTZ may be a substrate of the endogenous BBB efflux transport system, P-glycoprotein. These findings demonstrate that the primary mechanism governing the uptake of PTZ by the brain is carrier-mediated transport, not passive diffusion. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2346–2353, 2002

Transport characteristics of 17β-estradiol 17β-d-glucuronide (E217βG), a dual substrate of the transporters for cellular uptake (organic anion-transporting polypeptide 1 or oatp1) and cellular excretion (multidrug resistance-associated protein 1or MRP1), in the rat choroid plexus were studied in vivo and in vitro. The uptake of E217βG into isolated choroid plexus was mediated by an energy-dependent system with a Kmof 3.4 μM. Together with the previous finding that oatp1 is localized on the apical membrane of choroid plexus, these results suggest that oatp1 is responsible for the uptake of this ligand. After intracerebroventricular administration, elimination of E217βG from cerebrospinal fluid was probenecid sensitive and much more rapid than that of inulin; less than 2% of the administered E217βG and 40 to 50% of inulin remained in the cerebrospinal fluid 20 min after intracerebroventricular administration. In addition, the amount of E217βG associated with choroid plexus at 20 min was negligible, suggesting the presence of an efficient excretion system on the basolateral membrane of choroid plexus. Expression of MRP1 was detected in choroid plexus. Semiquantitative reverse transcription-polymerase chain reaction and Western blot analyses indicated that the expression level of MRP1 in choroid plexus is about four or five times higher than that in the lung, one of the tissues exhibiting high expression of MRP1. Together with the in vivo vectorial transport of E217βG, these results can be accounted for by assuming that there is basolateral localization of MRP1 in choroid plexus. Combined, oatp1 and MRP1 may synergistically mediate the efficient transcellular transport of E217βG across choroid plexus.

The effect of phenobarbitone on the steady state volume of distribution (Vdss) and the total body blood clearance (CLtot, b) of imipramine and the serum concentration of its metabolite, desipramine was examined. The serum disappearance of imipramine after an 8 mg kg−1i.v. dose followed a biexponential decline in both control and phenobarbitone-treated rats while the concentration of its metabolite increased in the phenobarbitone-treated rats then rapidly declined compared with that in control rats. Since CLtot, bwas nearly equal to the hepatic blood flow (QH), QHmay be the rate-determining step of imipramine elimination. In the control rats the Vdssof imipramine was large at 19.9 litre kg-1. In the phenobarbitone-treated rats the pharmacokinetic parameters, biological half-life (t 1/2) and Vdsssignificantly decreased to approximately 23-40% while CLtot, bincreased to 126% of those in the control rats, although the latter difference was not statistically significant. The blood-to-plasma concentration ratios (RB) of imipramine and desipramine decreased in the phenobarbitone-treated rats. The urinary excretion ratios of imipramine and desipramine, to the dose of imipramine over 8 h, were <1.5% in both groups. These ratios were not significantly changed in the phenobarbitone-treated rats. It was concluded that the significant decrease in t 1/2 of the phenobarbitone-treated rats may not be attributed to the changes in CLtot,band/or in the urinary excretion, but mainly to the decrease in Vdss.

Treatment of human serum with DEAE-cellulose in acid conditions almost completely removed α1-acid glycoprotein (α1-AG) with little change in the concentration of albumin and β-lipoprotein, while treatment with sulphosalicylic acid removed almost all the proteins except α1-AG. The binding of various drugs to serum treated as above was measured by equilibrium dialysis and the contribution of α1-AG to drug binding by human serum was assessed. Sulphosalicylic acid-treated serum exhibited a saturable binding for propranolol, which was considered to be due to the binding to α1-AG while DEAE-cellulose-treated serum mostly exhibited non-saturable binding, for which albumin and β-lipoprotein may be responsible. With this treated serum, α1-AG was estimated to contribute approximately 40% to the binding of therapeutic concentrations of propranolol, 15% to that of imipramine and 15-20% to that of desipramine, respectively, in serum samples pooled from healthy adults. However, no contribution of α1-AG was observed in the binding of salicylic acid to the serum. Dissociation constants of the propranolol binding to the high affinity site in control serum, sulphosalicylic acid-treated serum and purified α1-AG showed similar values (3.7-6.7 μM). These results suggest that treatment of serum with sulphosalicylic acid and DEAE cellulose is useful in assessing the contribution of α1-AG to the serum binding of basic drugs.

The effect of sulfaphenazole on the distribution of tolbutamide was examined by comparing the change in the steady-state volume of distribution (Vdss) determined from in vivoplasma elimination with the tissue-to-plasma concentration ratio of various tissues (Kp) in rabbits; this effect was compared with that previously reported in rats. In rabbits, the Kpvalues of six tissues studied (i.e., brain, heart, spleen, small intestine, muscle, and skin) increased in the presence of sulfaphenazole; except for brain, lung, and adipose tissue, the tissue-to-plasma unbound concentration ratio (Kp,f) of other tissues did show a significant decrease. This suggested that both the tissue and plasma protein binding of tolbutamide were affected by sulfaphenazole and that the increase in Kpwas due mainly to the displacement of plasma protein binding of tolbutamide by sulfaphenazole, which was greater than that of tissue binding, while no change in Kpwas due to a parallel change in both the plasma protein binding and tissue binding of tolbutamide. In both rabbits and rats, the Vdsscalculated from plasma concentration versustime curve was very close to that calculated from the Kpvalues and volumes of various tissues in the presence and absence of sulfaphenazole, respectively. The interspecies difference of the effect of sulfaphenazole on the tissue distribution of tolbutamide between rabbits and rats was elucidated from both in vivotissue distribution and in vitroplasma protein binding studies.

The possibility that the fluorescence probe, 1-anilino-8-naphthalenesulfonate (I), might be used for predicting the interindividual differences in the plasma protein binding of acidic drugs was examined. The interindividual differences in the free fraction of I (fI) were found not to be due to corresponding differences in plasma albumin concentration, but to those differences in binding constant. The binding constant of I to the plasma of 18 individual rats ranged from 1.75 × 106M−1to 2.3 × 107M−1. The free fraction of I had a highly significant statistical correlation with plasma concentration of free fatty acids, but had no significant correlation with the infinite fluorescence of I or the degree of the polarization of I. Each free fraction of five acidic drugs (warfarin, phenylbutazone, salicylic acid, indomethacin, and sulfaphenazole) was correlated with fIwith high statistical significance. Therefore, the simple and convenient method using I may predict the interindividual differences in the plasma protein binding of acidic drugs in rats.

The interaction of doxorubicin with nuclei isolated from rat liver and kidney was studied by fluorospectrometry. The nuclei had at least two different types of binding sites for the drug. Both Mg2+and Ca2+competitively inhibited the binding of doxorubicin to the nuclei, which showed a remarkable temperature dependency. No significant difference was observed between the numbers of binding sites (n=6.70 × 10−2mol/mol of DNA for liver; 6.41 × 10−2mol/mol of DNA for kidney) or the affinity constants (Ka=4.85 × 105M−1for liver; 5.41 × 105M−1for kidney) under quasi-physiological conditions. These results obtained from in vitrobinding experiments support previous suggestions that the differences in the in vivodistribution of doxorubicin among tissues are not due to differences in the nuclear binding of the drug. The amount of nuclei per gram of tissue is the primary determinant of the characteristic tissue distribution of doxorubicin.

The mechanism of interorgan and interspecies variations in the tissue distribution of doxorubicin was studied in rats, rabbits, and guinea pigs. The permeation properties of doxorubicin were investigated by using isolated rat erythrocytes as a model membrane. A significant dependency of the uptake rate constant on medium pH was observed, suggesting that only un-ionized doxorubicin is diffusible through the plasma membrane. The values of plasma unbound fraction (fp) of doxorubicin were 0.344 for rats, 0.415 for rabbits, and 0.529 for guinea pigs. The tissue DNA concentrations of rats were larger than those of rabbits and smaller than those of guinea pigs in corresponding organs or tissues. An in vitroorgan model that described the distribution behavior of doxorubicin in the whole body was constructed, and an equation was derived to estimate the tissue-to-plasma partition coefficients, (Kp) from in vitroexperiments. The in vitro Kpvalues showed comparatively good agreement with the in vivo Kpvalues for the various organs or tissues in all three species. Doxorubicin is exclusively bound to the nuclei in the cells. The variation of the Kpvalues in different organs depended on the amount of nuclei per gram of tissue. The primary determinant of the interspecies variation in the Kpvalues was the difference in tissue DNA concentrations among rats, rabbits, and guinea pigs, and a secondary determinant was the difference in fpvalues.

The dose dependency in the hepatic transport of an anionic fluorescent dye, 1-anilino-8-naphthalene sulfonate (ANS), was investigated by measuring the plasma disappearance and biliary excretion in rats. Bulk of the administered ANS distributed into the liver at 10 min after iv bolus injection. The plasma disappearance curves of ANS were then kinetically analyzed based on a two-compartment model, in which the ligand is eliminated only from the peripheral compartment (liver compartment). The total body clearance (CLtot) decreased with increasing dose of ANS. That is, the values of CLtotwere 4.06 and 1.98 ml/min/per kg at the doses of 3 and 100 ?mol/kg, respectively. The clearances of the uptake and sequestration processes (CLupand CLseq, respectively) for a total ligand were constant irrespective of dose, while the efflux clearance (CLeff) for a total ligand was increased by twofold with increasing dose. A mechanism for the increase in the CLeffvalue might be explained by a saturation of the ANS binding to the intracellular proteins. The hepatocellular distribution and the binding of ANS to cytosolic proteins were then determined. ANS mainly distributed to the cytosol fraction, and the unbound fraction in the cytosol increased from approximately 0.04 to 0.09 when the cytosolic concentrations of ANS increased from 40 to 900 ?M, respectively. In,spite of such increase in the unbound fraction in the cytosol, the CLseqvalues remained unchanged with increasing dose, suggesting that the saturation of sequestration clearance for unbound ANS might occur. Furthermore, the plasma disappearance curves of ANS at various doses were simultaneously analyzed based on three nonlinear kinetic models: Model I is a model incorporating both saturable intracellular binding and saturable sequestration; Model II is a model incorporating only saturable intracellular binding; Model III is the model incorporating only saturable sequestration. Goodness- of- fit evaluated by AIC value was best for Model I. Taken together, the nonlinearity in the plasma clearance of ANS was confirmed to be attributed to saturation of both its binding to cytosolic proteins and sequestration process.

□ The effect of quinidine on the distribution and elimination of digoxin was examined by comparing the change in the steady-state volume of distribution (Vd88),determined both from in vivoplasma elimination and tissue distribution and in vitroserum binding studies, with that in the total body clearance (CLtot) determined from biliary, renal, and metabolic clearances in guinea pigs. The plasma disappearance of digoxin after a 250-μg/kg iv dose followed a triexponential decline in both the control and quinidine-treated guinea pigs. In the quinidine-treated guinea pigs, the pharmacokinetic parameters Vd88and CLtotsignificantly decreased to approximately half of that for the control guinea pigs. The tissue-to-plasma partition coefficients (Kp) of all tissues studied, i.e.liver, heart, muscle, and brain, at 6 hr after bolus injection of digoxin decreased in the presence of quinidine. The serum free fraction and the plasma-to-blood concentration ratio of digoxin in the therapeutic range did not show a significant alteration in the presence of quinidine. This suggested that the decrease of Kpis due mainly to the inhibition of tissue distribution of digoxin by quinidine. The biliary clearance (CLB) and renal clearance (CLR) also significantly decreased in the presence of quinidine. It was concluded that quinidine caused a inhibition of digoxin in the tissue binding or uptake, which significantly decreased the Kpvalues of digoxin; this result may explain the significant decrease of Vdss. Moreover quinidine may be the cause of a reduction of biliary, renal, and metabolic clearances, which significantly decrease the CLtotof digoxin.

□ Uptake of the fluorescent probe, l-anilino-8-naphthalene-sulfonate (I) into isolated rat liver cells was studied using both fluorescence and filtration methods. The time course of the fluorescence enhancement of I after addition to the isolated liver cells was analyzed in terms of rapid, medium, and slow phases. The slow phase (half-time ~7min) was characteristic of viable cells. The fluorescence enhancement was proportional to the amount of I taken into the cells, as measured by the filtration method. The uptake of I followed Michaelis–Menten kinetics with an apparent Kmof 39μMand Vmaxof 1.4 nmole/106cells/min. The temperature coefficient (Q10) of the uptake of I was found to be ~1.9. No pH optimum was observed, and various metabolic inhibitors did not affect the uptake of I. Among the amino acid reagents used, only 2,4-dinitrofluorobenzene decreased the uptake of I (by −45%). The effects of various organic anions on the uptake of I were measured. The inhibition of the uptake of I by sulfobromophthalein could be analyzed in terms of competitive inhibition; the slight inhibition by sodium taurocholate could not. It is concluded that the uptake of I is a carrier-mediated facilitated process, and that the carrier is common to both I and sulfobromophthalein.

The enterohepatic cycling of glycyrrhizin was examined using rats with and without biliary fistulization. The plasma decay in the control rats without fistulization following an iv dose of 100mg/kg of glycyrrhizin, was generally biphasic. However, secondary peaks were observed in all rats in the elimination phase, i.e., 0.5 to 12h following dosing. The plasma concentrations in the rats with biliary fistulization administered the same dose showed a biexponential decline. The AUC and CLtotwere significantly higher and lower in the control rats, respectively. The biliary excretion was 80.6 ± 9.9% of the administered dose, and intestinal absorption was confirmed by using the bile collected after iv dosing. From these results, we concluded that glycyrrhizin was predominantly secreted from the liver into the bile, and that the secondary peaks in the elimination phase, the higher AUC, and the lower CLtotin the control rats were due to the effects of enterohepatic recycling of glycyrrhizin. Furthermore, the transport of the drug from the liver to the bile appears to be a saturable process.

This study was designed to determine the pH of the intestinal surface in the rat jejunum in an attempt to investigate the effect of potential damaging agents on the microclimate-pH. A significant pH gradient was observed between the mucosal surface and the bulk phase; however, the microclimate-pH usually ranged from 6.5 to 7.5, irrespective of the wide range in the bulk pH. When the bulk pH was either 7.3 or 4, the microclimate-pH was ∼6.7, while the microclimate-pH was ∼7.4 when the bulk pH was raised to 9. The distance of the PH gradient was found to be ∼900–1300 μm. After treatment with ouabain (10mM) or amiloride (8mM), or replacement of Na+with Li+, the microclimate-pH significantly increased compared with the control (buffer solution). Chlorpromazine (2mM) and ouabain also significantly increased the microclimate-pH, but at the same time, chlorpromazine decreased the bulk pH so that a more marked pH gradient was observed. Both aspirin (10mM) and acetazolamide (80mg/kg, iv) significantly decreased the microclimate-pH under the condition of the acidic bulk phase pH. N-Acetyl-L-cysteine (5%) induced marked changes in microclimate-pH, the direction depending upon the bulk phase pH. Neither taurocholic acid (10mM) nor glucose (10mM) significantly changed the microclimate-pH.

The effect of a uniform diffusional barrier on hepatic extraction of the parent drug by evenly or unevenly distributed unienzyme was quantitatively determined by the present simulation study. Five models of enzymic distribution were defined with regard to the hepatic blood flow path, and the extraction ratios were calculated or simulated under the various conditions of average intrinsic clearances and diffusion clearances across hepatocytes. Differences in the extraction ratios among the five models were evaluated by the “relative extraction ratios,” which are the extraction ratios in each model divided by that in the model where the enzymatic activity is evenly distributed. It was found that when a diffusion clearance was high compared to the intrinsic clearance, enzymic distribution was not an Important determinant of the extent of hepatic extraction. By contrast, when a diffusional barrier across hepatocytes exists, i.e., the diffusion clearance is low or intermediate compared to the intrinsic clearance, extraction ratios differed widely among the models of enzymic distribution, especially at intermediate average intrinsic clearances. In the presence of a diffusional barrier, the more skewed the distribution of the enzymatic activity is, the lesser the amount of drug eliminated at steady state. The most efficient metabolism occurred when the enzymatic activity was evenly distributed.

To study the transport system of propranolol (PL), a basic drug, in the blood-brain barrier, the uptake of PL into isolated bovine brain microvessels was investigated. The uptake of PL was a concentrative one via saturable process (Km=42.5 μM) that was decreased by hypothermia (Q10=2.2), but not by metabolic inhibitors (2,4-dinitrophenol, KCN, ouabain). Although basic drugs such as quinidine and imipramine decreased both the initial rate of uptake and the steady-state cell-to-medium concentration ratio (C/M) of PL, acidic drugs (phenobarbital, salicylic acid) did not affect them. These results suggest that PL is taken up by the endothelial cells of the isolated brain microvessels by facilitated diffusion which is specific for basic drugs and then binds to certain binding sites in the cells.

The tissue distribution of vincristine was correlated with the tubulin content in different mouse tissues. The concentration of tubulin in the mouse tissues was determined by estimation of the tissue binding capacities for colchicine. Significant differences in the tubulin concentration were observed among the tissues. The comparison of the apparent tissue-to-plasma partition coefficients (Kpapp) of vincristine (taken from the literature) with the tubulin concentration (expressed by the binding capacities for colchicine) showed a good correlation. These results suggest that the in vivo distribution of vincristine is predicted by the tissue tubulin concentration.

The Z-fraction in rat liver cytoplasm, defined first by Levi et al.[J. clin. Invest.48,2156 (1969)] as an organic anion binding fraction of small molecular weight (10,000–14,000) was further purified by the method of ion-exchange chromatography. The purification was characterized by the use of 1-anilino-8-naphthalenesulfonate (ANS) in sensitively detecting the organic anion binding protein in the Z-fraction. The Z-fraction was found to contain three protein fractions having fairly high binding activities for ANS and sulfobromophthalein (BSP). Among these fractions, the D 2-fraction, which eluted earlier on a DEAE-Sephadex column, had the highest binding affinity for these anions. This D 2-fraction was further purified by CM-Sephadex chromatography. The ANS binding protein, thus purified. was similar in physico-chemical properties to the aminoazodye-binding protein of S 20.w 1.6s (Form III) reported by Ketterer et al.[Biochem. J.155.511 (1976)]. The binding affinities for both ANS and BSP were little influenced by the purification. By the fluorescence method, about 30 per cent of the protein in the Z-fraction was found to be this binding protein. The binding stoichiometries for the high affinity sites were 0.7 for ANS and 0.4 for BSP.

The effects of temperature and protein concentration on the binding of thiopental to bovine and rat serum albumin and to rat plasma were examined using flow and equilibrium dialysis techniques. The effects of temperature and protein concentration were peculiar to each species. The binding of thiopental to rat serum albumin or rat plasma was sensitive to temperatures between 4 and 37°, whereas the binding to bovine serum albumin was sensitive to a protein concentration difference of 0.1-4.44%. Therefore, the effective protein fraction is affected by various sets of binding parameters of albumins determined under various temperatures and protein concentrations. Thus, a semi-predictive method for plasma or tissue binding may be unsuccessful unless proper binding parameters are used.

The effects of several phenothiazine derivatives (PTDs) and quinidine (QD) on the jejunal microclimate-pH in rats were studied using a microelectrode. Chlorpromazine, thioridazine, chlorpromazine sulfoxide (CPZSO), trifluoperazine, prochlorperazine, and QD at concentrations of 1 mM increased this microclimate-pH by 0.15–0.3 pH units, while 1 mM diethazine and 1 mM promethazine had little effect on it. The increases in the microclimate-pH caused by PTDs and QD were concentration dependent and reversible. We studied the effects of PTDs on the fluidity of intestinal brush border membranes and on the release of proteins from the intestinal tissue to the lumen. The PTD-induced changes in microclimate-pH could not be explained by either of these nonspecific effects on the membranes. Then, the effects of PTDs on Na+, K+-ATPase activity and Mg2+-ATPase activity were studied using the jejunal homogenate. Each PTD inhibited Na+, K+-ATPase and Mg2+-ATPase activity to some extent. The inhibitory effects on Na+, K+-ATPase and Mg2+-ATPase activity were compared with the PTD-induced increases in the microclimate-pH. No good correlation was obtained between the IC50values of PTDs for Na+, K+-ATPase activity and the concentrations required to increase the microclimate-pH by 0.1 pH unit, while IC50values of PTDs for Mg2+-ATPase activity showed a relatively good correlation, except for that of CPZSO. These findings suggest that the effects of PTDs on the microclimate-pH were not nonspecific, although the increases in the microclimate-pH caused by PTDs cannot be fully explained by the inhibitory effects of these compounds on either Na+, K+-ATPase activity or Mg2+-ATPase activity alone.

To characterize the efflux system of digoxin, a cardiac glycoside, from the brain to the blood through the blood–brain barrier and blood–cerebrospinal fluid (CSF) barrier, the accumulation of digoxin by the brain microvessel or the choroid plexus isolated from guinea pig brain was investigated. The accumulation of digoxin by the brain microvessel has a saturable component (Km= 0.163 μM, Vmax= 0.142 nmol/mL of tissue/min), with a nonsaturable component [Kd= 0.203 cell‐to‐medium (C:M) ratio/min] that was decreased by hypothermia (Q10= 2.9), sulfhydryl reagent, and quinidine, but not by a metabolic inhibitor [2,4‐dinitrophenol (DNP)]. It was concentration‐ and Na+‐ dependent. The accumulation of digoxin by the choroid plexus was also saturable (Km= 1.9 μM, Vmax= 3.8 nmol/mL of tissue/min), and was decreased by hypothermia (Q10= 4.4), sulfhydryl reagents, ouabain, and quinidine, but not by metabolic inhibitors (DNP, KCN); it was also concentration‐ and Na+‐dependent. The binding of digoxin to the homogenate of choroid plexus was one‐tenth of digoxin accumulation by the intact choroid plexus, suggesting that digoxin is transported into the cells and bound to the cytosol fraction. The value of (Vmax/Km+ Kd) multiplied by the total tissue weight of the microvessel per guinea pig is ∼10‐fold that of Vmax/Kmmultiplied by the tissue weight of the choroid plexus, although (Vmax/Km+ Kd) per milliliter of the microvessel is half the Vmax/Kmvalue of the choroid plexus. These findings suggest that digoxin can be excreted from both the brain and the cerebrospinal fluid to blood by a carrier‐mediated diffusion system which is inhibited by quinidine, and that a main route of digoxin efflux from the brain to the blood is not through the blood–CSF barrier, but through the blood–brain barrier.

Kinetic analysis of the tissue distribution of human epidermal growth factor (hEGF) in rats was performed in vivo. The plasma disappearance half-life of [125I]hEGF was prolonged by coadministration of unlabeled hEGF, indicating saturation of the mechanism for hEGF removal from the systemic circulation. To analyze the contribution of each tissue to the uptake of hEGF, the amount of [125I]hEGF taken up by each tissue was determined after coadministration of various amounts of unlabeled hEGF. Kinetic analysis of the data yielded the following results. (1) Among the tissues examined, the distribution of [125I]hEGF to the liver, kidney, small intestine, stomach, and spleen was much greater than that accounted for by the distribution to the extracellular space of each tissue. (2) The binding (or uptake) of hEGF by these tissues showed remarkable saturation, which may represent the receptor-dependent binding (or uptake) mechanism. (3) The apparent binding (or uptake) clearance per gram of tissue at the low dose (in the range of first-order kinetics), defined with regard to the arterial plasma concentration, was greatest in the kidney, followed by the liver and small intestine. The larger binding (or uptake) clearance of the kidney compared with that of the liver can be attributed to the higher plasma flow rate (per gram of tissue) in the kidney. However, the intrinsic ability to take up hEGF was much greater in the liver than that in the kidney. The hepatic binding (or uptake) of hEGF at the low dose was almost limited by the hepatic plasma flow rate. (4) In the whole animal, the bulk of the removal of hEGF from the systemic circulation was accounted for mainly by hepatic clearance, both at the low and high doses.

The hepatic transports of 4-methylumbelliferone (4-MU) and its conjugative metabolites, the glucuronide (4-MUG) and sulfate (4-MUS), were investigated in rats with various methods. The extraction ratio (E) was estimated with the multiple indicator dilution (MID) method using isolated perfused rat liver. The values of Efor 4-MUG and 4-MUS were much lower (<0.2) than that for the parent compound, 4-MU (0.89). In addition, we examined the simulation of the outflow curves of conjugates based on the “distributed” model in which we varied the permeability between the blood and hepatocytes. When the permeability was much smaller relative to the hepatic blood flow, the simulated curve was superimposed on the dilution curve. These results suggest that the influx permeabilities of these conjugates are so low that little extraction occurs during the passage through the liver. Measuring the unidirectional uptake of these conjugates into the liver with the in vitro centrifugal filtration method using isolated hepatocytes, we determined the influx permeabilities (PSinf(total)for the total ligands. The value of PSinf(total)determined with the in vitro method was extrapolated to that per gram of liver, assuming 1g of liver has 1.3 × 108cells. The values of PSinf(total)for 4-MU, 4-MUG, and 4-MUS were 4.8, 0.06, and 0.11 mL/min/g liver, respectively. Thus, the influx permeabilities for 4-MUG and 4-MUS were much smaller than the hepatic blood flow (1.6 mL/min/g liver), confirming the results of MID method.

To assess whether the secretory clearance of N1-methylnicotinamide (NMN), an endogenous organic cation, represents renal tubular secretion of the organic cation, the relationship between the secretory clearance of NMN, CLscn(NMN),and that of tetraethylammonium bromide (TEA), CLscn(TEA),was examined in normal and experimental renal failure (ERF) rats. TEA was selected as a representative organic cation secreted by the kidney. ERF was induced by glycerol, folate, salicylate, uranium, and gentamicin, substances which have been demonstrated to produce specific damage to the kidney by pathophysiological studies. Glomerular filtration rate (GFR), CLscn(NMN),and CLscn(TEA)decreased significantly in most of ERF rats, while blood urea nitrogen (BUN) increased significantly in all ERF rats. There was a statistically significant correlation (r=0.952, p<0.001) between the endogenous CLscn(NMN)and CLscn(TEA)in both the normal and ERF rats. Correlation analysis revealed that CLscn(NMN)was superior to GFR in the degree of relationship to CLscn(TEA),but BUN could not be used as an index for the secretion of NMN or TEA. Although the plasma concentration of NMN in most of the ERF rats was much higher than that in the normal rats, it affected neither the urinary clearance of NMN itself nor the excretion of TEA. From these findings, we propose that CLscn(NMN)can be used as an index to assess renal tubular function for the secretion of organic cations that are excreted by both filtration and secretion without reabsorption.