Notes on the inverse Gaussian distribution and choice of boundary conditions for the dispersion model in the analysis of local pharmacokinetics.

The dispersion model has been widely used to analyze local pharmacokinetics in the organs and the tissues since the 1980's. However, an ambiguity still remains in selecting the boundary conditions which are necessary to solve the basic equation of the model. In this note, theoretical considerations are given to this problem and we present here some deficiencies of the mixed boundary conditions. It seems that theoretical confusion exists in the literature for the mixed boundary conditions. It is well-known that the solution of the dispersion model with a bolus input is the inverse Gaussian distribution for the mixed boundary conditions. However, it is rarely recognized that the inverse Gaussian distribution requires an open boundary at either the inlet or the outlet. For the analysis of local pharmacokinetics, the use of the classical Danckwerts (or closed) boundary conditions is recommended.

Carrier-mediated hepatic uptake of peptidic endothelin antagonists in rats.

The endothelin antagonist BQ-123, an anionic cyclopentapeptide, is taken up by rat hepatocytes through active transport systems. Here, we have examined the hepatocellular uptake mechanism for several BQ-123 derivatives with anionic charges using isolated rat hepatocytes. BQ-485, a linear peptide, BQ-518, a cyclic peptide, and compound A, a cyclic peptide with a cationic moiety, were taken up by hepatocytes in a concentration-dependent manner. The uptake of BQ-485 was most efficient, whereas compound A showed comparable uptake with BQ-123. The uptake of these peptides was Na(+)- and energy-dependent, suggesting that active transport mechanisms are involved in their uptake into hepatocytes. BQ-485, BQ-518, and compound A can almost completely inhibit both the Na(+)-dependent and -independent uptake of [(3)H]BQ-123, with inhibition constants (K(i)) that are comparable to the Michaelis-Menten constants (K(m)) for their Na(+)-dependent and -independent uptake, respectively. Inhibition by BQ-485 was competitive, and the uptake of BQ-485 can be inhibited by BQ-123, with K(i) values that are comparable with the K(m) values for BQ-123 uptake. The uptake of BQ-123 by COS-7 cells transfected with either Na(+)-dependent taurocholate-cotransporting polypeptide (Ntcp) or Na(+)-independent basolateral organic anion-transporting polypeptide (oatp1) was minimal. Thus, these three peptides share the transporters that also recognize BQ-123 but appear to differ from Ntcp and oatp1.

Analysis of nonlinear hepatic clearance of a cyclopentapeptide, BQ-123, with the multiple indicator dilution method using the dispersion model.

PURPOSE: To bridge in vitro, in situ and in vivo kinetic analyses of the hepatic clearance of a cyclopentapeptide, BQ-123, by using dispersion models that assume nonlinear pharmacokinetics. METHODS: Rat livers were perfused by the multiple indicator dilution method with doses of BQ-123 ranging from 1-1000 microg. The outflow dilution curves were fitted to a two-compartment dispersion model that was solved numerically by the finite difference method. Further, in vivo plasma concentrations of BQ-123 after bolus injection were analyzed with a hybrid physiological model that incorporates the hepatic dispersion model. RESULTS: The calculated Michaelis-Menten constants (Km = 12.0 microM, Vmax = 321 pmol/min/10(6) cells, P(dif) = 1.2 microl/min/10(6) cells) were comparable to those obtained previously from the in vitro isolated hepatocyte experiment (Km = 9.5 microM, Vmax = 517 pmol/min/l0(6) cells, P(dif) = 1.1 microl/min/10(6) cells). The plasma concentrations of BQ-123 at doses of 1-25 mg/kg were explained well by the hybrid physiological model. CONCLUSIONS: These results suggest that carrier-mediated transport on the sinusoidal membrane was responsible for the in vivo hepatic elimination of BQ-123.

Hepatobiliary transport governs overall elimination of peptidic endothelin antagonists in rats.

The overall disposition and hepatobiliary transport of BQ-123, an anionic cyclopentapeptide, and three analogs were examined in rats in vivo. Total body clearance (CLtotal) and biliary excretion clearance (CLbile, p) exhibited 4- to 8-fold differences between the compounds, with those for BQ-485 and compound A having the highest and lowest values, respectively. The CLbile, p values of BQ-485, BQ-123, and BQ-518 were almost equal to the CLtotal, suggesting that hepatobiliary transport is the major elimination pathway for these compounds. Hepatic uptake clearance (CLuptake, vivo) and biliary excretion clearance (CLbile, h/fT), which was defined for the hepatic unbound concentration, were separately determined to examine the hepatic uptake and excretion processes, respectively. Both the CLuptake, vivo and CLbile, h/fT of BQ-485 were higher than those of BQ-123, whereas the corresponding values for BQ-518 were similar to those for BQ-123. The CLuptake, vivo and CLbile, h/fT of compound A were, respectively, approximately two thirds and one half those of BQ-123, suggesting that the lower CLbile, p value is due to the low efficiency of both the uptake and excretion processes. The CLuptake, vivo of these four peptides in vivo was similar to the extrapolated values based on the carrier-mediated transport activity previously assessed in vitro in isolated rat hepatocytes. The primary active transport previously assessed in an in vitro study in canalicular membrane vesicles was also highest for BQ-485 and lowest for compound A, similar to CLbile, h/fT in vivo. Thus, the transporters on both the sinusoidal and canalicular membranes determine the efficiency of the peptide overall elimination from the circulation.

Primary active transport of peptidic endothelin antagonists by rat hepatic canalicular membrane.

The biliary excretion mechanism of three derivatives of BQ-123, an anionic cyclopentapeptide, was examined using isolated canalicular membrane vesicles (CMVs) from Sprague-Dawley rats. The uptake by CMV of BQ-485, a linear peptide, BQ-518, a cyclic peptide, and compound A, a cyclic peptide with a cationic moiety, was stimulated by ATP. An "overshoot" phenomenon and saturation were observed for the ATP-dependent uptake of these three peptides. The Michaelis-Menten constants (Km) for the uptake of BQ-485 and BQ-518 were comparable to the inhibition constants (Ki) for their inhibitory effects on ATP-dependent [3H]BQ-123 uptake. The uptake of BQ-485 showed the highest value and was inhibited by BQ-123 with a Ki that was comparable to the Km for BQ-123 uptake. The ATP-dependent uptake of BQ-123, BQ-485, and BQ-518 was much lower in CMVs from Eisai hyperbilirubinemic rats, a strain having a hereditary defect of the canalicular multispecific organic anion transporter (cMOAT). These results suggest that both BQ-485 and BQ-518 principally share the cMOAT transporter with BQ-123. Compound A almost completely inhibited BQ-123 uptake, although its ATP-dependent uptake was much lower than that of the other three peptides. The ATP-dependent uptake of compound A was not very different in Sprague-Dawley rats and Eisai hyperbilirubinemic rats and was not inhibited by S-(2, 4-dinitrophenyl)-glutathione, a typical substrate for cMOAT. Thus, although compound A inhibits cMOAT-mediated transport, its own transport by cMOAT is minimal and mediated by another transporter. This low degree of primary active transport by cMOAT may be the principal reason for its relatively longer residence in the circulation.

Prediction of in vivo nonlinear first-pass hepatic metabolism of YM796 from in vitro metabolic data.

Recent cumulative evidence suggests the possibility of predicting the in vivo metabolic clearance and/or hepatic availability (Fh) from in vitro metabolism data under linear conditions. Under nonlinear conditions, however, it is essential to consider the rate constant for the absorption (ka) for predicting Fh after oral administration, because the time profiles for the portal vein concentration depends on ka. In our study, we numerically solved the dispersion model under nonlinear conditions to propose a method to predict Fh after oral administration by taking ka into consideration. As a model compound, (S)-(-)-2,8-dimethyl-3-methylene-1-oxa-8-azaspiro [4,5] decane-L-tartrate monohydrate (YM796) was used. After oral administration, we found that the dose-normalized AUC (AUCoral/dose) was markedly increased in rats from 5.0 x 10(-6) to 33 x 10(-6) hr/ml.kg as the dose increased from 1 to 10 mg/kg, whereas the same value was relatively constant in dogs (87.7 x 10(-6) to 105 x 10(-6) hr/ml.kg at 1 to 10 mg/kg) and in humans (1260 x 10(-6) to 1768 x 10(-6) hr/ml.kg at 5 to 60 mg/body). Kinetic analysis indicated that AUCoral could be accurately predicted at each dose if ka value was assumed as 0.07 min-1 for all animal species examined in our study. These results suggest that it is possible to predict Fh even if the metabolism is composed of non-linear process by considering the absorption rate into the portal vein.

Analysis of nonlinear and nonsteady state hepatic extraction with the dispersion model using the finite difference method.

A numerical calculation method for dispersion models was developed to analyze nonlinear and nonsteady hepatic elimination of substances. The finite difference method (FDM), a standard numerical calculation technique, was utilized to solve nonlinear partial differential equations of the dispersion model. Using this method, flexible application of the dispersion model becomes possible, because (i) nonlinear kinetics can be incorporated anywhere, (ii) the input function can be altered arbitrarily, and (iii) the number of compartments can be increased as needed. This method was implemented in a multipurpose nonlinear least-squares fitting computer program, Napp (Numeric Analysis Program for Pharmacokinetics). We simulated dilution curves for several nonlinear two-compartment hepatic models in which the saturable process is assumed in transport or metabolism, and investigated whether they could definitely be discriminated from each other. Preliminary analysis of the rat liver perfusion data of a cyclic pentapeptide, BQ-123, was performed by this method to demonstrate its applicability.

Hepatobiliary transport mechanism for the cyclopentapeptide endothelin antagonist BQ-123.

The hepatobiliary transport of an anionic cyclopentapeptide endothelin antagonist, BQ-123, was studied in rats. Biliary excretion of [3H]BQ-123 was extensive in vivo (approximately 75% of intravenous infusion rates). Liver-to-plasma and bile-to-liver concentration ratios at steady state were approximately 3 and 200, respectively, suggesting that hepatic uptake and biliary excretion are concentrative processes. The biliary excretion clearance exhibited a saturation at a hepatic concentration of > 100 nmol/g liver and was markedly reduced in Eisai hyperbilirubinemic rats, which have a hereditary defect of canalicular multispecific organic anion transporter. An ATP-dependent and saturable uptake of BQ-123 by isolated canalicular membrane vesicles was observed in vitro. Impaired transport of BQ-123 was also confirmed in canalicular membrane vesicles prepared from Eisai hyperbilirubinemic rats. These results demonstrate that the biliary excretion process is ATP-driven primary active transport. It is proposed that a canalicular multispecific organic anion transporter is mainly responsible for the biliary excretion of BQ-123.

Carrier-mediated active transport of BQ-123, a peptidic endothelin antagonist, into rat hepatocytes.

The hepatic uptake mechanisms and pharmacokinetics of BQ-123, an anionic cyclopentapeptidic endothelin ET(A) receptor antagonist, were studied in rats. Elimination of BQ-123 from plasma after intravenous injection of the compound was very rapid as evidenced by high total body clearance (CLtot, 50 ml/min/kg), which is comparable with hepatic blood flow rate. Within 1 hr after injection, 86% of the dose was excreted as its intact form in the bile. BQ-123 was taken up extensively by isolated rat hepatocytes both Na(+)-dependently and Na(+)-independently. The Na(+)-dependent system transported BQ-123 with higher affinity than did the Na(+)-independent system (Km; 6 and 12 microM, respectively), but its capacity was lower (Vmax; 140 and 390 pmol/min/10(6) cells, respectively). Both uptake systems were found to be active transport systems because of explicit inhibition by metabolic inhibitors. BQ-485, an anionic linear tripeptide, strongly inhibited BQ-123 uptake with Ki values of 1.6 and 2.5 microM for Na(+)-dependent and Na(+)-independent systems, respectively, whereas BQ-587, a cationic cyclopentapeptide, inhibited BQ-123 uptake only slightly. Considering this in vitro finding and the low in vivo CLtot of BQ-587 together, the carrier systems for BQ-123 seem to recognize negatively charged substances selectively. Both Na(+)-dependent and Na(+)-independent uptake of BQ-123 were competitively inhibited by a bile acid (taurocholate) and an organic anion (dibromosulfophthalein). The Ki values were comparable with the Km values of taurocholate and dibromosulfophthalein transport, which suggests that the Na(+)-dependent system corresponds to the bile acid transporter and that the Na(+)-independent system corresponds to the organic anion transporter.

The endothelin receptor is a major determinant for the nonlinear tissue distribution of the endothelin antagonist BQ-123.

The nonlinearity in the pharmacokinetics of the cyclopentapeptide endothelin antagonist BQ-123 was studied. Both the total body clearance and tissue-to-plasma concentration ratio (Kp) were investigated in rats under a wide range of steady-state plasma concentrations (Cpss) obtained by changing the intravenous infusion rate of BQ-123. The total body clearance was constant up to a Cpss level of 50 microM, although it was markedly decreased at higher Cpss values, which suggests the existence of a saturable elimination mechanism. A Cpss-dependent nonlinearity in the apparent Kp values (Kp,app) was clearly observed in many organs including lung, heart, spleen, pancreas, adrenal, stomach, intestine, colon, aorta, testis and muscle, where the endothelin ET(A) receptor is known to be localized. By fitting the saturation curves of the Kp,app values, a similar dissociation constant (Kd) was obtained for most organs at 5 to 10 nM, which is close to the reported Kd values of BQ-123 for the endothelin ET(A) receptor. The saturable portion of the Kp,app values observed in vivo showed a good correlation with reported values of the endothelin ET(A) receptor density. Binding of BQ-123 to isolated membrane fractions from several organs demonstrated clear saturability for the lung, heart, spleen and liver with Kd values of 1 to 3 nM. Such specific binding also showed a good correlation with the saturable portion of the Kp,app values. From these results, we concluded that the endothelin receptor(s) is responsible for the nonlinear tissue distribution of BQ-123 in rats.

Cyclic pentapeptide endothelin A receptor antagonists with attenuated in vivo clearance.

A series of analogues of BQ-123 (1), a potent cyclic pentapeptide endothelin A receptor antagonist, with amino acids linked to the side-chain of the Pro residue via an ester linkage was synthesized. All analogues synthesized exhibited potent endothelin A receptor binding affinity similar to that of 1. Of the synthesized analogues, the Lys, Arg and N alpha,N epsilon-dimethyllysine analogues, 9d-f, exhibited about a three-fold attenuation of in vivo clearance compared with 1. In rats, these analogues exhibited a 3-fold-higher plasma concentration and a longer retention time in plasma as compared with those of 1. The attenuated in vivo clearance was thought to be a consequence of decreased extraction of the compounds from the blood via the hepatic anion transport system, which efficiently extracts 1 from the blood.

Removal of imipenem and cilastatin by hemodialysis in patients with end-stage renal failure.

The removal of imipenem and cilastatin by hemodialysis was studied in 14 (for imipenem) and 6 (for cilastatin) subjects. Following intravenous infusion of imipenem and cilastatin at a combined concentration of 10 mg/kg of body weight, drug levels in plasma were determined serially during off- and on-hemodialysis periods, which were 2 and 4 h, respectively. The biexponential decay of the drug levels in plasma was evident in each subject for both imipenem and cilastatin. Hemodialysis accelerated the elimination of both imipenem and cilastatin: the mean elimination-phase half-life of imipenem was shortened from 200 to 78 min, and that of cilastatin was shortened from 445 to 115 min. Hemodialysis clearance of imipenem and cilastatin was calculated by five different methods, each with intrinsic assumptions. The mean hemodialysis clearance of imipenem was estimated to be 74.08 +/- 13.29 ml/min, and that of cilastatin was estimated to be 65.0 +/- 8.6 ml/min, after consideration of various methodological limitations. It was estimated that in a hypothetical anephric patient weighing 60 kg, a 4-h hemodialysis treatment would remove 54.8% of the imipenem and 62.9% of the cilastatin present in the body at the start of dialysis.

Absorption of a novel prodrug of L-dopa, L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine (NB-355). In vitro and in situ studies.

Absorption mechanism and absorption site of a prodrug of L-DOPA, L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine (NB-355, 1) was investigated using rats. Prodrug 1 (0.5 mM) was taken up by intestinal tissue segments time-dependently in vitro at pH 6.0. However, the rate of uptake was less than that of L-dopa. Inhibitors of the amino acid active transport system (L-Phe, dinitrophenol, ouabain) had no effect on the uptake of prodrug 1. In the intestinal tissue segments, prodrug 1 was extensively hydrolyzed by diisopropylfluorophosphate-sensitive esterase(s). To characterize the absorption site, gastrointestinal tracts were ligated to make acute loops in situ and prodrug 1 or L-dopa was injected into the loops. L-dopa disappeared rapidly from the lumen of the jejunum. In contrast, prodrug 1 disappeared rapidly from the ileum rather than the duodenum or jejunum. From these results, it was suggested that prodrug 1 was slowly absorbed primarily from the lower small intestine.

Hydrolysis and acyl migration of a catechol monoester of L-dopa: L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine.

Hydrolysis and acyl migration studies on L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine (1, NB-355), which produced long-lasting plasma L-dopa levels after oral dosing, have been conducted. Compound 1 exists as pure 4-O-pivaloyl-L-dopa in the solid state, but it converts rapidly to a mixture of the 3- and 4-O-isomers in solution. The rate of acyl migration increased with increases in pH and temperature, and the content of the 4-O-isomer in the equilibrium state was 53-59%. The hydrolysis rate of 1 to L-dopa (6) also increased with increases in pH and temperature, and accelerated steeply at neutral and alkaline pH. The rapid hydrolysis at neutral pH was not observed with O-pivaloyl-L-tyrosine (3), di-O-pivaloyl-L-dopa (4), or L-dopa methyl ester (5). Because of this chemical lability, 1 was hydrolyzed in rat plasma far faster than the other tested catechol esters. However, in rat intestinal homogenate at pH 6.0, 1 was hydrolyzed at the slowest rate among the tested esters, predominantly by a diisofluorophosphate (DFP)-sensitive esterase. Thus, 1 showed a unique in vitro profile on hydrolysis and acyl migration due to the existence of a neighboring hydroxyl group. The stability of 1 in the intestine might be essential for the long-lasting plasma L-dopa profile after oral dosing of 1.

Quantification of L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine (NB-355) by high-performance liquid chromatography using o-phthalaldehyde/N-acetyl-L-cysteine derivatization.

A new and rapid high-performance liquid chromatographic assay has been developed for the determination of L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine (NB-355,I), a novel prodrug of L-DOPA. The method involves precolumn derivatization of the drug in biological samples with o-phthalaldehyde (OPA) and N-acetyl-L-cysteine (NAC) in a triethanolamine buffer (pH 8.0), giving a fluorescent compound that is stable for 2 h at 4 degrees C. Use of an internal standard improved the assay in accuracy and reliability. A programmable injector allowed automatic derivatization of large numbers of samples. Chromatographic separation was performed on a reversed-phase column (Capcell Pak C18) in which the silica gel was coated with silicone polymer. The peaks corresponding to compound I and the internal standard were eluted within 16 min with a mobile phase of acetonitrile-phosphate buffer (pH 7.1). The reliable limit of quantification was 0.5 pmol per injection (0.05 micrograms equivalents of L-DOPA per ml in plasma). The method was successfully applied for the measurements of dog plasma concentrations after oral dosing of compound I.

A new potential prodrug to improve the duration of L-dopa: L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine.

L-3-(3-Hydroxy-4-pivaloyloxyphenyl)alanine (1, NB-355) is a novel L-dopa prodrug. After oral administration with carbidopa in rats, 1 demonstrated 2.3 times longer duration (MRT) and 1.4 times larger bioavailability (AUC) on plasma L-dopa concentrations than those of L-dopa itself. Similar results were obtained in dogs. The prolonged profile of L-dopa was parallel to that of carbidopa, and the intact ester was undetectable in rat plasma. After intravenous administration in rats, 1 was converted quickly and completely to L-dopa in the systemic circulation. It was also noted that the oral LD50 value of 1 was greater than 6 g/kg in mice. These data suggest that 1 will offer long-lasting L-dopa therapy for the treatment of Parkinson's disease with little concern about toxicity.