Kinetic analysis of the dose-dependent hepatic handling of 1-anilino-8-naphthalene sulfonate in rats

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 (CL_tot) decreased with increasing dose of ANS. That is, the values of CL_tot were 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 (CL_up and CL_seq, respectively) for a total ligand were constant irrespective of dose, while the efflux clearance (CL_eff) for a total ligand was increased by twofold with increasing dose. A mechanism for the increase in the CL_eff value 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 CL_seq values 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.     

Determination of the Rate-Limiting Step in the Hepatic Elimination of YM796 by Isolated Rat Hepatocytes

Purpose. The membrane permeability clearance and intrinsic metabolic clearance of a drug in the liver were estimated using isolated rat hepatocytes, and the rate-limiting step in the overall intrinsic clearance of the drug in vivo was investigated. For this purpose, an anti-dementia drug, (S)-(-)-2,8-dimethyl-3-methylene-l-oxa-8-azaspiro [4,5] de-cane-L-tartarate monohydrate (YM796) was used as a model drug. Methods. The parent drug and its metabolites in both medium and cells were separated by thin-layer chromatography (TLC). The total amount of drug taken up by hepatocytes and the total amount of metabolites were plotted against the AUC of YM796 in the medium or cells to obtain the kinetic parameters. Results. While the influx clearance (PS_int) through the sinusoidal membrane defined in terms of YM796 concentration in the medium was almost constant, irrespective of the concentration of YM796 in the medium, the intrinsic metabolic clearance (CL_int) and the efflux clearance (PS_eff) defined in terms of the total concentration of YM796 in the cells markedly decreased and increased, respectively, as the concentration of YM796 increased. The overall intrinsic metabolic clearance (Cl_int,all), defined in terms of the YM796 concentration in the medium, corresponding to the hepatic intrinsic clearance obtained from thein vivo pharmacokinetic data on the drug, was comparable with PS_inf at low concentrations of YM796. As the YM796 concentration increased, however, Cl_int,all fell markedly approaching CL_int. Conclusions. While, at low concentrations of YM796, CL_int,all was predominantly affected by membrane permeability clearance, at high concentrations it was completely rate-determined by the intrinsic metabolic clearance because of the marked reduction in CL_int resulting from the saturation of YM796 metabolism.     

A new approach to predicting human hepatic clearance of CYP3A4 substrates using monkey pharmacokinetic data

1. Focusing on the genetic similarity of CYP3A subfamily enzymes (CYP3A4 and CYP3A5) between monkeys and humans, we have attempted to provide a single-species approach to predicting human hepatic clearance (CL_h) of CYP3A4 substrates using pharmacokinetic parameters in cynomolgus monkeys following intravenous administrations.

2. Hepatic intrinsic clearance (CL_int,h) of six CYP3A4 substrates (alprazolam, clonazepam, diltiazem, midazolam, nifedipine, and quinidine), covering a wide range of clearance, in monkeys correlated well with that cited in literature for humans (R = 0.90) with a simple equation of Y = 0.165X (Y: human CL_int,h, X: monkey CL_int,h, represented in mL/min/kg).

3. To verify the predictability of human CL_int,h, monkey CL_int,h of a test set of CYP3A4 substrates cited in literature (dexamethasone, nifedipine, midazolam, quinidine, tacrolimus, and verapamil) was applied to the equation and human CL_int,h was calculated. The human CL_int,h of all the substrates was predicted within 3-fold error (fold error: 0.35–2.77).

4. The predictability of human CL_h by our method was superior to common in vivo prediction methods (allometry and liver blood flow method). These results suggest that human hepatic clearance of CYP3A4 substrates can be predicted by applying cynomolgus monkey CL_int,h obtained following intravenous administrations in each laboratory to the simple equation.     

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics. iii: mechanisms for sinusoidal efflux of 4-methylumbelliferone sulfate

1.?To elucidate the mechanisms involved in the sinusoidal efflux of sulfate and glucuronide metabolites of 4-methylumbelliferone (4MU), isolated rat liver perfusion studies were performed under several conditions.

2.?The effect of sodium azide on the hepatic handling of both conjugates was examined. The net sinusoidal efflux clearance (CL_eff) based on the unbound concentration in the liver did not change for 4MU glucuronide (4MUG) or significantly increase for 4MU sulfate (4MUS), suggesting that the sinusoidal efflux of both conjugates is not mediated by the transport systems dependent on adenosine triphosphate.

3.?Under Cl^?-depleted conditions, the CL_eff of 4MUG significantly decreased, but the saturation of its sinusoidal efflux rather than the transport system dependent on Cl^? might be involved because the hepatic concentration of 4MUG was extensively higher than that of the control study due to the extremely attenuated biliary excretion. The CL_eff of 4MUS also significantly decreased, but its hepatic concentration was not different from that in the control study, suggesting that the transport system using Cl^? is involved in the sinusoidal efflux of 4MUS.

4.?The effect of glutathione was examined. CL_eff of 4MUG was not affected by the additional glutathione, but CL_eff of 4MUS decreased significantly, suggesting that some transport system sensitive to glutathione is involved in the sinusoidal efflux of 4MUS, but not of 4MUG.

5.?Transporters such as Oatp1, Oatp2 and/or Npt1 might be involved in the sinusoidal efflux of 4MUS, but 4MUG is secreted from the sinusoidal membrane via the systems that are totally different from those for 4MUS.     

Drug pharmacokinetics and the carbon dioxide breath test

The interrelationship of the pharmacokinetics of a drug and the expiration of carbon dioxide formed as a metabolite have been studied. The pharmacokinetic characteristics of the drug that affect the usefulness of the carbon dioxide excretion as a measure of liver function were examined by means of computer simulations. The parent drug extraction ratio, fraction demethylated, volume of distribution, and absorption rate of an oral dosage form all contribute to the carbon dioxide breath test result. A drug that would be a useful substrate when the carbon dioxide breath test is used as a probe for changes in liver function should be at least 50% metabolized by demethylation, have a hepatic extraction ratio of 0.2–0.5, and be administered in a form that is rapidly absorbed.Appendix b. symbols CL _c net clearance of formaldehyde to carbon dioxide - CL _int,f intrinsic hepatic clearance of formation of formaldehyde from parent drug (bound and unbound to plasma proteins) - CL _int,p intrinsic hepatic clearance of total parent drug (bound and unbound to plasma proteins) - CL _sys,f systemic hepatic clearance of formation of formaldehyde from parent drug,Q _H CL _int,f /(Q _H +CL _int,p ) - CL _sys,p systemic hepatic clearance of parent drug,Q _H CL _int,p /(Q _H +CL _int,p ) - E extraction ratio,CL _int,p /(Q _H +CL _int,p - F I-E fraction escaping first-pass metabolism,Q _H/(Q _H +CL _int,p - fm fraction of parent drug metabolized by demethylation to formaldehyde,CL _int,f /CL _int,p - HCHO amount of formaldehyde - [HCHO] concentration of formaldehyde - _a absorption rate constant - M _i metabolite of P formed by routes other than demethylation - M ₁ metabolite of P formed by demethylation - P amount of parent drug in the body - [P] concentration of parent drug measured in arterial blood - P _A amount of parent drug at absorption site - P _L amount of parent drug in the liver - Q _H hepatic blood flow - V _F volume of distribution of formaldehyde - V _p volume of distribution of parent drug     

Utilization of Liver Microsomes to Estimate Hepatic Intrinsic Clearance of Monoamine Oxidase Substrate Drugs in Humans

Purpose

Monoamine oxidases (MAOs) are non-CYP enzymes that contribute to systemic elimination of therapeutic agents, and localized on mitochondrial membranes. The aim of the present study was to validate quantitative estimation of metabolic clearance of MAO substrate drugs using human liver microsomes (HLMs).

Methods

Three MAO substrate drugs, sumatriptan, rizatriptan and phenylephrine, as well as four CYP substrates were selected, and their disappearance during incubation with HLMs or mitochondria (HLMt) was measured. Metabolic clearance (CL) was then calculated from the disappearance curve.

Results

CL obtained in HLMs for sumatriptan and a typical MAO substrate serotonin was correlated with that obtained in HLMt among ten human individual livers. Hepatic intrinsic clearance (CL_int,vitro) estimated from CL in HLMs was 14–20 and 2–5 times lower than in vivo hepatic intrinsic clearance (CL_int,vivo) obtained from literature for MAO and CYP substrates, respectively. Utilization of HLMs for quantitatively assessing metabolic clearance of MAO substrates was further validated by proteomics approach which has revealed that numerous proteins localized on inner and outer membranes of mitochondria were detected in both HLMs and HLMt.

Conclusion

CL_int,vitro values of MAO substrate drugs can be quantitatively estimated with HLMs and could be used for semi-quantitative prediction of CL_int,vivo values.

     

Use of uptake intrinsic clearance from attached rat hepatocytes to predict hepatic clearance for poorly permeable compounds

1. We previously reported that the accuracy of clearance (CL) prediction could be differentiated by permeability. CL was drastically under-predicted by in vitro metabolic intrinsic clearance (CL_int) for compounds with low permeability (<5?×?10^?6 cm/s).

2. We determined apparent uptake CL_int by measuring initial disappearance from medium using attached rat hepatocytes and metabolic CL_int by measuring parent depletion in suspended rat hepatocytes (cells and medium).

3. Uptake and metabolic CL_int were comparable for highly permeable metabolic marker compounds. In contrast, uptake CL_int was 3- to 40-fold higher than metabolic CL_int for rosuvastatin, bosentan, and 15 proprietary compounds, which had low permeability, suggesting that uptake could be a rate-determining step in hepatic elimination for these poorly permeable compounds.

4. The prediction of hepatic CL was improved significantly when using uptake CL_int for the compounds with low permeability. The average fold error was 2.2 and 6, as opposed to >11 and >47 by metabolic CL_int, with and without applying a scaling factor of 4, respectively.

5. Uptake CL_int from attached hepatocytes can be used as an alternative approach to predict hepatic clearance and to understand the significance of hepatic uptake in elimination in an early drug discovery setting.

     

Relative Importance of Intestinal and Hepatic Glucuronidation—Impact on the Prediction of Drug Clearance

Purpose  To assess the extent of intestinal and hepatic glucuronidation in vitro and resulting implications on glucuronidation clearance prediction. Methods  Alamethicin activated human intestinal (HIM) and hepatic (HLM) microsomes were used to obtain intrinsic glucuronidation clearance (CL_int,UGT) for nine drugs using substrate depletion. The in vitro extent of glucuronidation (fm_UGT) was determined using P450 and UGT cofactors. Utility of hepatic CL_int for the prediction of in vivo clearance was assessed. Results  fm_UGT (8–100%) was comparable between HLM and HIM with the exception of troglitazone, where a nine-fold difference was observed (8% and 74%, respectively). Scaled intestinal CL_int,UGT (per g tissue) was six- and nine-fold higher than hepatic for raloxifene and troglitazone, respectively, and comparable to hepatic for naloxone. The remaining drugs had a higher hepatic than intestinal CL_int,UGT (average five-fold). For all drugs with P450 clearance, hepatic CL_int,CYP was higher than intestinal (average 15-fold). Hepatic CL_int,UGT predicted on average 22% of observed in vivo CL_int; with the exception of raloxifene and troglitazone, where the prediction was only 3%. Conclusion  Intestinal glucuronidation should be incorporated into clearance prediction, especially for compounds metabolised by intestine specific UGTs. Alamethicin activated microsomes are useful for the assessment of intestinal glucuronidation and fm_UGT in vitro.     

Kinetics of hepatic transport of 4-methylumbelliferone in rats. Analysis by multiple indicator dilution method

Hepatic elimination of 4-methylumbelliferone (4-MU), which has been used as a model compound for conjugative metabolism, was studied by means of a multiple indicator dilution (MID) method in the isolated perfused rat liver. Using this method, three intrinsic hepatic clearances, CLint,inf, CLint,eff, and CLint,seq, which represent the influx, efflux, and sequestration processes, respectively, were obtained. When the dose was increased from a low dose (50 micrograms/rat liver) to a high dose (3000 micrograms/rat liver), the hepatic availability of 4-MU increased from 0.11 to 0.73. With increasing dose, the CLint,eff value increased approximately two times, while the CLint,seq value decreased to approximately one-third. The remarkable dose dependence of hepatic availability was due to nonlinearity in both CLint,eff and CLint,seq values. However, the CLint,inf value was almost independent of dose. The dose-dependent change in CLint,seq might be explained by the saturation of conjugative metabolism of 4-MU, while the increase in the CLint,eff value with increasing dose might be partly explained by the nonlinear tissue binding of 4-MU, since the tissue unbound fraction determined by an ultrafiltration method using liver homogenate increased approximately 1.5 times at higher concentration of 4-MU compared to that at lower concentrations. In addition, based on a comparison of the individual intrinsic clearances, i.e., CLint,inf, CLint,eff, and CLint,seq, the major determining process of the apparent hepatic intrinsic clearance of 4-MU is thought to be the sequestration process at the high dose. However, at the low dose, the membrane transport process (influx and efflux processes) as well as the sequestration process also determine the apparent hepatic intrinsic clearance.     

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics II: Studies with isolated perfused rat liver

1.?To elucidate the determining factors for elimination pathways of sulfate and glucuronide metabolites of xenobiotics, a single-pass perfusion of 4-methylumbelliferone (4MU) or p-nitrophenol (pNP) was performed with an isolated rat liver preparation.

2.?Without bovine serum albumin in the perfusion system, clearance calculated based on the unbound concentration in the liver clearly showed that the net efflux clearances (CL_eff) of sulfates from the sinusoidal membrane were much higher than those of glucuronides and that the biliary excretion clearances (CL_b) of glucuronides were approximately two times larger than those of sulfates.

3.?The ratios of CL_eff to CL_b were much higher for sulfates than those for glucuronides. The bile-oriented elimination of glucuronides or sinusoidal efflux-oriented elimination of sulfates was observed even using the perfusate including 3% bovine serum albumin, but the sinusoidal efflux of sulfates was extensively enhanced by bovine serum albumin in the perfusate. The mechanisms behind this stimulatory effect remain to be elucidated.

4.?For both compounds, CL_b of glucuronide was comparable with CL_b of sulfate, meaning that CL_b is not responsible for the biliary excretion of glucuronides at extensively higher rate than sulfates.

5.?Higher concentration of glucuronides in the liver, partly caused by much lower CL_eff of glucuronides than that of sulfates, is likely responsible for the bile-oriented excretion of glucuronides. The extensive sinusoidal efflux of sulfates, leading to the urine-oriented excretion, is attributed to the substantially higher CL_eff than CL_b.

6.?In conclusion, the sinusoidal efflux is an important factor for determining elimination pathways of both sulfates and glucuronides, although further studies are needed to clarify the mechanisms of the sinusoidal efflux.     

Method for predicting human intestinal first-pass metabolism of UGT substrate compounds

1. As intestinal glucuronidation has been suggested to generate the low oral bioavailability (F) of drugs, estimating its effects would be valuable for selecting drug candidates. Here, we investigated the absorption and intestinal availability (F_aF_g) in animals, and intrinsic clearance via UDP-glucuronosyltransferase (UGT) in intestinal microsomes (CL_int,UGT) for three drug candidates possessing a carboxylic acid group, in an attempt to estimate the impact of intestinal glucuronidation on F and select potential drug candidates with high F in humans.

2. The F_aF_g values of the three test compounds were low in rats and monkeys (0.16–0.51), and high in dogs (≥0.81). Correspondingly, the CL_int,UGT values were high in rats and monkeys (101–731 µL/min/mg), and low in dogs (≤?59.6 µL/min/mg). A good inverse correlation was observed between F_aF_g and CL_int,UGT, suggesting that intestinal glucuronidation was a major factor influencing F_aF_g of these compounds.

3. By applying this correlation to F_aF_g in humans using human CL_int,UGT values (26.9–114 µL/min/mg), compounds 1–3 were predicted to have relatively high F_aF_g.

4. Our approach is expected to be useful for estimating the impact of intestinal glucuronidation on F in animals and semiquantitatively predicting human F for drug candidates.

     

Sinusoidal Efflux of Taurocholate Is Enhanced in Mrp2-Deficient Rat Liver

Purpose. It has been shown that plasma concentration and urinary excretion of bile acids is elevated under the cholestatic/hyperbilirubinemic conditions. Previously, it was demonstrated that the plasma concentration of bile acids was elevated in the multidrug resistance-associated protein 2 (Mrp2)-deficient rats. The purpose of the present study was to compare the sinusoidal efflux clearance of taurocholate (TC) between Mrp2-deficient Eisai hyperbilirubinemic rats (EHBR) and normal rats. Method. Hepatic disposition of the [³H]TC was examined in the perfused liver. Apparent efflux clearance (PS_{net, eff}) of [³H]TC from hepatocytes to outflow across the sinusoidal membrane was defined as the amount of [³H]TC excreted into the outflow from the liver divided by hepatic AUC of [³H]TC. Additionally, influx clearance (PS_inf) was also determined by multiple indicator dilution method because PS_{net, eff} is also affected by PS_inf. Results. PS_{net, eff} was significantly higher in EHBR than that in Sprague-Dawley (SD) rats (16.6 ±1.7 vs. 6.1 ± 1.3 L/min/g liver, P <0.01). In contrast, PS_inf was comparable between SD rats and EHBR. Kinetic analysis suggested that the intrinsic clearance for the efflux of [³H]TC across the sinusoidal membrane in EHBR was higher than that in SD rats (10.4 ± 1.0 v.s. 23.3 ± 1.7 L/min/g liver, P <0.01). Conclusions. Enhanced sinusoidal efflux of TC in EHBR may be related to the altered disposition of bile acids in the mutant rats. Because Mrp3 transports TC and its expression is induced on the basolateral membrane of Mrp2-deficient rats, the enhanced sinusoidal efflux of TC in EHBR may be accounted for, at least partially, by the increased expression of Mrp3.     

Effects of lansoprazole on pharmacokinetics and metabolism of theophylline

The effect of the new substituted benzimidazole proton pump inhibitor, lansoprazole, on pharmacokinetics and metabolism of theophylline has been studied in healthy adults given oral lansoprazole 30 mg once daily for 11 days. On Days 4 and 11 of 300 mg aminophylline was simultaneously administered orally and blood samples for theophylline analysis were taken over 24 h. Urine samples were collected for up to 24 h and were assayed for theophylline and its major metabolites 1,3-dimethyluric acid (1,3-DMU), 1-methyluric acid (1-MU) and 3-methylxanthine (3-MX). The pharmacokinetic parameters of theophylline were determined, and the urinary recovery of unchanged theophylline and its major metabolites were calculated.After administration of lansoprazole for 4 days, no significant alteration in the terminal elimination half-life (t _1/2) or the mean residence time (MRT) was detected. However, there was a significant decrease of about 13% in the area under the plasma concentration-time curve (AUC) and a significant increase of about 19% in the apparent clearance (CL_app). Lansoprazole treatment for 11 days caused a significant decrease of approximately 12% in t _1/2 and about 10% in the MRT of theophylline, although neither AUC nor CL_app showed a significant alteration. The excretion of 3-MX in the urine was significantly increased by about 20% after lansoprazole treatment for 4 and 11 days, although there was no significant alteration in the excretion of unchanged theophylline, 1,3-DMU or 1-MU.The results indicate that repeated administration of lansoprazole to humans induces the hepatic microsomal P-450-dependent drug oxidation system that mediates N-1-demethylation of theophylline, consequently increasing its metabolism.     

Correlation Between In Vivo Antipyrine Metabolite Formation and Theophylline Metabolism in Rats

Two model substrates for oxidative hepatic enzyme activity, viz. antipyrine (A) and theophylline (T), were given simultaneously to rats by iv administration. Blood concentrations of A and T were measured by a high-performance liquid chromatographic (HPLC) method. Urinary excretions of A, T, and the major metabolites arising from A—4-hydroxyantipyrine (OHA), norantipyrine (NORA), 3-hydroxymethylantipyrine (HMA), and 4,4-dihydroxyantipyrine (DOHA)—and from T—1-methyluric acid (1-MU) and 1,3-dimethyluric acid (1,3-DMU)—were also determined by HPLC. It was found that the pharmacokinetic parameters obtained after the simultaneous administration of A and T at relatively low dose levels (A, 5.0 mg; and T, 1.3 mg) were not different from those obtained after the separate administration of A or T at the same dose level. In order to investigate whether the metabolic pathways of A and T are mediated by the same or closely related forms of the cytochrome P-450 system, metabolic clearances of A (CL_A,M) and T (CL_T,M) and the clearances for production of their various metabolites, obtained in untreated rats and in rats pretreated with 3-methylcholanthrene (MC) or with MC followed by 9-hydroxyellipticine (E), were correlated. These two compounds are a selective cytochrome P-448 inducer and inhibitor, respectively. Strong correlations were found between CL_T,M and the clearances for production of OHA, NORA, and DOHA but not HMA. The best correlation, however, was observed between CL_T,M and CL_OHA, not only when all data points were taken into account (r = 0.99), but also in separate pretreatment groups (r ranging from 0.87 to 0.92). Moreover, the slopes of these correlation lines varied only slightly among groups, while the intercepts were not significantly different from zero. In the separate pretreatment groups, the correlation coefficients for the correlations between CL_T,M and the clearance for production of the other metabolites of A were considerably lower, while the slopes of the correlation lines varied substantially. Clearances for production of the metabolites of T were strongly correlated with each other (r = 0.99) and with CL_OHA (r = 0.95). It can be concluded that theophylline metabolism and formation of OHA are mediated by the same or very similar forms of cytochrome P-450, whereas formation of the other major metabolites of A is not or only partly. The study of the various pathways of metabolism after simultaneous administration of drugs is a powerful tool in the study of correlations in drug metabolism in vivo.     

Relationships between the Hepatic Intrinsic Clearance or Blood Cell-Plasma Partition Coefficient in the Rabbit and the Lipophilicity of Basic Drugs

The relationships between drug lipophilicity and hepatic intrinsic clearance (CL_int,h) or red blood cell-plasma partition coefficients (D) have been elucidated for ten highly lipophilic basic drugs with apparent octanol-water partition coefficients at pH 7.4 (P_{app, oct}) of 150 or above. The true octanol-water partition coefficients of the non-ionized drugs (P_oct) were used to determine CL_int,h and D for the unbound drugs (CL_int,h,f and D_f, respectively), and CL_int,h,f and D_f for the non-ionized and unbound drugs (CL_int,h,fu and D_fu, respectively). The total clearance values were determined at steady state by infusion studies of individual drugs in rabbits. There was better correlation between log P_oct, and log CL_int,h,fu (r = 0.974) than between log P_oct, and log CL_int,h,f (r = 0.864). The D values were calculated from the blood-plasma concentration ratio. There was a better correlation between log P_oct and log D_fu (r = 0.944) than between log P_oct, and log D_f (r = 0.612). The regression equations obtained were CL_int,h,fu = 0.0875 × P_oct^1.338 and D_fu = 0.0108 × P_oct^0.970, respectively. These results show that the CL_int,h and D of highly lipophilic basic drugs can be predicted from P_oct by taking f_u into consideration. By applying these parameters to a physiologically based pharmacokinetic model it might be possible to predict the pharmacokinetics of unknown basic drugs.     

Functional Impairment of Sinusoidal Membrane Transport of Organic Cations in Rats with CCl4-Induced Hepatic Failure

Purpose. The effect of CCl₄-induced experimental hepatic failure(EHF) on the sequential hepatobiliary transport of model organiccations (OCs), triethylmethylammonium (TEMA), and tributylmethylammonium (TBuMA), was investigated in rats. Methods. EHF was induced by an i.p. injection of CCl₄ at a dose of1 ml/kg 24 hr prior to the transport study. The cumulative in vivobiliary excretion, in vitro hepatic uptake by isolated hepatocytes,in vitro efflux (i.e., release) from hepatocytes, andin vivo hepatobiliary excretion clearance were measured for normal and CCl₄-EHF rats. Results. The CCl₄-EHF decreased the apparentin vivo biliary clearance (CL_o) and the in vitromaximum uptake rate (V_{max, uptake}) of TBuMA by 66 and 48%,respectively. The CCl₄-EHF had no effect on the CL_oof TEMA, but decreased both the V_{max, uptake} (59%) and thein vitromaximum hepatic efflux rate (Vmax, efflux) of TEMA (80%). On thecontrary, the CCl₄-EHF had no influence on the in vivohepatobiliaryexcretion clearance (CL_exc) of both OCs. Conclusions. Transport systems for the OCs on the sinusoidal membrane(uptake and/or efflux), rather than those on the bile canalicularmembrane (excretion) appear to be prone to damage by the CCl₄-EHF.     

Effect of a protein binding change on unbound and total plasma concentrations for drugs of intermediate hepatic extraction

For substances eliminated from blood by the liver, the effect of a change in unbound fraction of drug (fu _b )on steady state total (C _b )and unbound (Cu _b )blood concentrations has hitherto only been considered for the two limiting cases, i.e., at the upper and lower extremes of hepatic intrinsic clearance (CL _int ).For a substance of very low CL _int ,if fu _b changes, C _t will change and Cu _b will remain constant, whereas if CL _int isvery high, Cu _b will change and C _b will remain constant.The present study defines the effects of a change in fu _b on C _b and Cu _b over the whole CL _int range. Computer simulations were undertaken which predicted that, for a given change in fu _b ,absolute and relative changes in C _b would decreasenonlinearly with increasing CL _{int, twhile the relative change in} Cu _{b would} increasewith CL _int .The absolute change in Cu_b would be independent of CL _int .Significant changes in C_b and Cu _{b would be observed at intermediate values of} CL _{int not just at the high and low extremes. These theoretical predictions were investigated experimentally in the isolated perfused rat liver by examining the effects of a change in} fu _{b of sodium taurocholate a substance with intermediate} CL _int (such that at fu _b =0.27,hepatic extraction ratio=0.71) induced by concurrent administration of sodium oleate. Sodium 24- ¹⁴ C-taurocholate (specific activity 52 Ci/mmol) was infused into the reservoir in a recycling system at 30 mol/hr for 105 min (n=6). At 45 min a bolus dose of sodium oleate (50 mmol) was administered to the reservoir, followed by a constant infusion of 143 mmol/hr for 1 hr. Following the administration of oleate, taurocholate fu _{b fell promptly by 55% (0.27–0.12). There was a relative increase of taurocholate} C _{b of 22.7% and a relative decrease in} Cu _{b of 45.4%, in accordance with the simulations (p<0.05). We conclude that important changes in unbound steady-state concentration, the pharmacologically active moiety, can occur upon changes in unbound fraction with compounds of intermediate hepatic intrinsic clearance}.This study was supported by the National Health and Medical Research Council of Australia.     

In vitro-in vivo correlation for intrinsic clearance for CP-409,092 and sumatriptan: a case study to predict the in vivo clearance for compounds metabolized by monoamine oxidase

1. Oxidative deamination of the GABA_A partial agonist CP-409,092 and sumatriptan represents a major metabolic pathway and seems to play an important role for the clearance of these two compounds.

2. Similar to sumatriptan, human mitochondrial incubations with deprenyl and clorgyline, probe inhibitors of monoamine oxidase B and monoamine oxidase A (MAO-B and MAO-A), respectively, showed that CP-409,092 was metabolized to a large extent by the enzyme MAO-A.

3. The metabolism of CP-409,092 and sumatriptan was therefore studied in human liver mitochondria and in vitro intrinsic clearance (CL_int) values were determined and compared to the corresponding in vivo oral clearance (CL_PO) values. The overall objective was to determine whether an in vitro-in vivo correlation (IVIVC) could be described for compounds cleared by MAO-A.

4. The intrinsic clearance, CL_int, of CP-409,092 was approximately 4-fold greater than that of sumatriptan (CL_int, values were calculated as 0.008 and 0.002?ml/mg/min for CP-409,092 and sumatriptan, respectively). A similar correlation was observed from the in vivo metabolic data where the unbound oral clearance, CL(u)_PO, values in humans were calculated as 724 and 178?ml/min/kg for CP-409,092 and sumatriptan, respectively.

5. The present work demonstrates that it is possible to predict in vivo metabolic clearance from in vitro metabolic data for drugs metabolized by the enzyme monoamine oxidase.

     

Effects of Escherichia coli lipopolysaccharide on the metformin pharmacokinetics in rats

1. The time-dependent (2-h, 24-h, and 96-h) effects of Escherichia coli lipopolysaccharide (ECLPS) on the intravenous (100?mg kg^?1) and oral (100?mg kg^?1) metformin pharmacokinetics were evaluated in rats.

2. After the intravenous administration of metformin to 24-h and 96-h ECLPS rats, the total area under the plasma concentration–time curve from time zero to time infinity (AUCs) and time-averaged non-renal clearances (CL_NRs) of metformin were significantly greater and slower, respectively, than the controls. However, after the oral administration of metformin, the AUCs of metformin were comparable among four groups of rats.

3. The greater (slower) intravenous AUCs (CL_NRs) of metformin in 24-h and 96-h ECLPS rats were due to the slower hepatic intrinsic clearance (CL_int) because of a decrease in the protein expression of hepatic cytochrome P450 (CYP) 2C11 and/or CYP3A subfamily than controls. The comparable oral AUCs among four groups of rats were mainly due to the comparable gastrointestinal metabolism (CL_int).

     

Interplay of transporters and enzymes in the Caco‐2 cell monolayer: I. effect of altered apical secretion

Interactions are expected between transporters and enzymes that compete for the substrate within the cell, and controversy exists for data interpretation on the interplay between transporters and enzymes. In the Caco‐2 cell monolayer, the increase in mean residence time (MRT) of drug accompanying increased secretion has been construed as the reason for increased metabolism, whereas others hold an opposite view that increased secretion would evoke decreased metabolism in this closed system. A catenary Caco‐2 cell model was used to simulate the effects of altered secretion on metabolism, estimated as the fraction of dose metabolized (f_met) or the extraction ratio (ER). The simulations showed that both f_met and ER varied inversely with the transporter‐mediated intrinsic clearance for apical secretion (CL_int,sec) under linear conditions. Under non‐linear conditions of saturable metabolism, apical absorption, basolateral influx or efflux, the simulated f_met consistently bore a reciprocal relationship with CL_int,sec. For saturable apical absorption or basolateral efflux, a reciprocal relationship between the ER and CL_int,sec was also found. However, under conditions of saturable metabolism or basolateral influx, the pattern of change in the ER became dependent on the administration sites, showing increasing patterns for apical dosing but decreasing patterns for basolateral dosing with increasing values of CL_int,sec. In general, f_met consistently demonstrated an inverse relationship with CL_int,sec, whereas ER, failing to include the drug in the donor side, would not show the same pattern of change in metabolism especially for apical dosing, since a substantial amount of drug was back‐secreted into the apical compartment. Copyright © 2010 John Wiley & Sons, Ltd.