Daniel Y. Hung

Physiologically based synthetic models of hepatic disposition.

Current physiologically based pharmacokinetic (PBPK) models are inductive. We present an additional, different approach that is based on the synthetic rather than the inductive approach to modeling and simulation. It relies on object-oriented programming. A model of the referent system in its experimental context is synthesized by assembling objects that represent components such as molecules, cells, aspects of tissue architecture, catheters, etc. The single pass perfused rat liver has been well described in evaluating hepatic drug pharmacokinetics (PK) and is the system on which we focus. In silico experiments begin with administration of objects representing actual compounds. Data are collected in a manner analogous to that in the referent PK experiments. The synthetic modeling method allows for recognition and representation of discrete event and discrete time processes, as well as heterogeneity in organization, function, and spatial effects. An application is developed for sucrose and antipyrine, administered separately and together. PBPK modeling has made extensive progress in characterizing abstracted PK properties but this has also been its limitation. Now, other important questions and possible extensions emerge. How are these PK properties and the observed behaviors generated? The inherent heuristic limitations of traditional models have hindered getting meaningful, detailed answers to such questions. Synthetic models of the type described here are specifically intended to help answer such questions. Analogous to wet-lab experimental models, they retain their applicability even when broken apart into sub-components. Having and applying this new class of models along with traditional PK modeling methods is expected to increase the productivity of pharmaceutical research at all levels that make use of modeling and simulation.

Cytoplasmic binding and disposition kinetics of diclofenac in the isolated perfused rat liver

The binding kinetics of diclofenac to hepatocellular structures were evaluated in the perfused rat liver using the multiple indicator dilution technique and a stochastic model of organ transit time density. The single‐pass, in situ rat liver preparation was perfused with buffer solution (containing 2% albumin) at 30 ml min−1. Diclofenac and [14C]‐sucrose (extracellular reference) were injected simultaneously as a bolus dose into the portal vein (six experiments in three rats). An analogous series of experiments was performed with [14C]‐diclofenac and [3H]‐sucrose. The diclofenac outflow data were analysed using three models of intracellular distribution kinetics, assuming (1) instantaneous distribution and binding (well‐mixed model), (2) ‘slow’ binding at specific intracellular sites after instantaneous distribution throughout the cytosol (slow binding model), and (3) ‘slowing’ of cytoplasmic diffusion due to instantaneous binding (slow diffusion model). The slow binding model provided the best description of the data. The rate constants for cellular influx and sequestration were 0.126±0.026 and 0.013±0.009 s−1, respectively. The estimated ratio of cellular initial distribution volume to extracellular volume of 2.82 indicates an almost instantaneous distribution in the cellular water space, while the corresponding ratio of 5.54 estimated for the apparent tissue distribution volume suggests a relatively high hepatocellular binding. The non‐instantaneous intracellular equilibration process was characterized by time constants of the binding and unbinding process of 53.8 and 49.5 s, respectively. The single‐pass availability of diclofenac was 86%. The results obtained with [14C]‐diclofenac and [3H]‐sucrose were not statistically different.

Hepatic pharmacokinetics of taurocholate in the normal and cholestatic rat liver

1 The disposition kinetics of [3H]taurocholate ([3H]TC) in perfused normal and cholestatic rat livers were studied using the multiple indicator dilution technique and several physiologically based pharmacokinetic models. 2 The serum biochemistry levels, the outflow profiles and biliary recovery of [3H]TC were measured in three experimental groups: (i) control; (ii) 17α‐ethynylestradiol (EE)‐treated (low dose); and (iii) EE‐treated (high dose) rats. EE treatment caused cholestasis in a dose‐dependent manner. 3 A hepatobiliary TC transport model, which recognizes capillary mixing, active cellular uptake, and active efflux into bile and plasma described the disposition of [3H]TC in the normal and cholestatic livers better than the other pharmacokinetic models. 4 An estimated five‐ and 18‐fold decrease in biliary elimination rate constant, 1.7‐ and 2.7‐fold increase in hepatocyte to plasma efflux rate constant, and 1.8‐ and 2.8‐fold decrease in [3H]TC biliary recovery ratio was found in moderate and severe cholestasis, respectively, relative to normal. 5 There were good correlations between the predicted and observed pharmacokinetic parameters of [3H]TC based on liver pathophysiology (e.g. serum bilirubin level and biliary excretion of [3H]TC). In conclusion, these results show that altered hepatic TC pharmacokinetics in cholestatic rat livers can be correlated with the relevant changes in liver pathophysiology in cholestasis.

Hepatic structure‐pharmacokinetic relationships: The hepatic disposition and metabolite kinetics of a homologous series of O‐acyl derivatives of salicylic acid

1 The hepatic disposition and metabolite kinetics of a homologous series of O‐acyl (acetyl, propionyl, butanoyl, pentanoyl, hexanoyl and octanoyl) esters of salicylic acid (C2SA, C3SA, C4SA, C5SA, C6SA and C8SA, respectively) was determined using a single‐pass, in‐situ rat liver preparation. 2 The hepatic venous outflow profiles for the parent esters and the generated metabolite, salicylic acid (SA) were analysed by HPLC. Non‐parametric moments analysis was used to determine the area under the curve (AUC′), mean transit time (MTT) and normalized variance (CV2) for the parent esters and generated SA. 3 Pregenerated SA ([14C]‐salicylic acid) was injected into each liver with the parent ester to determine its distribution characteristics. 4 The overall recovery of ester plus metabolite was 89% of the ester dose injected and independent of the ester carbon number, suggesting that ester extraction was due to hepatic metabolism to salicylic acid. 5 The metabolite AUC′ value increased directly with the lipophilicity of the parent ester (from 0.12 for C2SA to 0.95 for C8SA). By contrast, the parent AUC′ decreased with the lipophilicity (from 0.85 for C2SA to zero for C8SA). The metabolite MTT value also showed a trend to increase with the lipophilicity of the parent ester (from 15.72 s for C3SA to 61.97 s for C8SA). However, the parent MTT value shows no significant change across the series. 6 The two‐compartment dispersion model was used to derive the kinetic parameters for parent ester, pregenerated SA and generated SA. Consequently, these parameters were used to estimate the values of AUC′, MTT and CV2 for the parent ester and metabolite. The moments values obtained using the two‐compartment dispersion model show similar trends to the corresponding moments values obtained from the outflow profiles using a non‐parametric approach. 7 The more lipophilic aspirin analogues are more confined to the portal circulation after oral administration than aspirin due to their more extensive hepatic elimination avoiding systemic prostacyclin inhibition. Given that aspirins selectivity as an anti‐thrombotic agent has been postulated to be due to selective anti‐platelet effects in the portal circulation, the more lipophilic and highly extracted analogues are potentially more selective anti‐thrombotic agents than aspirin.

Kinetic analysis of saturable hepatic uptake of digoxin and its inhibition by rifampicin

Although the organic anion transporter Oatp2 plays a critical role in determining the hepatic clearance of some drugs, little quantitative information exists about its functional characteristics in relation to inhibition of sinusoidal drug uptake. We investigated the uptake kinetics of the Oatp2 substrate digoxin in the isolated perfused rat liver. In the single-pass perfused liver three consecutive digoxin doses of 15, 30 and 45 micorg were administered in the presence or absence of rifampicin (100 micorM), an inhibitor of Oatp2. Digoxin was determined in the outflow samples by HPLC and all data were analyzed by simultaneous nonlinear regression assuming a Michaelis-Menten uptake mechanism. Hepatocellular uptake of digoxin was concentration-dependent with a Michaelis constant (K(M)) of 577.8 ng/ml. Rifampicin significantly reduced uptake (K(M) increased 2.5-fold) without affecting other parameters.

Synthesis, identification, characterization, stability, solubility, and protein binding of ester derivatives of salicylic acid and diflunisal

O-Acyl esters were prepared from salicylic acid and diflunisal by esterification with the appropriate acyl anhydride (in the presence of sulfuric acid at 80°C) or acyl chloride (in the presence of pyridine at 0°C). Synthesis, identification and characterization of these compounds is described. In vitro hydrolysis, solubility and protein binding studies of these O-acyl esters were performed. For the diflunisal esters, the melting points fell as the side chain was increased from ethyl to pentyl. The melting points showed no significant difference as the length of the side chain was increased from pentyl to heptyl. The aspirin analogues showed a similar trend. The relationship between solubility and carbon chain length agreed closely with that for the melting points with carbon chain length. In vitro non-enzymatic hydrolysis studies concluded that: (1) hydrolysis rate constants generally decreased with carbon chain length; (2) the diflunisal esters have shorter half lives compared with their salicylate counterparts; and (3) the in vitro hydrolysis of these compounds was retarded by the presence of bovine serum albumin. Protein binding experiments showed that the strength of binding of the aspirin and diflunisal analogues to bovine serum albumin increased with carbon chain length.

Focused antithrombotic therapy: Novel anti-platelet salicylates with reduced ulcerogenic potential and higher first-pass detoxification than aspirin in rats☆

The use of aspirin as an anti-platelet drug is limited by its propensity to induce gastric injury and by its adverse effect on vascular prostacyclin formation. Two phenolic non-steroidal anti-inflammatory drugs (salicylic acid and diflunisal) were modified by esterification with a series of O-acyl moieties. The short-term ulcerogenic in vitro and in vivo anti-platelet properties, pharmacodynamic profiles, and extent of hepatic extraction of these phenolic esters were compared with aspirin (acetylsalicylic acid). The more lipophilic esters (longer carbon chain length in O-acyl group) show significantly less gastrotoxicity in stressed rats than does aspirin after a single oral dose. The in vitro and in vivo anti-platelet studies show that these phenolic esters inhibited (1) arachidonate-triggered human platelet aggregation and (2) thrombin-stimulated rat serum thromboxane A2 production by platelets in the clotting process almost as effectively as aspirin. The hepatic extractions of these O-acyl derivatives are significantly higher than those of aspirin. The pharmacodynamic studies show that these O-acyl derivatives of salicylic acid and diflunisal probably bind to, or combine with, the same site on the platelet cyclooxygenase as aspirin. Replacing the O-acetyl group with longer chain O-acyl moiety in this series of phenolic esters markedly reduced the potential of these agents to induce short-term gastric injury but did not lessen their activity as inhibitors of platelet aggregation. These non-acetyl salicylates may therefore represent a novel class of anti-platelet drugs with less ulcerogenic potential.

The effect of protein binding on the hepatic first pass of O-acyl salicylate derivatives in the rat

In this work the in‐situ perfused rat liver has been used to examine the effect of changing the protein content of the perfusate on the hepatic extraction of O‐acyl esters of salicylic acid. The hepatic availability (F) of these solutes was studied at a flow‐rate of 30 mL min−1 with perfusate albumin concentrations of 0, 2, and 4% w/v.

Therapeutic effects and possible mechanisms of a snake venom preparation in the fibrotic rat liver

The effects of a Chinese snake venom preparation from Agkistrodon halys pallas, used for treatment of hepatic fibrosis/cirrhosis in China, was investigated in an {in vivo} rat model and using in situ hepatic perfusion. Four groups were used in the experiments: (i) healthy, (ii) healthy/venom-treated, (iii) carbon tetrachloride (CCl4)-treated, and (iv) CCl4/venom-treated. Treatment effects were assessed by determining hepatic histopathology, biochemistry and fibrosis index parameters, bile production, biliary taurocholate recovery, hepatic mRNA expression of four bile salt transporters (Ntcp, Bsep, Oatp-1, and Oatp-3), comparison of hepatic microcirculation, fibrinolytic activity, and antithrombotic effects. Liver histopathology, biochemistry, and fibrosis index showed a dramatic improvement in venom-treated animals. There were significant differences in bile production between healthy/venom-treated and all other experimental groups and between CCl4/venom-treated and CCl4-treated animals, but no significant differences were found between CCl4/venom-treated and healthy animals. Biliary taurocholate recovery was significantly increased in healthy/venom-treated and CCl4/venom-treated animals. The expression of mRNA levels of the four bile salt transporters showed an increase after venom treatment. The hepatic microcirculation studies showed normalized sinusoidal beds in CCl4/venom-treated animals compared to healthy animals, whereas CCl4-treated animals showed abnormal profiles to the healthy and the CCl4/AHPV-treated animals. The fibrinogen and plasma thromboxane B2 levels of healthy rats decreased with increasing dose after venom treatment. It was concluded that snake venom treatment may be therapeutic in treatment of hepatic fibrosis/cirrhosis by possibly a combination of increased bile flow and improved hepatic microcirculation, changes in bile salt transporter expression, and fibrinolytic and antithrombotic effects of the snake venom preparation.

Assessing the cellular transmembrane electrical potential difference on the hepatic uptake of palmitate

Understanding the driving forces for the hepatic uptake of endogenous and exogenous substrates in isolated cells and organs is fundamental to describing the underlying hepatic physiology/pharmacology. In this study we investigated whether uptake of plasma protein-bound [3H]-palmitate across the hepatocyte wall is governed by the transmembrane electrical potential difference (PD). Uptake was studied in isolated hepatocytes and isolated perfused rat livers (IPL). Protein-binding and vasoactive properties of the different perfusates were determined using in vitro heptane/buffer partitioning studies and the multiple indicator dilution (MID) technique in the IPL, respectively. Altering hepatocyte PD by perfusate ion substitution resulted in either a substantial depolarization (−14 ± 1 mV, n = 12, mean ± S.E., substituting choline for Na+) or hyperpolarization (−46 ± 3 mV, n = 12, mean ± S.E., substituting nitrate for Cl−). Perfusate ion substitution also affected the equilibrium binding constant for the palmitate–albumin complex. IPL studies suggested that, other than with gluconate buffer, hepatic [3H]-palmitate extraction was not affected by the buffer used, implying PD was not a determinant of extraction. [3H]-Palmitate extraction was much lower (p < 0.05) when gluconate was substituted for Cl− ion. This work contrasts with that for the extraction of [3H]-alanine where hepatic extraction fraction was significantly reduced during depolarization. Changing the albumin concentration did not affect hepatocyte PD, and [3H]-palmitate clearance into isolated hepatocytes was not affected by the buffers used. MID studies with vascular and extravascular references revealed that, with the gluconate substituted buffer, the extravascular volume possibly increased the diffusional path length thus explaining reduced [3H]-palmitate extraction fraction in the IPL. (Mol Cell Biochem 270: 115–124, 2005)