The thesis is concerned with differences in drug and solute pharmacokinetics and distribution in perfused organs under varying pathological conditions. It falls in two parts, and two organ systems are considered: The perfused (rat) liver and the perfused (rat and human) limb. Two important different aspects of drug and solute disposition have been determined: basic drug/solute binding, metabolism and clearance (in the healthy and the diseased liver) and drug effects and distribution in healthy and diseased tissue (oncological treatment of tumours of the limb).
The first part of the thesis (Chapters 1-6) is concerned with the disposition (and effects) of solutes and drugs in a variety of different perfused rat liver models. The solutes and drugs investigated are palmitate, taurocholate, propranolol (and its optical isomers), atenolol, antipyrine and a snake venom preparation.
In Chapter 1, the hepatic palmitate disposition was analyzed using models such as clofibrate treatment, pregnancy, and different genders. In the study the level of liver fatty acid binding protein, microsomal protein, albumin, and glutathione S-transferase and the outflow profiles of [3H]palmitate and metabolites were measured in four experimental groups: (i) male; (ii) clofibrate-treated male; (iii) female; and (iv) pregnant female rats. A slow diffusion/bound model was found to best describe the hepatic disposition of unchanged [3H]palmitate. Order of L-FABP levels was: pregnant female > clofibrate-treated male > female > male. The levels of other intra-hepatic proteins did not differ significantly. The hepatic extraction ratio and mean transit time for unchanged palmitate, as well as the production of metabolites of palmitate and their retention in the liver, increased with increasing L-FABP levels. Palmitate metabolic clearance, permeability-surface area product, retention of palmitate by the liver, and cytoplasmic diffusion constant for unchanged [3H]palmitate also increased with increasing L-FABP levels. It was concluded that the variability in hepatic pharmacokinetics of unchanged ['H]palmitate and its metabolites in perfused rat livers is related rather to levels of L-FABP and not to other intra-hepatic proteins.
In Chapter 2, the hepatic disposition of palmitate and its metabolites was analyzed using a rat model of estradiol-induced steatosis and studied using the multiple indicator dilution technique and a physiologically based slow diffusion/bound pharmacokinetic model. Steatosis was established by administration of 17α-ethynylestradiol to female Wistar rats. Presence of steatosis was assessed using biochemistry markers and histology. The steatotic group showed a significantly higher ALT/AST ratio, lower levels of liver fatty acid binding protein and cytochrome P450 as well as microvesicular steatosis with an enlargement of sinusoidal space. Hepatic extraction for unchanged ['H]palmitate and production of metabolites were significantly decreased in the steatotic group. Pharmacokinetic analysis suggested that the reduced extraction and sequestration for palmitate and its metabolites was mainly attributed to a reduction in liver fatty acid binding protein in steatosis.
In Chapter 3, the disposition of taurocholate was determined in healthy and cholestatic (estradiol-treated) isolated perfused rat livers using the multiple indicator dilution technique and several physiologically based pharmacokinetic models. The bilirubin serum level, the outflow profiles and biliary recovery of taurocholate were measured in three experimental groups: (i) control (untreated); (ii) 17α-ethynylestradiol (EE)-treated (low dose); and (iii) EE-treated (high dose) rats. EE treatment caused cholestasis in a dose-dependent manner. A hepatobiliary taurocholate transport model, which recognizes capillary mixing, active cellular uptake and active efflux into bile and plasma best described the disposition of taurocholate in the normal and cholestatic livers. Severe cholestasis reduced taurocholate extraction through biliary clearance being reduced to 1/18 of normal and a 3 fold increase in hepatocyte to plasma efflux clearance. This in turn led to an estimated 11-fold increase in hepatocellular taurocholate concentrations. There were good correlations between the predicted and observed pharrnacokinetic parameters of taurocholate based on liver pathophysiology (e.g. serum bilirubin level and biliary excretion of taurocholate. The results showed a correlation between altered hepatic taurocholate pharmacokinetics in cholestatic rat livers cholestasisinduced pathophysiological changes.
In Chapter 4, the disposition and respective contribution of ion-trapping in acidic compartments and microsomal binding to hepatic basic drug retention, and the relationship to the respective physico-chemical properties was studied using model cationic drugs (propranolol, its optical isomers, atenolol and antipyrine) in the isolated perfused rat liver. The ionophore monensin was used to abolish the vesicular proton gradient and thus allow an estimation of ion-trapping by acidic hepatic vesicles of cationic drugs. In vitro microsomal studies were used to independently estimate microsomal binding and metabolism. Hepatic vesicular ion-trapping, intrinsic elimination clearance, permeability-surface area product and intracellular binding were derived using a physiologically based pharrnacokinetic model. Modeling showed that the ion-trapping was significantly lower after monensin treatment for atenolol and propranolol, but not for antipyrine. However, no changes induced by monensin treatment were observed in intrinsic clearance, permeability or binding for the three model drugs. Monensin did not affect binding or metabolic activity in vitro for the drugs. The observed ion-trapping was similar to theoretical values estimated using the pHs and fractional volumes of the acidic vesicles and the pKas of drugs. Lipophilicity and pKa determined hepatic drug retention: a drug with low pKa and low lipophilicity (e.g. antipyrine) distributes as unbound drug, a drug with high pKa and low lipophilicity (e.g. atenolol) by ion-trapping and a drug with a high pKa and high lipophilicity (e.g. propranolol) is retained by ion-trapping and intracellular binding. In conclusion, monensin inhibits the ion-trapping of high pKa basic drugs leading to a reduction in hepatic retention but with no effect on hepatic drug extraction.
In Chapter 5, the determinants of the linear hepatic disposition kinetics of propranolol optical isomers were defined in the isolated perfused rat liver using the ionophore monensin to abolish the lysosomal proton gradient and thus to allow an estimation of propranolol ion-trapping by hepatic acidic vesicles. Independent estimates for microsomal binding and intrinsic clearance were obtained from in vitro studies. Hepatic extraction and mean transit time were determined from outflow-concentration profiles using a non-parametric method. Kinetic parameters were derived from a physiologically based pharmacokinetic model. Modeling showed an approximate 34-fold decrease in ion-trapping following monensin treatment. The observed model-derived ion-trapping was similar to estimated theoretical values. No differences in ion-trapping values was found between the two optical isomers. Hepatic propranolol extraction was sensitive to changes in liver perfusate flow, permeabilitysurface area product and intrinsic clearance. Jon-trapping, microsomal and non-specific binding and distribution of unbound propranolol accounted for 47.4%, 47.1% and 5.5% of the sequestration of propranolol in the liver, respectively. It was concluded that the physiologically more active S(-)-propranolol differs from the R(+) isomer in higher permeability-surface area product, intrinsic clearance, and intracellular binding site values. In Chapter 6, the therapeutic effects of a traditional Chinese snake venom preparation preparation from Agkistrodon halys pallas used for treatment of hepatic fibrosis/cirrhosis in China on the steatotic/fibrotic/cirrhotic rat liver were experimentally determined in a CCl4- induced diseased rat liver model. The four experimental groups used in the experiments were: (i) healthy; (ii) healthy/venom-treated; (iii) carbon tetrachloride (CCl4)-treated; (iv) CCl4/venom-treated. Treatment effects were assessed via hepatic histopathology, biochemistry and fibrosis index, 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 anti-thrombotic effects. Liver histopathology, biochemistry and fibrosis index showed dramatic improvement of venomtreated animals. There were significant differences in bile production between healthy/venomtreated versus different animals and CCl4/venom-treated versus 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 rnRNA levels of the four bile salt transporters showed an increase that was significant for Oatp-1 and Oatp-3 comparing healthy untreated and healthy/venom-treated animals. The hepatic microcirculation studies showed normalized sinusoidal beds in CCl4/venom-treated animals as compared to healthy animals, whereas CC14-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 snake venom treatment is likely to 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 anti-thrombotic effects of the snake venom preparation.
In the second part of this work the pharmacokinetics and effects of one specific cytotoxic drug (melphalan) were determined in the perfused limb (in both a rat model and human patients). In Chapter 7 dose-response relationships for the drug were analyzed in the perfused hind limb of the nude melanoma-bearing rat. Rats were treated by Isolated Limb Perfusion with increasing doses (7.5 to 400 ɥg/ml) of melphalan. Tumour response to treatment at the end of the observation period was graded as complete response (CR), partial response (PR), no change (NC) and progressive disease (PD). No linear relationship between dose of melphalan and tumour response was detected. All doses above a threshold (15 ɥg/ml) achieved a PR or CR. The achievement of CR was not related to increased dose.
Two major implications arose from this work. Firstly, the typically 2-3-fold increase of cytotoxic drug concentration given in high dose chemotherapy compared with standard drug concentration may be considered insufficient to produce the expected increase in tumour response to treatment, and secondly, an increase of melphalan dose above a certain threshold does not greatly increase tumour response combination therapies could be more promising and beneficial for patients.
In Chapter 8 the pharmacokinetics of melphalan in the perfused limb were determined in a cohort of human patients undergoing isolated limb perfusion for various malignancies. Isolated limb infusion has a lower morbidity in treating localized recurrences and in transit metastases of the limb for tumours and allows administration of higher concentrations of cytotoxic agent preferred cytotoxic agent for the treatment of melanoma (melphalan) to the affected limb. Pharmacokinetic data from 12 patients treated by Isolated Limb Infusion (ILI) for tumours of the limb were studied. The kinetics of drug distribution in the limb were calculated using a two-compartment vascular model (well-stirred). Analysis of melphalan concentrations in the perfusate during ILI showed good agreement between the values measured and concentrations predicted by the model. Recirculation and wash-out flow rates, tissue concentrations and the permeability surface area product (PS) were calculated. Correlations between the PS value and the drug concentrations in the perfusate and tissue were supported by the results. The data contribute to a better understanding of the distribution of melphalan during ILI in the limb.
In Chapter 9 the distribution of melphalan in different tissues of the isolated perfused limb was analyzed using microdialysis to relate clinical and biochemical responses to the time course of melphalan concentrations in the subcutaneous interstitial space and in tumour tissue in patients undergoing regional chemotherapy by ILI Nineteen patients undergoing ILI for treatment of various limb malignancies were monitored for intra-operative melphalan concentrations in plasma and, using microdialysis, in subcutaneous and tumour tissues. Peak and mean concentrations of melphalan were significantly higher in plasma than in subcutaneous or tumour microdialysate. No significant difference between drug peak and mean concentrations in interstitial and tumour tissue was detected, indicating that there was no preferential uptake of melphalan into the tumours.
The time course of melphalan in the microdialysate could best be described by a pharmacokinetic model which assumed melphalan distribution from the plasma into the interstitial space. The model also accounted for the vascular dispersion of melphalan in the limb. Tumour response in the whole group to treatment was partial response: 53.8% (n=7); complete response: 33.3% (n=5); no response: 6.7% (n=1). There was a significant association between tumour response and melphalan concentrations measured over time in subcutaneous microdialysate (P<0.01).
No significant relationship existed between the severity of toxic reactions in the limb or peak plasma creatine phosphokinase levels and peak melphalan microdialysate or plasma concentrations.
Microdialysis as a technique is apparently well suited for measuring concentrations of cytotoxic drug during ILI. The combination of predicting tissue concentrations and monitoring in microdialysate of subcutaneous tissue could potentially help optimize ILI with regard to post operative limb morbidity and tumour response.