Ruthenium(II) Complex Containing a Redox-Active Semiquinonate Ligand as a Potential Chemotherapeutic Agent: From Synthesis to In Vivo Studies.

Chemotherapy remains one of the dominant treatments to cure cancer. However, due to the many inherent drawbacks, there is a search for new chemotherapeutic drugs. Many classes of compounds have been investigated over the years to discover new targets and synergistic mechanisms of action including multicellular targets. In this work, we designed a new chemotherapeutic drug candidate against cancer, namely, [Ru(DIP)2(sq)](PF6) (Ru-sq) (DIP = 4,7-diphenyl-1,10-phenanthroline; sq = semiquinonate ligand). The aim was to combine the great potential expressed by Ru(II) polypyridyl complexes and the singular redox and biological properties associated with the catecholate moiety. Experimental evidence (e.g., X-ray crystallography, electron paramagnetic resonance, electrochemistry) demonstrates that the semiquinonate is the preferred oxidation state of the dioxo ligand in this complex. The biological activity of Ru-sq was then scrutinized in vitro and in vivo, and the results highlight the promising potential of this complex as a chemotherapeutic agent against cancer.

Novel quinazolin-4-one derivatives as potentiating agents of doxorubicin cytotoxicity.

We report the design, synthesis and biological evaluation of 17 novel 8-aryl-2-morpholino-3,4-dihydroquinazoline derivatives based on the standard model of DNA-PK and PI3K inhibitors. Novel compounds are sub-divided into two series where the second series of five derivatives was designed to have a better solubility profile over the first one. A combination of in vitro and in silico techniques suggested a plausible synergistic effect with doxorubicin of the most potent compound 14d on cell proliferation via DNA-PK and poly(ADP-ribose) polymerase-1 (PARP-1) inhibition, while alone having a negligible effect on cell proliferation.

The effects of ß-caryophyllene oxide and trans-nerolidol on the efficacy of doxorubicin in breast cancer cells and breast tumor-bearing mice.

BACKGROUND: One approach to improve effect of chemotherapy is combination of classical cytostatic drugs with natural compounds, e. g. sesquiterpenes. In our previous study, sesquiterpenes ß-caryophyllene oxide (CAO) and trans-nerolidol (NER) improved the anti-proliferative effect of doxorubicin (DOX) in intestinal cancer cell lines. PURPOSE: The present study was designed to evaluate the effect of CAO and NER on DOX efficacy, focusing on cell proliferation, migration, apoptosis and DOX accumulation in breast cancer cells MDA-MB-231 and MCF7 in vitro and in mice bearing solid Ehrlich tumors (EST) in vivo. METHODS: The impact of cytotoxic effect was assessed by the neutral red uptake test. The ability to migrate was tested using real-time measurement in x-CELLigence system. Expressions of molecules were examined using western blot analysis. The accumulation of DOX inside the cells using time lapse microscopy was observed. The mice with inoculated EST cells were treated repeatedly with DOX and DOX+CAO or DOX+NER and the growth of tumors were monitored. DOX concentrations in plasma and tumor were assayed using HPLC. RESULTS: In MDA-MB-231, combination of DOX with CAO enhanced anti-proliferative effect and acted strongly synergistic. NER increased accumulation of DOX inside the cells; moreover combination DOX with NER suppressed migration ability in vitro. In vivo, apoptosis was activated especially in group treated with DOX and CAO. However, none of tested sesquiterpenes was able to improve DOX accumulation in tumors and DOX-mediated inhibition of tumor growth. CONCLUSION: In conclusion, sesquiterpenes CAO and NER increased the efficacy of DOX in breast cancer cells in vitro, but did not improve its effect in vivo, in Ehrlich solid tumor bearing mice.

Anticancer potential of Amaryllidaceae alkaloids evaluated by screening with a panel of human cells, real-time cellular analysis and Ehrlich tumor-bearing mice.

In this study, twenty-two Amaryllidaceae alkaloids were screened for their anticancer potential. All isolates were evaluated for antiproliferative activities on a panel of 17 human cell types of different tissue origin using WST-1 assay. In addition, we determined the antiproliferative effect with a real-time cell analysis xCELLigence system. Thereafter, to evaluate the barely known in vivo anticancer potential of the most potent molecule haemanthamine, a preliminary study was performed using an Ehrlich tumor-bearing mice model. The results showed that haemanthamine, lycorine and haemanthidine exerted the highest antiproliferative activity. The mean growth percent (GP) value after a single-dose 10 µM treatment was for haemanthamine 21%, for lycorine 21% and for haemanthidine 27% that of untreated control cells (100%). Furthermore, haemanthamine, lycorine and haemanthidine exhibited significant cytotoxicities against all the tested cell lines with individual IC50 values in the micromolar range. Dynamic real-time measures of impedance by xCELLigence indicated that these three compounds suppress cell proliferation after 10 h of treatment at a concentration of 10 µM or higher. Regrettably, in a follow-up in vivo antitumor activity study, haemanthamine showed no statistically significant reduction in the tumor size with no prolongation of survival time of Ehrlich tumor-bearing mice. Taken together, these results provide a new clue and guidance for exploiting Amaryllidaceae alkaloids as anticancer agents.

Boldine Inhibits Mouse Mammary Carcinoma In Vivo and Human MCF-7 Breast Cancer Cells In Vitro.

Boldine is an aporphine alkaloid widely consumed in the folk medicine of some regions. Its anticancer potential has been shown but not yet elucidated. We compared the antitumor effect of orally and parenterally applied boldine in mice bearing solid Ehrlich tumor. We also explored the effects of boldine on breast adenocarcinoma MCF-7 cells in vitro. Repeated i. p. injections of 30, 60, or 90 mg boldine/kg, either alone or combined with doxorubicin, slowed tumor growth in vivo. The latter two doses also prolonged the post-therapeutic survival of the mice. When fed food supplemented with boldine at a dose of 90 mg/kg, the tumor-bearing mice survived significantly longer, but there was no effect on tumor size. Interestingly, continuous p. o. administration did not produce detectable levels of boldine in plasma or tissue samples, in contrast to high but short-lived concentrations after i. p. injections. There was neither antagonism nor synergism between boldine and doxorubicin, except a possible synergism of i. p. boldine 90 mg/kg combined with doxorubicin when compared with doxorubicin alone.Boldine was cytotoxic to MCF-7 cells and reduced their viability and proliferation in vitro. Exposure to boldine decreased bromodeoxyuridine incorporation and histone H3 phosphorylation but did not induce apoptosis. Boldine treatment resulted in p38, ERK, and JNK activation in the mitogen-activated protein kinase pathway in a dose-dependent manner. Since bioavailability in mice seems to be different from that reported in rats, pharmacokinetic studies in humans are needed to evaluate the role of boldine in the beneficial effects of Boldo infusions.

In-vitro and in-vivo evaluation of the anticancer activity of diruthenium-2, a new trithiolato arene ruthenium complex [(η6-p-MeC6H4Pri)2Ru2(µ-S-p-C6H4OH)3]Cl.

In the present study, we investigated the anticancer action of the trithiolato arene ruthenium complex, [(η-p-MeC6H4Pr)2Ru2(µ-S-p-C6H4OH)3]Cl, named diruthenium-2, both in vitro and in vivo. The mechanism of antiproliferative, cytotoxic, and DNA-damaging activity, and the effect on expressions of cell cycle regulatory proteins were investigated using a WST-1-based proliferation assay, lactate dehydrogenase leakage assay, comet assay, flow cytometry, and western blot analysis. In-vivo anticancer activity was evaluated using Ehrlich tumor-bearing NMRI mice. Diruthenium-2 inhibited the growth of all cancer cell lines used, the most sensitive being gastric (AGS), breast cancer (BT-549, MCF-7, MDA-MB-231), and leukemic (HL-60, MOLT-4) cells. In MCF-7 cells, it caused a G1/S cell cycle arrest, along with an increase in the expression of protein p21 and cyclin B1. We also observed increased levels of MRN complex proteins, which, together with the results from the comet assay, indicate the formation of DNA double-strand breaks. In tumor-bearing mice, diruthenium-2 at doses of 3 and 5 mg/kg inhibits the growth of solid Ehrlich tumor, although weaker than cisplatin. However, it did not prolong the post-therapeutic survival. Our results suggest the in-vitro potential of diruthenium-2 should be further evaluated in studies using other in-vivo models.

Chlorambucil conjugates of dinuclear p-cymene ruthenium trithiolato complexes: synthesis, characterization and cytotoxicity study in vitro and in vivo.

Four diruthenium trithiolato chlorambucil conjugates have been prepared via Steglich esterification from chlorambucil and the corresponding trithiolato precursors. All conjugates are highly cytotoxic towards human ovarian A2780 and A2780cisR cancer cell lines with IC50 values in the nanomolar range. The conjugates exhibit selectivity towards A2780 cells as compared to non-cancerous HEK293 cells, while being only slightly selective for RF24 and A2780cisR cells. In vivo, the conjugate [10]BF4 suppressed the growth of a solid Ehrlich tumor in immunocompetent NMRI mice but did not prolong their overall survival. The reactivity of the chlorambucil conjugates with glutathione, a potential target of the dinuclear ruthenium motive, and with the 2-deoxyguanosine 5'-monophosphate (dGMP-a model target of chlorambucil) was studied by mass spectrometry and NMR spectroscopy. The conjugates did not show catalytic activity for the oxidation of glutathione nor binding to nucleotides, indicating that glutathione oxidation and DNA alkylation are not key mechanisms of action. Four highly cytotoxic diruthenium trithiolato chlorambucil conjugates have been prepared. All conjugates exhibit selectivity towards A2780 cells as compared to HEK293 cells, while being only slightly active in RF24 and A2780cisR cells. In vivo, the best candidate suppressed the growth of a solid Ehrlich tumor in immunocompetent NMRI mice but did not prolong their overall survival.

The Steroidal Glycoalkaloids from Solanaceae: Toxic Effect, Antitumour Activity and Mechanism of Action.

Steroidal glycoalkaloids present in Solanaceae are toxic compounds biosynthesised for the protection of the plants. However, many health benefits of these compounds have been reported so far. One of their promising targets might be cancer, as demonstrated in a large number of studies. However, the main mechanism of action seems to be unclear. It could include the induction of apoptosis or trigger a necrosis with a subsequent inflammatory response. The relatively high systemic toxicity of steroidal compounds is another effect that must be taken into account in anticancer research. The main aim of this work was to summarise the recent progress in the investigation of the mechanisms of their antitumour action and to discuss their potential.

Boldine enhances bile production in rats via osmotic and farnesoid X receptor dependent mechanisms.

Boldine, the major alkaloid from the Chilean Boldo tree, is used in traditional medicine to support bile production, but evidence to support this function is controversial. We analyzed the choleretic potential of boldine, including its molecular background. The acute- and long-term effects of boldine were evaluated in rats either during intravenous infusion or after 28-day oral treatment. Infusion of boldine instantly increased the bile flow 1.4-fold in healthy rats as well as in animals with Mrp2 deficiency or ethinylestradiol induced cholestasis. This effect was not associated with a corresponding increase in bile acid or glutathione biliary excretion, indicating that the effect is not related to stimulation of either bile acid dependent or independent mechanisms of bile formation and points to the osmotic activity of boldine itself. We subsequently analyzed bile production under conditions of changing biliary excretion of boldine after bolus intravenous administration and found strong correlations between both parameters. HPLC analysis showed that bile concentrations of boldine above 10 µM were required for induction of choleresis. Importantly, long-term pretreatment, when the bile collection study was performed 24-h after the last administration of boldine, also accelerated bile formation despite undetectable levels of the compound in bile. The effect paralleled upregulation of the Bsep transporter and increased biliary clearance of its substrates, bile acids. We consequently confirmed the ability of boldine to stimulate the Bsep transcriptional regulator, FXR receptor. In conclusion, our study clarified the mechanisms and circumstances surrounding the choleretic activity of boldine.

Ferns and lycopods--a potential treasury of anticancer agents but also a carcinogenic hazard.

Many species of seedless vascular plants-ferns and lycopods-have been used as food and folk medicine since ancient times. Some of them have become the focus of intensive research concerning their anticancer properties. Studies on the anticancer effect of crude extracts are being increasingly replaced by bioactivity-guided fractionation, as well as detailed assessment of the mechanism of action. Numerous compounds-especially flavonoids such as amentoflavone and protoapigenone, and also simpler phenolic compounds, steroids, alkaloids and terpenoids-were isolated and found to be cytotoxic, particularly pro-apoptotic, or to induce cell cycle arrest in cancer cell lines in vitro. In in vivo experiments, some fern-derived compounds inhibited tumour growth with little toxicity. On the other hand, many ferns-not only the well-known Bracken (Pteridium)-may pose a significant hazard to human health due to the fact that they contain carcinogenic sesquiterpenoids and their analogues. The objective of this review is to summarise the recent state of research on the anticancer properties of ferns and lycopods, with a focus on their characteristic bioactive constituents. The carcinogenic hazard posed by ferns is also mentioned.

The cytotoxic effect of α-tomatine in MCF-7 human adenocarcinoma breast cancer cells depends on its interaction with cholesterol in incubation media and does not involve apoptosis induction.

In recent years, α-tomatine has been studied for its anticancer activity. In the present study, we focused on the cytotoxic effect of α-tomatine in the MCF-7 human breast adenocarcinoma cell line, its mechanism of action, biotransformation and stability in the culture medium. We observed an inhibition of cell proliferation and viability at concentrations of 6 and 9 µM but then a recovery of cells occurred. The recovery was not caused by the biotransformation of α-tomatine in MCF-7 cells, but by a substantial decrease in the concentration of α-tomatine in the culture medium due to its binding with cholesterol. Regarding the mechanism of action of α-tomatine, we observed no DNA damage, no changes in the levels of the proteins p53 and p21(WAF1/Cip1), and no apoptosis (neither activated caspase-8 and -9, nor sub-G1 peak, or morphological signs). We found a loss of ATP in α-tomatine-treated cells. These results support the conclusion that α-tomatine does not induce apoptosis in the MCF-7 cell line.

Development of an HPLC fluorescence method for determination of boldine in plasma, bile and urine of rats and identification of its major metabolites by LC-MS/MS.

Boldine belongs to the group of aporphine alkaloids isolated from Boldo tree. In contrast with numerous reports on the pharmacological effects of boldine, the data about its pharmacokinetics and biotransformation are scarce. No validated bioanalytical method of sufficient sensitivity has so far been described in the literature which could be used for quantification of boldine in various body fluids collected in pharmacokinetic studies. This work presents, for the first time, the assay for boldine in the plasma, bile and urine of rats. It includes liquid-liquid extraction/back-extraction of boldine, its chromatographic separation and sensitive fluorescence detection. Separation was carried out on a pentafluorophenyl core-shell column (Kinetex PFP, 150×3mm, 2.6µm) in gradient elution mode with solvent system consisting of an acetonitrile-ammonium formate buffer (5mM, pH=3.8). Fluorimetric detection (λEX=320nm, λEM=370nm) was used for quantitative work. Validation according to the EMEA guideline proved the assay LLOQ (0.1µmolL(-1)), linearity over a broad range of 0.1-50µmolL(-1), precision (intra- and inter-day CVs less than 4.5% and 6.1%, respectively) and accuracy (relative errors between -5.8% and 4.8%). In a pilot pharmacokinetic experiment, the concentration-time profiles were described for boldine (single i.v. bolus 50mgkg(-1)) in plasma and bile and cumulative excretion in urine was investigated. The major metabolites identified by means of LC-MS(n) were boldine-O-glucuronide, boldine-O-sulphate and disulphate, boldine-O-glucuronide-O-sulphate and N-demethyl-boldine-O-sulphate.

In vivo effect of oracin on doxorubicin reduction, biodistribution and efficacy in Ehrlich tumor bearing mice.

BACKGROUND: The limitation of carbonyl reduction represents one possible way to increase the effectiveness of anthracycline doxorubicin (DOX) in cancer cells and decrease its toxicity in normal cells. In vitro, isoquinoline derivative oracin (ORC) inhibited DOX reduction and increased the antiproliferative effect of DOX in MCF-7 breast cancer cells. Moreover, ORC significantly decreases DOX toxicity in non-cancerous MCF-10A breast cells and in hepatocytes. The present study was designed to test in mice the in vivo effect of ORC on plasma and tissue concentrations of DOX and its main metabolite DOXOL. The effect of ORC on DOX efficacy in mice bearing solid Ehrlich tumors (EST) was also studied. METHODS: DOX and DOX + ORC combinations were iv administered to healthy mice. Blood samples, livers and hearts were collected during the following 48 h. DOX and DOXOL concentrations were assayed using HPLC. The mice with inoculated EST cells were treated repeatedly iv with DOX and DOX + ORC combinations, and the growth of tumors was monitored. RESULTS: ORC in combination with DOX significantly decreased DOXOL plasma concentrations during four hours after administration, but this significantly affected neither DOX plasma concentrations nor DOX or DOXOL concentrations in the liver and heart at any of intervals tested. In EST bearing mice, ORC did not significantly affect DOX efficacy on tumor growth. However, EST was shown to be an improper model for the testing of ORC efficacy in vivo, as ORC did not inhibit DOXOL formation in EST. CONCLUSIONS: In vivo, ORC was able to retard DOXOL formation but was not able to improve DOX efficacy in EST-bearing mice.

The anticancer activity of alpha-tomatine against mammary adenocarcinoma in mice.

AIM: To evaluate the anticancer effect of alpha-tomatine (i.p.) either alone or in combination with doxorubicin (i.v.) in a mouse tumour model. METHODS: We studied the effect of repeated alpha-tomatine (0.1 - 9 mg/kg) and/or doxorubicin (2 mg/kg) on the growth and mitotic activity of the solid Ehrlich tumour in vivo, as well as on the survival of the tumour-bearing mice. RESULTS: Monotherapy with alpha-tomatine had a significant dose-dependent anticancer effect which peaked at 1 mg/kg. This was shown by both slowed tumour growth and reduced tumour cell proliferation. We also provide the first evidence that the combination alpha-tomatine (1 mg/kg) and doxorubicin (2 mg/kg) had a synergistic effect and significantly prolonged the survival of the mice. Neither alpha-tomatine nor doxorubicin influenced the infiltration of tumours with CD3+ lymphocytes; nor were we able to find an in vivo modulation of the key molecules of two regulatory pathways reported in vitro as the principal anti-cancer mechanisms of alpha-tomatine, i.e. iNOS and phosphorylated ERK2. However, alpha-tomatine still led to intracellular DNA inhibition and protein synthesis in Ehrlich tumour cells in a short-term culture ex vivo with IC50 values of 8.7 and 6.6 µM. CONCLUSIONS: The results suggest that ΤΟΜ, especially in combination with doxorubicin, may be a promising agent for the treatment of malignant solid tumours. Despite growing knowledge of the mechanisms of ΤΟΜ action in cancer cells, most aspects remain unclear. Parallel organ toxicity, especially potential liver effects, requires careful attention when performing in vivo studies in the future.

Cholestatic effect of epigallocatechin gallate in rats is mediated via decreased expression of Mrp2.

Epigallocatechin gallate (EGCG) has been shown to be protective in various experimental models of liver injury, although opposite effects have also been reported. Since its effect on biliary physiology has not been thoroughly investigated, the present study evaluated effect of EGCG on bile flow and bile acid homeostasis in rats. Compared to controls, EGCG treatment decreased bile flow by 23%. Hepatic paracellular permeability and biliary bile acid excretion were not altered by EGCG administration, but biliary glutathione excretion was reduced by 70%. Accordingly, the main glutathione transporter on the hepatocyte canalicular membrane, multidrug resistance-associated protein 2 (Mrp2), was significantly decreased at the protein level. EGCG administration also doubled plasma bile acid levels compared to controls. While protein levels of the main hepatic bile acid transporters were unchanged, the rate-limiting enzyme in the bile acid synthesis, Cyp7a1, was significantly increased by EGCG. Enhanced bile acid synthesis in these animals was also confirmed by a 2-fold increase in plasma marker 7α-hydroxy-4-cholesten-3-one. In contrast, EGCG markedly downregulated major bile acid transporters (Asbt and Ostα) and regulatory molecules (Shp and Fgf15) in the ileum. When EGCG was coadministered with ethinylestradiol, a potent cholestatic agent, it did not show any additional effect on the induced cholestasis. This study shows ability of EGCG to raise plasma bile acid concentrations, mainly through Cyp7a1 upregulation, and to decrease bile production through reduction in Mrp2-mediated bile acid-independent bile flow. In conclusion, our data demonstrate that under certain conditions EGCG may induce cholestasis.

Boldine and related aporphines: from antioxidant to antiproliferative properties.

Plant and folk medicine represent nowadays a source of either new therapeutic substances or substrates for drug synthesis. One such promising group for possible further exploitation is the family of aporphine alkaloids containing boldine and related compounds. In this mini-review we focus on boldine and its newly described effects, which predominantly arise from its antioxidant properties. Moreover, we try to compare its antiproliferative properties with other better known members of the aporphine group.

Pravastatin modulates liver bile acid and cholesterol homeostasis in rats with chronic cholestasis.

BACKGROUND AND AIM: The administration of pravastatin to patients with cholestatic liver disease has suggested the potential of the drug with regard to reducing raised plasma cholesterol and bile acid levels. Information about the mechanisms associated with this effect is lacking. Thus, the aim of the present study is to evaluate pravastatin effects on the liver bile acid and cholesterol homeostasis in healthy and cholestatic rats. METHODS: Control sham-operated and reversibly bile duct-obstructed (BDO) rats were treated with pravastatin (1 or 5 mg/kg) or the vehicle alone for 7 days after surgery. RESULTS: Lower doses of pravastatin reduced bile acid plasma concentrations in cholestatic animals. The effect was associated with reduced liver mRNA expression of Cyp7a1, Cyp8b1, Mrp2, Ugt1a1 and the increased expression of Bsep. In addition, BDO-induced increase in the liver content of cholesterol was normalized by pravastatin. The change was accompanied by the reduced liver expression of Hmg-CoA reductase, LDL receptor, and Acat2, and induced the expression of Abca1 and Mdr2. These changes corresponded with the upregulation of nuclear receptors LXRα and PPARα, and the downregulation of FXR, CAR, SREBP-2 and HNF1α. High doses of pravastatin lacked any positive effects on bile acids and cholesterol homeostasis, and blocked bile formation through the reduction of the biliary excretion of bile acids. CONCLUSIONS: Pravastatin rendered a positive reduction in BDO-induced increases in plasma bile acid concentrations and cholesterol liver content, mainly through the transcriptionally-mediated downregulation of genes involved in the synthesis of these compounds in the liver.

Evaluation of the antineoplastic activity of L-rhamnose in vitro. A comparison with 2-deoxyglucose.

The effect of unsubstituted deoxyhexoses, 2-deoxy-D-glucose (2-DG) and L-fucose, on tumor cells has been reported in several papers throughout the last decades. That of a similar deoxysugar, L-rhamnose, which is synthesized in bacteria and plants but not in animal cells, has until today not been explored. In the present study, we examined the effect of L-rhamnose on DNA and protein synthesis, growth and the potential induction of apoptosis of tumor cells in vitro. Using 2-DG for comparison, we studied the effect of L-rhamnose in concentrations up to 20 (32 resp.) mmol/l on the initial velocity of the incorporation of labeled precursors of DNA and proteins in short term cultures of both mouse Ehrlich ascites tumor (EAT) and human HL-60 cells in vitro, and further, on cell proliferation and apoptosis induction in HL-60 cells. Neither cytotoxic nor cytostatic effects of L-rhamnose were observed with the exception of slightly pronounced inhibition of DNA synthesis in EAT cells. From the lacking inhibition of the protein synthesis it can be considered that L-rhamnose does not interfere with energy metabolism, at least not in a similar manner as 2-DG.

Increased melibiose/rhamnose ratio in bile of rats with acute cholestasis.

BACKGROUND AND AIM: Melibiose/rhamnose permeability test is used for noninvasive intestinal mucosa barrier testing. However, the possible escape route of the absorbed saccharides through either intact or impaired blood-biliary barriers has not so far been explored. The objective of the present study was therefore two-fold: First, to describe in detail the biliary pharmacokinetics of melibiose and rhamnose in rats; second, to evaluate the changes of both sugars' pharmacokinetics upon impairment of the blood-biliary barrier by acute extrahepatic cholestasis in rats. METHODS: Bile duct obstructed (BDO), sham-operated and intact (unoperated) male Wistar rats were administered, 24 h after the appropriate intervention, with a single intravenous dose of melibiose and rhamnose, and a 4-h pharmacokinetic study was performed. RESULTS: In intact animals, the biliary excretion of melibiose and rhamnose was only 0.06% and 0.4% of the administered dose, respectively, while the urinary excretion accounted for 70.6% and 61.7%, respectively. In BDO animals, the biliary excretion rate of both saccharides, especially that of melibiose, was increased with a consequent 4.4-fold rise of the biliary melibiose/rhamnose ratio, the accepted paracellular permeability indicator. Both, the renal clearance of melibiose and the urinary melibiose/rhamnose ratio remained uninfluenced by cholestasis. CONCLUSION: The present study is the first to describe in detail pharmacokinetic parameters and the biliary excretion of melibiose and rhamnose in healthy and cholestatic rats. The altered melibiose/rhamnose biliary excretion ratio in BDO rats indicates that the test is able to detect the impairment of the blood-biliary barrier in acute extrahepatic cholestasis.