Leos Fuksa

Morphological and functional changes in P-glycoprotein during dexamethasone-induced hepatomegaly

1 The effect of dexamethasone on hepatic and renal P‐glycoprotein (P‐gp) expression, localization and activity was investigated in rats after 4 days oral administration of two dose regimens (1 or 25 mg/kg per day). Simultaneous increases in liver weight were evaluated by quantitative histological examination. 2 In the liver, dexamethasone pretreatment produced hepatomegaly as a consequence of extensive periportal fat accumulation, which was quantified by densitometry of oil red O‐stained liver sections. Quantitative immunohistochemical analysis revealed preferential periportal zonation of P‐gp in control animals. Dexamethasone pretreatment resulted in spatially disproportional induction of P‐gp protein expression within the liver acinus characterized by preferential increase in pericentral areas, with consequent uniform panlobular distribution. Western blot analysis confirmed these results, showing increases in P‐gp protein. Quantitative reverse transcription–polymerase chain reaction analysis revealed no statistically significant change in liver mdr1b mRNA expression after either dexamethasone treatment regimen. The expression of mdr1a mRNA was significantly decreased by 85–87%. 3 In the kidney, dexamethasone reduced mdr1a mRNA expression by 69–89%, whereas mdr1b mRNA expression was increased in a dose‐dependent manner. However, despite tendencies, no significant increases in P‐gp expression were observed at the protein level. 4 The in vivo function of P‐gp was evaluated by measuring renal and biliary secretion of rhodamine‐123 (Rho123) under a steady state plasma concentration. The biliary, renal and tubular secretory clearance of Rho123 was significantly increased only after high‐dose dexamethasone. 5 In conclusion, the present study suggests that drug interactions observed during corticosteroid therapy may be mediated, at least in part, through increased biliary, and also renal, excretion of P‐gp substrates. Expression of P‐gp in the liver showed primary periportal zonation with differential changes during induction. Accompanying hepatomegaly may be explained by severe microvesicular steatosis selectively localized to the periportal areas.

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.

Dexamethasone reduces methotrexate biliary elimination and potentiates its hepatotoxicity in rats

Increased hepatotoxicity of methotrexate has been reported during dexamethasone therapy in humans. Despite the observed inducing effect of dexamethasone on some methotrexate transporting proteins in the liver, the kinetic aspects of this interaction have not been studied yet. Thus, the aim of the present study was to evaluate the influence of dexamethasone on the hepatic and overall pharmacokinetics of methotrexate. Pharmacokinetics of methotrexate was evaluated in rats during an in vivo steady-state clearance study after either single intravenous dose of dexamethasone or its four-day oral administration in a dose optimized for transport proteins induction. Dexamethasone oral pretreatment reduced biliary clearance of methotrexate by 53%. Although liver tissue concentration of methotrexate increased only slightly in these animals, a significant increase in liver weights produced by dexamethasone pretreatment revealed a marked increase in liver content of the drug. An evaluation of plasma liver enzyme activities measured before and after methotrexate administration demonstrated a potentiation of corticosteroid hepatotoxicity by the cytostatic. Analysis of methotrexate transporter expression in the liver showed up-regulation of Mrp2, Oatp1a4, and Oat2, and down-regulation of Mrp3. These observations comply with increased biliary excretion and reduced plasma concentrations of their endogenous substrate, conjugated bilirubin. In contrast, single intravenous bolus of dexamethasone did not influence any pharmacokinetic parameter of methotrexate. In conclusion, these results indicate that hepatocellular impairment associated with reduced biliary elimination of methotrexate, and its raised liver content may contribute to increased hepatotoxicity of the drug when co-administered with dexamethasone. Moreover, an influence of dexamethasone on protein expression of anionic drugs transporters in the liver and kidney was demonstrated.

Up-regulation of renal Mdr1 and Mrp2 transporters during amiodarone pretreatment in rats

Although amiodarone (AMD) is known to produce drug-drug interactions through inhibition of transporter-mediated excretion of drugs, its impact on these mechanisms during chronic treatment has not been described yet. Therefore, the aim of this study was to investigate the influence of AMD pretreatment on the main multidrug transporting proteins, Mdr1 and Mrp2, in the liver and kidney. The expression of the transporters and pharmacokinetics of their substrates, rhodamine-123 (Rho123) and endogenous conjugated bilirubin (CB), were evaluated in rats after either AMD oral pretreatments (4-14 days) or single intravenous bolus. AMD pretreatment of all durations up-regulated renal Mdr1 and Mrp2 protein expression to 155-190% and 152-223% of the control values, respectively. In agreement, we observed a corresponding increase in renal clearance of both substrates. Hepatic expression was increased only for Mdr1 to 234-270% of controls, which was associated with increased biliary elimination of amiodarone without change in Rho123 biliary clearance. Interestingly, hepatic expression of another Mdr transporter, Mdr2, was progressively decreased by amiodarone administration. Acute administration of AMD reduced Rho123 biliary clearance by 64%. Our results indicate that repeated administration of AMD to rats is associated with significant increase in hepatic and renal expression of Mdr1 and Mrp2 transporters, which may contribute to variability in pharmacokinetics of AMD and simultaneously applied drugs.

Zonation of multidrug resistance‐associated protein 2 in rat liver after induction with dexamethasone

Background and Aim:  The present study was aimed to evaluate the hepatic zonation of multidrug resistance‐associated protein 2 (mrp2), an important drug transporter, and its potential changes during the induction of its expression by known inducer, dexamethasone (DEX).

P-glycoprotein function and expression during obstructive cholestasis in rats.

Objectives The present study was aimed at evaluation of in vivo biliary and renal excretion of rhodamine 123 (Rho123), a P-glycoprotein (P-gp) substrate, in rats during either acute or chronic cholestasis induced by bile duct obstruction (BDO). Methods The Rho123 clearance study was performed either one (BDO1) or seven (BDO7) days after BDO. Bile flow was reconstituted, and bile and urine were collected after steady-state plasma concentration of Rho123 was attained. Tissue expression of P-gp was evaluated by quantitative immunohistochemistry, and immunoblotting. Results Significant up-regulation of the liver P-gp protein was observed in acute and chronic cholestasis. Primary periportal location of P-gp was enlarged also to pericentral areas. In the kidneys, immunohistochemistry showed pancellular increase in P-gp after 1 day of BDO, which subsided after 7 days of BDO. Nevertheless, biliary and renal clearances (CLBile and CLR) of Rho123 did not reflect the induction of P-gp expression. While CLBile was reduced one day after cholestasis and restored on the seventh day, the CLR was preserved in BDO1 group and reduced in BDO7 group without change in glomerular filtration rate. In parallel, biliary and renal clearances of conjugated bilirubin were significantly reduced in both cholestatic groups compared with controls. Conclusion These findings suggest that extrahepatic cholestasis causes time-dependent changes in elimination of Rho123 which do not exactly reflect alteration of P-gp expression in the rat liver and kidney. These data may help to explain impaired elimination of P-gp substrates after short-term cholestasis that may commonly occur in clinical practice.

Amiodarone modulates pharmacokinetics of low‐dose methotrexate in rats

Clinical studies of low‐dose methotrexate (LDMTX) pharmacokinetics document increased plasma concentrations of MTX after co‐administration of the drug with amiodarone or macrolide antibiotics. As drug–drug interactions may increase the toxicity of LDMTX, a rat model was used to follow renal and biliary elimination of MTX during its constant‐rate i.v. infusion and concomitant single bolus i.v. injections of amiodarone or azithromycin. The mean steady‐state plasma concentration of 1.7±0.1 µmol/l was reached and the total clearance achieved 17.7±1.0 ml/min/kg. Administration of amiodarone decreased the biliary clearance of MTX to 73% of the control values (p<0.05). Correspondingly, the total clearance decreased to 72% and plasma MTX concentrations were augmented to 2.5±0.4 µmol/l (p<0.05). Amiodarone‐treated rats exhibited a 3.3‐fold decrease in the renal clearance (p<0.05) of conjugated bilirubin, which was associated with its increased plasma concentration. In contrast, azithromycin did not alter any of the MTX pharmacokinetic parameters. In conclusion, this is the first report describing the impairment of MTX hepatic elimination during co‐administration with amiodarone. This study also provides new insight into acute amiodarone‐induced hyperbilirubinaemia, where increased bilirubin production and decreased renal clearance may contribute to this effect. Importantly, azithromycin seems to be a safe co‐medication during LDMTX therapy. Copyright

Methotrexate released in vitro from bone cement inhibits human stem cell proliferation in S/G2 phase

Methotrexate (MTX) released from bone cement showed a useful local effect in animal models of bone tumours. However, local toxic reactions such as impaired wound healing were observed in areas surrounding the MTX-loaded implant. Therefore, we hypothesised that MTX released from bone cement would have harmful effects on human mesenchymal stem cells (MSC)—one of the basic components of bone marrow and tissue reparatory processes. Moreover, elution of MTX was calculated from implants prepared either with liquid or powdered MTX. During the 28-day incubation, the cement compounded with liquid MTX showed the highest elution rate of the drug. MTX released from pellets produced a significant decrease in proliferation of MSC as a consequence of a blockade of their cell cycle in the S/G2 phase. These findings indicate impairment of stem cell function in marginal areas surrounding the MTX-loaded cement and may help to explain problems with regeneration of tissues in these locations.RésuméLe Methotrexate (MTX) libéré par le ciment acrylique a un effet certain sur les tumeurs osseuses développées sur un modèle animal. Cependant, des réactions toxiques locales telles que défauts de cicatrisation ont été observées à proximité de l’implantation de ce ciment. Nous faisons donc l’hypothèse que le Methotrexate MTX libéré par le ciment a un effet nocif néfaste sur les cellules mésenchyméteuses humaines MSC qui sont l’un des composants de base de la moelle osseuse et des phénomènes de la réparation tissulaire. De plus, la dynamique de pénétration du méthotrexate a été analysée à partir d’implants préparés avec du liquide ou de la poudre de méthotrexate. Au cours des 28 jours d’incubation, le ciment préparé avec du méthotrexate liquide montre un taux important de pénétration de la drogue. Le méthotrexate libéré des billes de ciment entraîne une diminution significative de la prolifération des cellules mésenchyméteuses avec un bloquage cellulaire à la phase S/G2. Ces travaux montrent que la fonction cellulaire est altérée au voisInage du méthotrexate pouvant expliquer les problèmes de régénétration tissulaire.

Adherence to oral bisphosphonates: 30 more minutes in dosing instructions matter

Abstract Objectives Low adherence to treatment with bisphosphonates significantly impedes its effectiveness. The objectives were: (1) to compare adherence to oral weekly and monthly bisphosphonates with emphasis on dosing instructions; and (2) to study associations between adherence and beliefs about the bisphosphonate treatment among women ≥ 55 years. Methods A multicenter survey was performed in secondary-care patients with osteoporosis. Osteoporosis Specific Morisky Medication Adherence Scale (OS-MMAS), questions on compliance with five dosing instructions and Beliefs about Medicines Questionnaire (BMQ) Specific were used. Results As many as 363 questionnaires (response rate 95%) were analyzed. Respondents (mean age 69 years) were treated with weekly bisphosphonates (37%) or monthly ibandronate (63%). Based on OS-MMAS, 67% of respondents showed high adherence with no differences between the subgroups. Only 44% of respondents were compliant with all dosing instructions. Compliance with dosing instructions concerning time interval (fasting and staying upright) was 71% in weekly and 52% in monthly subgroups, respectively (p < 0.001). Compliance with dosing instructions correlated positively with education (p = 0.009). The mean BMQ necessity score of 18.4 was greater than the mean BMQ concerns score of 13.3. OS-MMAS score correlated with necessity (p = 0.010). Persistence derived from OS-MMAS correlated with both necessity (p = 0.014) and concerns (p = 0.041). Conclusion Despite relatively high adherence to the treatment, most patients do not follow dosing instructions. Reduced bioavailability, particularly of monthly ibandronate, can be expected in clinical practice. Adherence-related outcomes are associated with beliefs about the oral treatment with bisphosphonates.

Modification of hepatic iron metabolism induced by pravastatin during obstructive cholestasis in rats.

AIMS To evaluate iron biochemistry and contributing liver mechanisms during obstructive cholestasis and pravastatin treatment in rats. MAIN METHODS A rat model of cholestasis induced by bile duct ligation (BDL) was used for the study. The detection of iron and the expression of relevant molecules were performed one week after surgery in the control, and cholestatic animals after treatment with either saline or pravastatin (1mg/kg/day). KEY FINDINGS Saline-administered BDL rats showed, in comparison to sham-operated animals, a significant increase in plasma iron concentration, increased liver protein content of heme oxygenase-1 (HO-1) and a decline in the expression of hepcidin. Ferroportin 1 expression was increased with a simultaneous reduction in intrahepatic iron concentration. The administration of pravastatin to BDL animals attenuated proliferation changes in liver parenchyma, prevented HO-1 induction, restored hepatic mRNA hepcidin expression to control levels and induced the expression of ferritin, transferrin receptors (TfR1/2) and divalent metal transporter-1. This was accompanied by an increased content of intrahepatic iron when compared to the BDL animals, and a reduction of hyperbilirubinemia. SIGNIFICANCE Cholestasis-induced increase in plasma and decrease in hepatic iron levels were associated with up-regulation of liver HO-1 and ferroportin 1. Pravastatin alleviated cholestatic liver impairment and raised liver iron content by modulation of heme catabolism and an increase of hepatic iron uptake and storage capacity.