Penetration of piperacillin into the vitreous after intravenous administration.

PURPOSE: Penetration of piperacillin into the vitreous cavity after intravenous administration was investigated in humans. METHODS: Forty-five eyes undergoing vitrectomy between November 1993 and December 1994 were included. Each patient received a single intravenous dose of piperacillin 4 g 2 hours before ocular incision. A 0.2-mL vitreous sample was aspired at the beginning of vitrectomy, a blood sample was obtained, and piperacillin level was assessed. RESULTS: There were no detectable drug concentrations in 25 eyes. Mean vitreous drug concentration in the remaining evaluable eyes (n = 14) was 2.33 microg/mL (+/-0.76). We divided samples into two groups: 23 uninflamed (Group 1) and 16 inflamed (Group 2) eyes. Mean vitreous drug concentration was 0.42 microg/mL in Group 1 and 4.95 microg/mL in Group 2 (P < 0.2). Piperacillin concentration was higher than the minimum inhibitory concentration for gram-positive bacteria in 13% of uninflamed and 69% of inflamed eyes (P < 0.001). CONCLUSION: Studies show that intravenously administered piperacillin can penetrate the vitreous cavity in rabbits. Our study suggests that a single dose of piperacillin can produce intravitreal concentrations sufficient to kill gram-positive bacteria in inflamed eyes. The poor intraocular penetration of systemically administered piperacillin in uninflamed eyes raises questions about its usefulness as a prophylactic agent in ophthalmic surgery.

Toxic effects of tacrine on primary hepatocytes and liver epithelial cells in culture.

Administration of tacrine (THA) for the treatment of Alzheimer's disease results in a reversible hepatotoxicity in 30-50% of patients, as indicated by an increase in transaminase levels. However, the intracellular mechanisms underlying such a toxicity have not yet been elucidated. In this study, we performed short-term and long-term in vitro treatments on primary human and rat hepatocyte cultures as well as on nonparenchymal rat liver epithelial cells (RLEC), known as CYP1A-deficient cells. Cell ultrastructure was analyzed under different conditions and the release of lactate dehydrogenase (LDH) was used to evaluate cytotoxicity. The effects of THA on protein synthesis, intermediary metabolism and reduced glutathione (GSH) level were also determined in rat hepatocytes. THA induced dose-dependent toxic effects in liver parenchymal and nonparenchymal cells, with human hepatocytes being less sensitive. This toxicity appeared to be unrelated to metabolism of THA since similar effects were observed in rat hepatocytes and RLEC, in which THA metabolism was found negligible. Ribosome aggregation appeared only at high concentrations (> 1 mmol/L) and was not specific to hepatocytes. Therefore, the THA-induced decrease in protein synthesis observed at lower concentrations was likely not related to this alteration. ATP and glycogen levels as well as GSH content were reduced upon THA. However, while glycogen level decreased at THA doses similar to those inducing an increase in LDH release, the fall in ATP and GSH contents occurred at higher doses. Thus, glycogen level in hepatocytes appeared to be a more sensitive indicator of THA toxicity than were ATP and GSH levels. We also found that protein synthesis started to decrease at THA doses that were still ineffective on LDH release. This might suggest that the decrease in synthesis of one or several proteins upon THA treatment represents the early signal leading cells to death.

Use of heterologously expressed human cytochrome P450 1A2 to predict tacrine-fluvoxamine drug interaction in man.

The aim of the present study was to evaluate the use of recombinant human cytochrome P-450 1A2 (rH-CYP1A2) in studies performed in vitro in order to predict metabolic drug-drug interactions occurring in man. In vitro metabolism of tacrine (a CYP1A2 probe) in the presence and absence of fluvoxamine, a CYP1A2 inhibitor, was investigated in human liver mircrosomes and with different rH-CYP. Vmax, Km and Ki determined with human liver microsomes were compared with those observed using rH-CYP1A2, assuming that 1 mg of liver microsomes contains, on average, 69 pmol of CYP1A2. The extent of tacrine metabolism inhibition procured by fluvoxamine with rH-CYP1A2, was compared with previous results observed in man. The Vax and Km for 1-hydroxytacrine formation rates obtained with rH-CYP1A2 were in good agreement with those observed in human liver microsomes (175+/-9 versus 140+/-60 pmol/min/mg for Vmax and 14+/-2 versus 16+/-2 microM for Km, respectively. The Ki of fluvoxamine on 1-hydroxytacrine formation rate observed with rH-CYP1A2 was similar to that observed with human liver microsome (0.35+/-0.05 versus 0.20+/-0.20 microM, respectively). Using the Km, Vmax and Ki determined with rH-CYP1A2, we calculated that fluvoxamine produced an inhibition of 1-, 2- and 4-hydroxytacrine formation rate of 91, 87 and 88%, respectively, in the range of tacrine and fluvoxamine concentrations observed in man. These percentages of inhibition calculated in vitro were in agreement with the percentage of fluvoxamine-dependent decrease in tacrine apparent oral clearance previously observed in man (83+/-13%). We conclude that human CYP1A2 expressed in yeast is a powerful tool to predict and to quantify drug-drug interactions in man.

Influence of the CYP1A2 inhibitor fluvoxamine on tacrine pharmacokinetics in humans.

OBJECTIVE: Tacrine is extensively metabolized by cytochrome P4501A2 (CYP1A2). Fluvoxamine, a potent CYP1A2 inhibitor, may be coadministered with tacrine. The aim of this study was to examine the influence of fluvoxamine administration on the disposition kinetics of single-dose tacrine administration. METHODS: Thirteen healthy volunteers participated in this double-blind, randomized crossover study, which compared the effects of fluvoxamine (100 mg/day during 6 days) and placebo on the pharmacokinetics of a single oral dose of tacrine (40 mg). RESULTS: Fluvoxamine caused a significant increase in tacrine area under the plasma concentration versus time curve (AUC): arythmetic mean, 27 (95% confidence interval [CI], 19 to 38) ng.hr/ml versus 224 (95% CI, 166 to 302) ng. hr/ml. Fluvoxamine caused a decrease in the apparent oral clearance of tacrine from 1683 +/- 802 to 200 +/- 106 L/hr (mean +/- SD), which was explained by a decrease in its nonrenal clearance. Five subjects had gastrointestinal side effects during fluvoxamine administration. Fluvoxamine administration was associated with significant increases in the plasma AUC values of three monohydroxylated tacrine metabolites and in the total urinary recovery measurements of tacrine and its metabolites (9.1% +/- 4.6% versus 24.0% +/- 2.6% of recovery). These results may be attributable to fluvoxamine-dependent inhibition of CYP1A/, which is responsible of the biotransformation of tacrine into its monohydroxylated metabolites and further into dihydroxylated and reactive metabolites. CONCLUSION: Fluvoxamine inhibits the metabolism of tacrine. CYP1A2 may be the target of this inhibition. Fluvoxamine may modulate the hepatotoxicity of tacrine, depending on the relative contribution of tacrine and its reactive metabolites to this toxicity.

Uncoupling of rat and human mitochondria: a possible explanation for tacrine-induced liver dysfunction.

BACKGROUND & AIMS: Tacrine administration (1-3 mg/kg) may lead to sinusoidal concentrations in the micromolar range and produce liver dysfunction in 50% of recipients. The aim of this study was to determine the cellular effects of tacrine that account for liver dysfunction. METHODS: The effects of tacrine on mitochondrial function were determined in isolated rat liver mitochondria, cultured rat hepatocytes, and isolated human lymphocytes. RESULTS: In vitro, tacrine was taken up by rat liver mitochondria, decreased their membrane potential, and stimulated their respiration. Ex vivo, respiration was increased in rat mitochondria isolated 30 minutes after the administration of 2 mg of tacrine per kilogram. After 7 days of culture, tacrine (2.5 mumol/L) decreased rat hepatocyte adenosine triphosphate levels. Ten micromolar decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium reduction and neutral red uptake without modifying cell glutathione, the morphology of the endoplasmic reticulum, or protein synthesis. Tacrine (1.25 mumol/L) decreased levels of adenosine triphosphate in human lymphocytes. CONCLUSIONS: The weak base tacrine exerts a protonophoric effect in mitochondria that wastes energy and decreases levels of adenosine triphosphate in rat and human cells. These effects are visible after clinically relevant doses of tacrine and might be involved in tacrine-induced liver dysfunction.

A cellular model for drug interactions on hematopoiesis: the use of human umbilical cord blood progenitors as a model for the study of drug-related myelosuppression of normal hematopoiesis.

A cellular model of hematopoiesis which would be more convenient than bone marrow (BM) progenitors and directly relevant to human pathology is needed in order to investigate xenobiotic toxicity. Human umbilical cord blood (HCB), previously shown to be able to repopulate BM, provides a powerful in vitro model of normal human hematopoiesis. In order to validate the use of normal HCB progenitors as targets for dose-related myelosuppression, we used clonogenic assays and expansion in a liquid culture of progenitor-enriched cell suspensions from HCB. A series of 8 reference molecules, doxorubicin, cytosine-arabinoside, 5-fluorouracil, 3'-azido-3'-deoxythymidine, acetylsalicylic acid, sodium valproate and two cephalosporin antibiotics, were tested. In vitro 50% inhibition concentrations (IC50) were compared to those observed or reported with BM progenitors, and to the values of plasma concentrations from treated patients. HCB progenitors as in vitro targets for cytotoxic molecules were easy to access and handle, and their use was sensitive, specific and reproducible. They gave results similar to BM progenitors and allowed a qualitative approach to cellular metabolism and toxicity using morphological, flow cytometric and chromatographic methods.

[Effect of S9788, cyclosporin A and verapamil on intracellular accumulation of doxorubicin, daunorubicin and daunorubicinol in primary rat hepatocyte culture]. / Effet du S9788, de la ciclosporine A et du vérapamil sur l'accumulation intracellulaire de la doxorubicine, de la daunorubicine et du daunorubicinol dans des hépatocytes de rat en culture primaire.

The aim of this study was to compare the effect of three MDR modulators, cyclosporine A, S9788 and verapamil on the efflux of two anthracyclines, doxorubicin and daunorubicin and of daunorubicinol, the C-13 alcohol metabolite of daunorubicin. Rat hepatocyte primary cultures were used as a model of P-gp expression. They allow to study MDR at different levels of P-gp expression which increases in parallel with culture time. Furthermore, hepatocytes are able to metabolize drugs and enable determination of the role of P-gp on metabolite efflux. Hepatocytes grown for 4 or 48 hours were incubated for 6 hours in the presence of a combination of each modulator and one of the two anthracyclines (0.5 microM). Modulator concentrations used were 1, 5 and 15 microM when associated with DOX, and 1 and 15 when associated with DNR. In fresh hepatocytes, the three MDR modulators did not induce an increase in intracellular retention of the two anthracyclines compared to controls without MDR modulators. At 48 hours of culture, the three tested drugs increased intracellular accumulation of DOX. However, daunorubicin retention was not modified but that of its metabolite was increased. The activity rank order was cyclosporine A > S9788 > verapamil. Cyclosporine A and S9788 were active in simultaneous as well as in sequential combinations with anthracyclines. Verapamil was only effective when co-incubated with anthracyclines.

P-glycoprotein expression and function in rat hepatocytes in culture.

Expression of P-glycoprotein, which confers multidrug resistance to a broad range of anticancer drugs, was studied in rat hepatocytes in culture. P-glycoprotein was localized in the plasma membrane by immunohistochemical staining and was evaluated by western blotting with C219 as primary antibody and quantification of the coloured spots. The conditions of culture (time in culture, cell density at seeding) had a strong effect on the expression of P-glycoprotein. Expression increases with time in culture. At 10 x 10(6) cells/75 cm2 flasks, which is the normal density seeding for hepatocytes in culture, the increase of P-glycoprotein was 17% between 4 and 24 hr in culture, 52% between 24 and 48 hr and 37% between 48 and 96 hr. At low density cell seeding (2 x 10(6) cells/75 cm2), the expression of P-glycoprotein was higher than at normal density from the first day in culture (+20%). This difference of expression was maintained until 96 hr of culture and was maximum at 48 hr (+44%). This P-glycoprotein was functional and this overexpression was correlated with a decrease of doxorubicin retention in hepatocytes.

Modulation of anthracycline accumulation and metabolism in rat hepatocytes in culture by three revertants of multidrug resistance.

The aim of this study was to compare the action of three multidrug resistance (MDR) modulators, cyclosporine A, S 9788, and verapamil, on the efflux of two anthracyclines, doxorubicin and daunorubicin, and of daunorubicinol, the C-13 alcohol metabolite of daunorubicin. Rat-hepatocyte primary cultures have been used as a model of P-glycoprotein (Pgp) expression. This model allows the study of MDR at different levels of Pgp expression, which increases in parallel with the time in culture; furthermore, the hepatocytes are capable of metabolizing drugs, which enables the determination of the role of Pgp on metabolite efflux. All modulators tested were incubated for 6 h at concentrations of 1, 5, and 15 microM with doxorubicin (0.5 microM) and at 1 and 15 microM with daunorubicin (0.5 microM) on hepatocytes grown for 4 and 48 h in culture. Daunorubicinol (0.5 microM) was tested with modulators at 48 h of culture. In fresh hepatocytes, the three MDR modulators did not induce an increase in the intracellular retention of anthracycline as compared with controls (no MDR modulator). At 48 h of culture, the three test drugs increased doxorubicin intracellular accumulation. In contrast, daunorubicin retention was not modified, but that of its metabolites was increased. Within the concentration range tested, cyclosporine was the most potent modulator without dose-dependent activity. The activity rank order was cyclosporine > S 9788 > verapamil. Cyclosporine and S 9788 were as active in coincubation as in preincubation with anthracyclines. Verapamil had no action when incubated before the addition of anthracyclines. Cyclosporine and S 9788 had an effect on the intracellular retention of daunorubicinol used alone whereas verapamil did not. The action of cyclosporine and S 9788 on the retention of daunorubicinol proves that at least a part of the efflux of C-13 alcohol metabolites of anthracyclines is mediated by Pgp. This study shows that S 9788, cyclosporine, and verapamil are MDR modulators in hepatocytes with high-level Pgp expression. This study also demonstrates that hepatocytes are a potent tool for the study of the action of new MDR modulators on cytostatic drugs as well as on their metabolites.

Metabolism of doxorubicin, daunorubicin and epirubicin in human and rat hepatoma cells.

The metabolism of three anthracyclines, namely daunorubicin, doxorubicin and epirubicin, was studied in two human (HepG2 and HBG2) and one rat (FAO) hepatoma cell lines. Both species and substrate differences in anthracycline metabolism were retained by these hepatoma cell lines with regard to the aldoketoreductase pathway. However, glucuronidation of epirubicin normally found in vivo and in human hepatocyte cultures was not recovered in human hepatoma cells, further indicating that these cells express only some of the liver functions. Nevertheless, these cell lines could be a suitable model to investigate the still poorly understood aldoketoreductase activity.

Solid phase extraction and HPLC determination of spiramycin in plasma and vitreous concentrations.

A method for extracting spiramycin by an octadecylsilica cartridge is described for plasma or vitreous samples. The macrolide antibiotic is then measured by reversed-phase HPLC with UV detection. The limit of detection is estimated to be 50 ng/mL. The coefficient of variation for the procedure is 6.1% and 5.2% for the range of concentrations 0.2 micrograms/mL and 10 micrograms/mL respectively. By this method, pharmacokinetic profiles were performed for five adult patients. Spiramycin could be accurately measured in the vitreous humour, allowing the determination of antibiotic at its site of action.

Study of doxorubicin photodegradation in plasma, urine and cell culture medium by HPLC.

An HPLC method was used to monitor the chemical stability of doxorubicin-HCl (DXR) to light in plasma, urine and cell culture medium at room temperature. The results indicated that DXR was very unstable in cell culture medium and urine when exposed to light. It was more stable in plasma under the same conditions. In all the cases, the decrease in the amount of DXR is greatly dependent on light intensity (no noticeable degradation was observed after 8 h in the dark). These observations may be important for the correct interpretation of the effects and the toxicity of doxorubicin on cells incubated in cell medium, and for determination of urinary or plasma pharmacokinetic parameters.

Different cytotoxicity and metabolism of doxorubicin, daunorubicin, epirubicin, esorubicin and idarubicin in cultured human and rat hepatocytes.

Both cytotoxicity and metabolism of five anthracyclines, namely doxorubicin, daunorubicin, epirubicin, esorubicin and idarubicin, were investigated in primary cultures of both rat and human adult hepatocytes and, for comparison, in a rat liver epithelial cell line. Toxicity was assessed by morphological examination and measurement of lactate dehydrogenase leakage after 24 hr of treatment. The rank order of toxicity for both rat and human hepatocytes was esorubicin greater than doxorubicin = epirubicin greater than or equal to idarubicin greater than daunorubicin, and for rat epithelial cells: esorubicin greater than or equal to epirubicin greater than idarubicin = daunorubicin = doxorubicin. Human cells were around 2-fold less sensitive than rat hepatocytes to all anthracyclines. Anthracyclines and their metabolites were analyzed by HPLC. Differences in both the percentages and routes of metabolism were demonstrated between rat and human hepatocytes. The main metabolite was the 13-dihydro-derivative (-ol derivative) in both species from daunorubicin, idarubicin and esorubicin. Glucuronides of epirubicin and epirubicinol were found only in human hepatocytes. In addition, several unidentified metabolites were detected of esorubicin, idarubicin and daunorubicin in rat hepatocytes. In human hepatocytes, only one unknown metabolite from daunorubicin and doxorubicin was found to be formed by cells from a different donor. In spite of variations between individuals, human hepatocytes generally metabolized anthracyclines more actively than did rat hepatocytes. Rat liver epithelial cells were only able to convert daunorubicin and idarubicin, the two molecules which have the best affinity for the non-specific NADPH-dependent aldoketoreductase system. Three compounds (doxorubicin, epirubicin and esorubicin) were present in large amounts in the cells as the parent drug, another (idarubicin) as the 13-dihydro-derivative. This comparative study on cytotoxicity and metabolism of five anthracyclines in rat and human hepatocyte cultures emphasises species differences and the importance of this in vitro model system for further analysis of the metabolism and effect of anthracyclines.

[Kinetics of tobramycin in newborn infants in intensive care]. / Cinétique de la tobramycine chez le nouveau-né en réanimation.

Maximal and minimal tobramycin concentrations were studied in 16 preterm and 4 fullterm neonates on the last day of a four-day course of tobramycin (infusion of 2 mg/kg twice a day). Maximal concentrations were under 6 mg/l in all patients and minimal concentrations were above 2 mg/l in one-third of the preterm babies. Next, tobramycin pharmacokinetic parameters were studied in 4 fullterm and 4 preterm neonates following a first infusion of 2 mg/kg. Half-life was 4 hours in term babies and 7.75 hours in preterm babies. Mean clearance was 0.118 l/h/kg and 0.06 l/h/kg in fullterm and preterm babies respectively (significant difference). Our results show that the 2 mg/kg dosage is too low and that, in preterm neonates, the 12-hour interval between infusions is too short.