Cetuximab directly inhibits P-glycoprotein function in vitro independently of EGFR binding.

PURPOSE: Cancer chemotherapy typically combines anticancer drugs from different mechanisms of action. However, cancer cells could become resistant to chemotherapy via P-gp or other ATP binding cassette proteins. The objective of this study was to evaluate whether cetuximab, monoclonal antibody directed toward epidermal growth factor receptor, could increase intracellular concentration of conventional chemotherapy by interacting with P-gp. METHODS: Two human ovarian carcinoma (IGROV1) and two human embryonary kidney (HEK) cell lines, overexpressing or weakly expressing P-gp, were used. Their EGFR expressions were compared. Cetuximab effect on P-gp functionality was evaluated by measuring doxorubicin (P-gp fluorescent substrate) intracellular accumulation. Cetuximab ability to increase doxorubicin cytotoxicity was evaluated by MTT test. A quaternary structure model of the P-gp-Cetuximab complex was established. RESULTS: Exposure of cetuximab in therapeutic concentrations range with doxorubicin led to significant doxorubicin accumulation and reversion of doxorubicin resistance in P-gp expressing cells lines. Molecular modeling of P-gp-cetuximab interactions showed that cetuximab is able to bind P-gp extracellular part. CONCLUSIONS: Cetuximab increases a P-gp substrate intracellular accumulation in both P-gp expressing cell lines, independently of their EGFR expression. One hypothesis is that cetuximab binding on P-gp could hamper the conformational changes that occur during drugs efflux. Our results offer new possibilities of research on monoclonal antibodies influence in MDR phenomena.

Influence of the multidrug transporter P-glycoprotein on the intracellular pharmacokinetics of vandetanib.

Efflux transporters play an important role in the resistance of tumor cells against anticancer agents. Interaction between these transporters, including P-glycoprotein (P-gp), and drugs might influence their pharmacological properties and toxicities. The aim of this study was to investigate whether vandetanib (Caprelsa(®)), a small tyrosine kinase inhibitor, could interact with the multidrug transporter P-gp. Interaction of vandetanib with the P-gp was investigated using the parental cell line (IGROV1) and the P-gp doxorubicin-resistant (IGROV1-DXR) cell line, derived from the parental drug-sensitive IGROV1 cells. Cytotoxicity tests were assessed in both cell lines to examine the impact of P-gp on the cell survival after a vandetanib treatment. The effects of P-gp on vandetanib intracellular pharmacokinetics were investigated. To this aim, we developed a quantitative liquid chromatography tandem mass spectrometry to quantify vandetanib in cell medium. Results showed that overexpression of P-gp confers resistance to vandetanib in the IGROV1-DXR cell line. Using a LC-MS/MS assay validated in cell medium, cellular pharmacokinetic studies revealed that in cells overexpressing the P-gp intracellular concentrations of vandetanib were decreased compared to parental cell line. For the first time, vandetanib is described as a substrate of P-gp. In tumor cells, P-gp could be responsible for cellular resistance to vandetanib. It may be relevant to the clinical efficacy of vandetanib. Moreover, interaction of vandetanib with P-gp could modify the pharmacodynamics of other conventional chemotherapeutics, substrates of P-gp. It could impact on the overall response to anticancer therapy.

[Influence of hospitalisation on drug prescription in arterial hypertension and chronic conditions]. / Influence de l'hospitalisation sur la prescription médicamenteuse dans l'hypertension artérielle et les maladies chroniques.

OBJECTIVES: To evaluate the influence of hospitalization on drug prescription in chronic conditions. METHODS: Admission and discharge prescriptions from 92 patients consecutively admitted in a specialized department of the Assistance Publique-Hôpitaux de Paris hospital were recorded in a prospective two-month study. A Qualitative Therapeutical Score (QTS) was calculated as an estimation of qualitative modifications in the prescription. RESULTS: Patients admitted for an hospital stay of over 24h have more lines of prescription than patients admitted for an hospitalization shorter than 24h (5.7±4.2/d vs 2.9±2.5/d, P<0.01). For all the patients enrolled, the hospital stay is not associated with any change in the global number of treatments. However, in patients treated with antihyperstensive drug, the number of drug intakes decreases (2.6±1.5/d vs 1.9±1.4/d, P<0.05) as a consequence of an increase in the prescription of fixed-dose combinations. In patients with cardiovascular diseases, the QTS is higher and qualitative modifications are more often found in patients admitted for an hospital stay of over 24h than for those admitted for a an hospitalization shorter than 24h (0.57 vs 0.11; P<0.01 and 31% vs 11%; P<0.05 respectively). Antihypertensive drugs are the most represented drugs within these qualitative modifications. CONCLUSION: In patients with drug treatments for arterial hypertension or chronic conditions, hospitalization is not associated with quantitative but with qualitative modifications, especially for an over 24h hospital stay.

Inhibition of P-glycoprotein functionality by vandetanib may reverse cancer cell resistance to doxorubicin.

P-glycoprotein belongs to the ATP binding cassette transporters, responsible for the multidrug resistance of cancer cells. These transporters efflux hydrophobic drugs outside cells and decrease their therapeutic efficacy. The aim of this study was to investigate the effect of vandetanib, an oral tyrosine kinase inhibitor of EGFR, VEGFR 2 and RET kinases, on the functionality of P-gp after a 24h-treatment at therapeutic concentration (2µM), and its ability to increase the cytotoxicity of chemotherapeutic agents in multidrug resistance cancer cells. In this study we found that IGROV1-DXR and IGROV1-CDDP cells were resistant to doxorubicin and cisplatin respectively, compare to parental cell line IGROV1. The parental sensitive and the two resistant cell lines similarly expressed MRP1 and did not express BCRP. Moreover, in contrast to the IGROV1 and IGROV1-CDDP cells, IGROV1-DXR cell line overexpressed P-gp. Functional activity studies demonstrated that MRP1 was not functional and the MDR phenotype in IGROV1-DXR cells was linked to P-gp functionality. Results also showed that vandetanib reversed resistance to doxorubicin in IGROV1-DXR cells, but not to cisplatin in IGROV1-CDDP cells. After 24h of treatment, vandetanib increased the accumulation of rhodamine 123 and calcein AM, demonstrating a functional inhibition of the transporter. In IGROV1-DXR cell line, vandetanib reverse resistance to doxorubicin by inhibiting the functionality of P-gp. In conclusion, vandetanib should be an option for drug combination in patients already developing a P-gp mediated multidrug resistance.

[Contributions of the ex vivo human perfused placenta in the study of placental transfer of drugs]. / Apports de la perfusion ex vivo du cotylédon humain dans l'étude du passage placentaire des médicaments.

Perfused human placental lobule was developed during the 1970s. Only this model respects the anatomical features of the human placenta. This approach allows different technical conditions (concentrations of drugs…) without ethical problems. Limitations of this ex vivo model are detailed in this review, also its recent contributions in better understanding of placental passage of drugs.

Comparative effects of drugs on P-glycoprotein expression and activity using rat and human trophoblast models.

The drug efflux transporter P-glycoprotein (P-gp) is an active component of the placental barrier which protects the fetus against maternal xenobiotics. The goal of the study was to compare species difference between human and rat in terms of susceptibility to drugs at the level of the placental barrier using in vitro models in order to improve translation from rat to human. Effects of selected drugs (Aspirin, Methadone, a Cardiovascular Proprietary Compound, Thalidomide) on cytotoxicity and P-gp expression and activity were compared using human and rat trophoblast cultures. No direct cytotoxicity of drugs on trophoblasts was noted in both invitro models, but for Thalidomide a proliferative effect on human trophoblast primocultures was observed. All tested drugs induced changes towards P-gp; for each drug the same profile was noted in both human and rat trophoblast models except for Thalidomide. Observation of this similar response between these two in vitro trophoblast models is promising for assessment between P-gp expression and activity of drugs towards placental function.

Disposition of everolimus in mdr1a-/1b- mice and after a pre-treatment of lapatinib in Swiss mice.

The aim of this study was to document the in vivo transport of everolimus (inhibitor of mTOR) by P-glycoprotein (P-gp), and to investigate the influence of lapatinib (inhibitor of P-gp) on everolimus disposition. Pharmacokinetics of everolimus (0.25mg/kg) has been investigated after oral administration in mdr1a-/1b- mice compared to the wild type. Also, everolimus pharmacokinetics was characterized after oral administration on Swiss mice either alone or after 2 days of pre-treatment of lapatinib (200mg/kg). The influence of lapatinib pre-treatment on intestinal P-gp expression was investigated by Western blot analysis. The non-compartimental analysis was performed using Winonlin professional version 4.1 software (Pharsight, Mountain View, CA). The areas under the plasma concentration-time curve (AUC) were compared using Bailer's method. A significant 1.3-fold increase of everolimus AUC observed in mdr1a-/1b- mice suggested that everolimus is transported in vivo by intestinal P-gp in mice. In addition, a 2.6-fold significant increase of everolimus AUC with lapatinib pre-treatment as compared with the everolimus alone group was noticed. The elimination half-life was comparable (t(1/2)=5.3h vs. t(1/2)=4h). A 38.5% significant decrease of P-gp expression was observed in duodenum segment in lapatinib pre-treated group as compared with control group. In conclusion, lapatinib enhanced everolimus absorption by decreasing intestinal P-gp expression. An inhibition of CYP 450 could not be excluded. These results confirm the necessity of a therapeutic monitoring of everolimus combined with an inhibitor of the P-gp and CYP 450 like lapatinib in a future anti-tumor treatment.

Development and characterisation of a new model of rat trophoblasts.

The placenta plays a key role during pregnancy. In vitro models have proven to assess the role of placental transporters in the exchange of nutrients, waste products and the distribution of drugs between the maternal and fetal compartments. Therefore, a primoculture of Wistar rat trophoblasts from the labyrinth zone was developed and characterised. Expression of placental transporters including P-glycoprotein (P-gp) and bcrp was evaluated by western blot and their activity using different inhibitors. A time-dependent increase in P-gp expression was noted from primocultures Day 2 to Day 4 followed by a plateau thereafter, whereas bcrp expression was stable throughout the culture period. P-gp and bcrp expression was maintained after seven passages in primocultures and in cryopreserved trophoblasts (up to 3 freezings and 10 passages). Activity of efflux transporters was confirmed in both placental primocultures and cryopreserved trophoblasts by an approximately 60% inhibition with cyclosporin A and valspodar for P-gp and 55% with elacridar for bcrp. In sum, this new in vitro model seems promising for a better understanding of the role of P-gp and bcrp in the toxicity of drugs during pregnancy and could be considered as an additional step towards the minimization of animal testing during drug development.

Increased expression of MDR1 mRNAs and P-glycoprotein in placentas from HIV-1 infected women.

P-glycoprotein transports several compounds including protease inhibitors, actually used in the clinical management of HIV-1 infection. Since P-glycoprotein is expressed in placental trophoblasts, its efflux activity could interfere with placental transfer of antiretrovirals. The purpose of this study was to investigate the expression of P-gp-encoding MDR1 gene and P-gp itself in full-term placentas from uninfected (n=35) and HIV-1 infected women (n=24). MDR1 transcripts were quantified by real-time PCR using relative (MDR1 normalized upon 28S levels) and absolute (copy number) determinations. P-glycoprotein localization and expression were evaluated by immunohistochemistry and western blot analysis, respectively. Relative or absolute PCR quantification showed a significant 3.3-fold (p<0.0009) or 3.7-fold (p<0.0002) mean increase in MDR1 placental transcription in HIV-infected compared to non-infected women, respectively. Ratios of individual HIV-positive values to HIV-negative mean ranged from 0.1 to 21.8. Moreover a significant 2.5-fold increased expression of immunoreactive P-glycoprotein was evidenced in placentas from HIV-infected women (p<0.0001). This MDR1 overexpression was observed in a similar extent in placentas from pregnant women treated with Zidovudine alone or in combination with Nelfinavir and/or Lamivudine. Our findings suggest that P-glycoprotein in placentas from HIV-infected women would contribute to modulate the materno-fetal transport of antiretrovirals across the placental barrier and consequently diminish fetal exposure to these compounds.

Evaluation of pharmacokinetic interaction between cetirizine and ritonavir, an HIV-1 protease inhibitor, in healthy male volunteers.

BACKGROUND: Serious adverse effects have been observed with some non-sedative H1-antihistamines (terfenadine and astemizole) when they were associated with drugs known to inhibit their metabolism. However, this is not a class effect, and this interaction should be considered on a case-by-case basis. The aim of this study was to evaluate the potential of pharmacokinetic interaction between cetirizine and ritonavir, the most potent cytochrome P450 (CYP) inhibitor. METHODS: An open-label, single-center, one-sequence crossover pharmacokinetic study was conducted in three running periods: cetirizine (CTZ) alone, ritonavir (RTV) alone and then CTZ plus RTV. For each period, steady-state pharmacokinetics were obtained. RTV and CTZ plasma concentrations were determined using validated liquid chromatography methods. The statistical method was based on a 90% confidence interval (CI) for the ratio of population geometric means (combination/drug alone) for each drug and for each parameter [area under the plasma concentration versus time curve (AUC(0-tau,ss)), value of maximum plasma concentration (C(max,ss))] and compared to bioequivalence ranges 80-125% and 70-143% for AUC(0-tau,ss) and C(max,ss), respectively. RESULTS: Among the 17 male subjects enrolled (26.4 +/- 8.6 years), 16 completed the study (1 withdrawal after the first period). The RTV pharmacokinetic parameter values were not affected by CTZ co-treatment. With RTV, a 42% increase in the CTZ AUC(0-tau,ss) (3406 versus 4840 microgh/l, 90% CI of 128-158%), a 53% increase in the CTZ elimination half-life (7.8 h versus 11.9 h, P = 0.001), a slight increase (15%) in the CTZ apparent volume of distribution (V(d,ss)/f) (34.7 l versus 39.8 l, P = 0.035), a 29% decrease in the CTZ apparent total body clearance (49.9 ml/min versus 35.3 ml/min, P < 0.001) and bioequivalent C(max,ss) (374 microg/l versus 408 microg/l) were observed. No serious drug related adverse effects were notified. CONCLUSIONS: CTZ does not significantly affect the pharmacokinetic parameters of RTV, and the association does not, thus, require a modification of the dosage of the protease inhibitor. The increased extent of exposure to CTZ in healthy subjects, in the presence of RTV administered at high doses, remained in the same range as previously observed in the elderly or in mildly renally impaired subjects.

P-glycoprotein expression of the human placenta during pregnancy.

OBJECTIVE: To investigate whether the placental expression of P-glycoprotein shows a quantitative difference during pregnancy. STUDY DESIGN: Villous tissue was collected from chorionic villus samples (13-14 weeks of gestation; n = 3 and 20-25 weeks of gestation; n = 4) and from full-term placentas (38-41 weeks of gestation; n = 28). P-glycoprotein was detected by western blot analysis and quantified by densitometry. RESULTS: We showed for the first time a significant and progressive two-fold decrease in the mean expression of P-glycoprotein between early and late samples, with a major overlap of values. CONCLUSION: As P-glycoprotein appears to be involved in drug extrusion, these data suggest that the placenta's ability to protect the fetus from xenobiotics is greater in early pregnancy than at term.

A 6-month prospective survey of cutaneous drug reactions in a hospital setting.

BACKGROUND: Few prospective studies are available on the incidence and analysis of the characteristics of adverse cutaneous drug reactions in hospital settings. OBJECTIVES: A 6-month prospective study was managed in our hospital among hospitalized patients to: (i) evaluate the incidence of cutaneous allergic reactions from systemic drugs; (ii) study characteristics of patients with cutaneous drug reactions; (iii) describe the adverse cutaneous reactions; and (iv) evaluate drug reaction imputability and preventability. METHODS: All suspected allergic cutaneous reactions to systemic drugs were collected during a 6-month period (November 2000 to May 2001). Exhaustivity of recording was ensured by regular dissemination of information about this study to the practitioners; a simple method for inclusion by fax was established. Inclusion criteria were suspected cutaneous allergic reactions induced by systemic drugs responsible for hospitalization or developed during hospitalization. A physical examination was done by a dermatologist who completed a standardized questionnaire. Requested information included patient characteristics (associated disorders, severity score), drug intake (list and chronology of the drug intake during the 3 weeks preceding the adverse reaction) and characteristics of the skin reaction (type, course). A group comprising dermatologists and pharmacologists evaluated the drug imputability and preventability. RESULTS: Forty-eight cases were collected. A prevalence of 3.6/1000 among hospitalized patients was estimated. The prevalence rate was higher in patients hospitalized in medical departments (0.5%) than in surgical departments (0.01%) (P < 0.001). The cases were mostly recruited in departments of infectious diseases and dermatology. The most frequent associated disorders were: human immunodeficiency virus (HIV) infection (19%), connective tissue disease (10%) and viral or autoimmune hepatitis (12%). Of these patients, 31% had had a previous immunological drug reaction. Adverse cutaneous drug reactions were principally exanthematous (56%). Reactions were considered severe in 34% of cases because they were responsible for hospitalization (18%), increased the duration of hospitalization (14%) or were life threatening (2%). Principal imputable drugs were antibiotics, mainly penicillins. An imputability score was likely in 56% of cases, but it was only possible to conclude definitively in 44% of patients. In 15% of the cases, the side-effect was considered to be preventable. CONCLUSIONS: This study finds a lower incidence than other studies that reported an incidence of 2% of cutaneous drug reactions in hospitalized patients, but only allergic adverse cutaneous reactions induced by systemic drugs were collected in this study. The previous studies were principally done in selected patients hospitalized only in general internal medicine or medical divisions. Our results confirm some data already known about skin drug reactions: HIV infection as a risk factor (P < 0.0001), high prevalence of adverse cutaneous reactions due to antibiotics, and difficulty in ascertaining the imputability of a drug. A high proportion (34%) of these reactions was severe and 15% were avoidable; these two facts justify the development of an intensive programme of clinical pharmacology.

Comparison of lithium concentrations in red blood cells and plasma in samples collected for TDM, acute toxicity, or acute-on-chronic toxicity.

BACKGROUND: Lithium salts (Li+) are still one of the most appropriate treatments in manic-depressive disorders. Since Li+ has a narrow therapeutic index, plasma levels must be closely monitored to verify maintenance pharmacotherapy, to prevent side effects, to evaluate compliance and to avoid increasing rates of relapse. Although it has been reported that Li+ concentrations in red blood cells (RBC) should be a better indicator of brain levels, therapeutic drug monitoring (TDM) of Li+ is not based on its routine assessment. OBJECTIVE: The aim of this retrospective study was to compare Li+ concentrations in RBC and plasma and the calculated ratio (LiR= RBC/plasma concentrations) in the three groups of patients. METHODS: During the past 3 years, 309 Li+ measures were collected corresponding to 165 patients classified into three subgroups (TDM, acute or acute-on-chronic intoxication). Li+ plasma (Cplasma) and RBC (CRBC) concentrations were determined by atomic absorption spectrophotometry. RESULTS: Results showed that Li+ concentrations in plasma are significantly correlated to Li+ concentration in RBC (r=0.81, P<0.0001). Although a wide inter- and intra-variability was found, Cplasma, CRBC and LiR were statistically different in the three groups. Compared with TDM, Cplasma was more elevated in cases of acute intoxication whereas Li+ accumulated preferentially in RBC in cases of acute-on-chronic intoxication. CONCLUSION: This study shows the interest of determining Li+ in RBC and plasma for TDM, and that LiR could be a sensitive marker of intoxication and of Li+ impregnation.

Decrease of intestinal P-glycoprotein activity by 2n-propylquinoline, a new oral treatment for visceral leishmaniasis.

Drugs currently available for visceral leishmaniasis treatment are potentially toxic, have to be administered by parenteral route and frequently give rise to drug resistance, due to the involvement of P-glycoproteins (P-gp) in Leishmania. The purpose of this study was to investigate a possible inhibitory effect of 2n-propylquinoline (2nPQ) on P-gp activity. 2nPQ is a new oral anti-leishmanial drug that has demonstrated its efficacy in BALB/c infected mice with Leishmania donovani [Antimicrob. Agents Chemother. 37 (1993) 859]. Rat everted gut sacs and human intestinal Caco-2 cell lines were used to study the effect of 2nPQ on P-gp activity. Our results demonstrate an inhibitory effect of 2nPQ on the P-gp activity with two P-gp substrates (rhodamine 123 and digoxin), two P-gp inhibitors (cyclosporin A and verapamil), and in two different species. Alone or associated with other active drugs, 2nPQ would be very useful to control Leishmania Multi-Drug-Resistance and intestinal P-gp in humans with kala-azar.

Determination of the human cytochrome P450s involved in the metabolism of 2n-propylquinoline.

1 2n-Propylquinoline (2nPQ) is a newly developed drug for visceral antileishmaniasis and its activity has been previously evaluated in mice following oral administration. The study was carried out to investigate the kinetic formation of 2nPQ metabolites and to characterize the human liver CYP forms involved in its oxidative metabolism. 2. The inhibition of 2nPQ metabolite formation by specific substrates or inhibitors of CYP forms and correlation studies were performed in human liver microsomes. 2nPQ biotransformation was then studied in human lymphoblasts expressing specific CYPs and microsomal epoxide hydrolase. 3. Three major metabolites were produced by human liver microsomes and their structures were identified by ESI-LC/MS: dihydroxy-2n-propylquinoline, 3'-hydroxy-2n-propylquinoline and 1'-hydroxy-2n-propylquinoline. An intermediary metabolite, epoxy-2n-propylquinoline, formed by CYP was also biotransformed by microsomal epoxide hydrolase into dihydroxy-2n-propylquinoline. 4. 2nPQ oxidation follows Michaelis-Menten kinetics. In human liver microsomes, its metabolism was extremely inhibited by pilocarpine, coumarin and diethyldithiocarbamate. From a panel of 12 human liver microsome samples, the rate of 2nPQ oxidation was highly correlated with the activities of CYP2A6 and CYP2E1. Human lymphoblasts expressing specific CYPs showed the involvement of CYP2A6, CYP2E1 and CYP2C19. 5. The results indicate that 2nPQ metabolites are 3'- and 1'-hydroxylated by human liver microsomes and an epoxy-2n-propylquinoline is biotransformed into a dihydroxy-2n-propylquinoline by microsomal epoxide hydrolase.

[Medicinal iatrogenics in hospitals. A survey on a given day]. / Iatrogénie médicamenteuse à l'hôpital. Enquête un jour donné.

OBJECTIVE: Medicinal iatrogenics are responsible for hospital admissions but also occur in hospitals. In view of the lack of knowledge, prevalence and nature of the adverse drug-related events (ADE) in the Bichat-Claude Bernard hospital group in Paris, and because of the potential severity of the latter, the Local drug committee has decided to develop a policy to manage these risks. METHOD: The first stage consisted in a transversal study on a given day in the departments in which patients are hospitalised for more than 24 hours, in order to assess the prevalence, severity and preventability of ADE and to search for factors of risk. RESULTS: 107 ADE were observed in 89 patients on the day of the survey (9.9% global prevalence of ADE [CI 95%: 8.8% - 11.0%]). Among the latter, 57 patients had exhibited at least one adverse event during their hospitalisation, i.e., a prevalence of 6.3% ([CI 95%: 4.7% - 7.9%] ). Two thirds of these patients were hospitalised in medical departments. These nosocomial ADE (nosocomial adverse drug events) were serious or severe in 73% of cases and 25% could have been avoided. The only clearly identified risk factor was the number of drugs prescribed. CONCLUSION: This review has drawn the attention of the medical and paramedical community to the need to define vigilance markers, and has provided some elements of response that should be further completed by a prospective cohort study.

Chronic administration of verapamil, ketoconazole and carbamazepine: impact on immunological parameters.

Inhibitors of P-glycoprotein (P-gp) (verapamil) or cytochrome P-450 (ketoconazole) may reduce IL2 production and T lymphocyte proliferation in vitro. We have examined the effects of chronic oral administration of these drugs and of the cytochrome P450 inductor, carbamazepine, on the hematological and immunological parameters of mice. We found no changes after giving the mice 0.12 mg verapamil, 0.85 mg ketoconazole, or 0.514 mg carbamazepine per mouse for 4 weeks (5 days/week). But giving the drugs for an additional 7 weeks at 0.6 mg (verapamil), 4.25 mg (ketoconazole) or 2.57 mg/mouse (carbamazepine), resulted in significant decreases in monocytes in the verapamil treated group (-51%) and in CD4+ cells in the carbamazepine group (-35%). Chronic oral administration of these drugs reduced the lymphocyte counts of mice by 10-18% and their NK counts by 10-16%. These changes could be due to changes in P-gp function in the transport of IL2, with decreases caused by verapamil and ketoconazole.

Determination of N-acetylation phenotype using caffeine as a metabolic probe and high-performance liquid chromatography with either ultraviolet detection or electrospray mass spectrometry.

A rapid, sensitive method using liquid chromatography-electrospray mass spectrometry (LC-ES-MS) was developed and evaluated for the simultaneous quantitative determination of caffeine metabolites 1U, 1X and AAMU in human urine. This method involved a simple dilution of urine samples. The chromatographic separation was achieved on a C18 reversed-phase column using a gradient of acetonitrile in 2 mM, pH 3.0 ammonium formate as mobile phase. After ionisation in an electrospray source, mass spectrometric detection was performed in the negative ion, selected ion monitoring mode. This method yielded acceptable accuracy and precision within the range 0.25-50 microg/ml. This analytical method was applied to investigate the N-acetylator phenotype of HIV-infected patients and compared with high-performance liquid chromatography with UV detection. Its specificity was better, which appeared to be absolutely necessary to prevent errors in metabolic ratios and phenotype interpretation.

Maternal-fetal transfer of saquinavir studied in the ex vivo placental perfusion model.

OBJECTIVE: This study was undertaken to determine the placental transfer of the human immunodeficiency virus protease inhibitor saquinavir. STUDY DESIGN: An ex vivo perfused human placental cotyledon model was used. Ten placental perfusion studies were performed, with concentrations of saquinavir in the maternal compartment ranging from 322 to 2197 ng/mL (within reference therapeutic ranges). Drug concentrations were determined by high-performance liquid chromatography. RESULTS: The mean (+/- SD) fetal transfer rate of saquinavir was 1.8% +/- 1.6%, and the mean (+/- SD) clearance index was 0.05 +/- 0.05. A mean (+/- SD) of 1.6% +/- 3.1% of the perfused saquinavir was retained by the cotyledon. The small amount of saquinavir that crossed the placenta corresponded to the fraction not bound to human serum albumin. CONCLUSION: The low rate of placental transfer of saquinavir suggests that use of this antiretroviral drug by pregnant women may not lead to significant exposure of the fetus.

Effect of chronic renal failure on the expression and function of rat intestinal P-glycoprotein in drug excretion.

BACKGROUND: In chronic renal failure, the renal excretion of certain drugs is dramatically reduced. To determine whether other routes of drug elimination, such as secretion through the intestinal barrier by intestinal P-glycoprotein can be altered, we compared P-glycoprotein activity, P-glycoprotein protein content, and P-glycoprotein mRNA levels in intestine of control and chronic renal failure rats. METHODS: Chronic renal failure was surgically induced in rats by partial (7/8) nephrectomy. After 5 weeks, intestinal transport of rhodamine 123, a P-glycoprotein substrate, was carried out using an in vitro model of everted gut sacs. P-glycoprotein protein content was quantified by enzyme-linked immunosorbent assay and P-glycoprotein mRNA expression was evaluated by semi-quantitative reverse transcriptase polymerase chain reaction. RESULTS: A decrease of intestinal rhodamine 123 transport was observed in chronic renal failure rats, pointing to an inhibition of P-glycoprotein activity. Transport was inhibited in both sham-operated rats and rats with chronic renal failure by verapamil and cyclosporin A, but relative inhibition vs baseline was less marked in chronic renal failure than in sham-operated rats. In contrast, no significant differences in levels of P-glycoprotein protein or mRNA were observed between the two groups. CONCLUSIONS: Intestinal secretion of rhodamine 123 is mainly mediated by P-glycoprotein. It was reduced in rats with chronic renal failure, reflecting reduced intestinal drug elimination via a decrease in P-glycoprotein transport activity rather than via protein underexpression.