Atomistic Picture of Fluorescent Probes with Hydrocarbon Tails in Lipid Bilayer Membranes: An Investigation of Selective Affinities and Fluorescent Anisotropies in Different Environmental Phases.

By reverting to spectroscopy, changes in the biological environment of a fluorescent probe can be monitored and the presence of various phases of the surrounding lipid bilayer membranes can be detected. However, it is currently not always clear in which phase the probe resides. The well-known orange 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbo-cyanine perchlorate (DiI-C18(5)) fluorophore, for instance, and the new, blue BODIPY (4,4-difluoro-4-bora-3 a,4 a-diaza- s-indacene) derivative were experimentally seen to target and highlight identical parts of giant unilamellar vesicles of various compositions, comprising mixtures of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM), and cholesterol (Chol). However, it was not clear which of the coexisting membrane phases were visualized (Bacalum et al., Langmuir. 2016, 32, 3495). The present study addresses this issue by utilizing large-scale molecular dynamics simulations and the z-constraint method, which allows evaluating Gibbs free-energy profiles. The current calculations give an indication why, at room temperature, both BODIPY and DiI-C18(5) probes prefer the gel (So) phase in DOPC/DPPC (2:3 molar ratio) and the liquid-ordered (Lo) phase in DOPC/SM/Chol (1:2:1 molar ratio) mixtures. This study highlights the important differences in orientation and location and therefore in efficiency between the probes when they are used in fluorescence microscopy to screen various lipid bilayer membrane phases. Dependent on the lipid composition, the angle between the transition-state dipole moments of both probes and the normal to the membrane is found to deviate clearly from 90°. It is seen that the DiI-C18(5) probe is located in the headgroup region of the SM/Chol mixture, in close contact with water molecules. A fluorescence anisotropy study also indicates that DiI-C18(5) gives rise to a distinctive behavior in the SM/Chol membrane compared to the other considered membranes. The latter behavior has not been seen for the studied BODIPY probe, which is located deeper in the membrane.

Is there a benefit of mechanical thrombectomy in patients with large stroke (DWI-ASPECTS ≤ 5)?

BACKGROUND AND PURPOSE: Whether to withhold mechanical thrombectomy when the diffusion-weighted imaging (DWI) lesion exceeds a given volume is undetermined. Our aim was to identify markers that will help to select patients with large DWI lesions [DWI-Alberta Stroke Program Early Computed Tomography Score (DWI-ASPECTS) ≤ 5] that may benefit from thrombectomy. METHODS: From May 2010 to November 2016, 82 acute ischaemic stroke patients with DWI-ASPECTS ≤5 (43 men, 64.6 ± 14.4 years, National Institutes of Health Stroke Scale 18.4 ± 5.4) treated with state-of-the-art mechanical thrombectomy were studied. Thrombectomy alone was performed in 28 (34%) and bridging therapy in 54 (66%) patients. Recanalization was defined as a thrombolysis in cerebral infarction score 2B-3 and significant hemorrhagic transformation as parenchymal haematoma type 2 (European Cooperative Acute Stroke Study 3 classification). Pretreatment variables were compared between patients with a good (modified Rankin Scale 0-2) and a poor (modified Rankin Scale 3-6) neurological outcome at 3 months. RESULTS: Overall, 28 patients (34%) achieved good neurological outcome at 3 months. Recanalizers were significantly more likely to achieve good outcome (61% vs. 7.3%, P < 0.0001), had lower mortality (24% vs. 49%, P = 0.03) and similar rates of parenchymal haematoma type 2 (9.8% vs. 7.3%, P = 1) compared to non-recanalizers. Regression modelling identified DWI-ASPECTS >2 [odds ratio (OR) 6.93; 95% confidence interval (CI) 1.05-45.76, P = 0.04), glycaemia ≤6.8 mmol/l (OR 4.05; 95% CI 1.09-15.0, P = 0.03) and thrombolysis (OR 3.67; 95% CI 1.04-12.9, P = 0.04) as independent predictors of good neurological outcome. CONCLUSIONS: In patients with DWI-ASPECTS ≤5, two-thirds of patients experienced good neurological outcome when recanalized by state-of-the-art thrombectomy, whilst only one in 14 non-recanalizers achieved similar outcomes. Pretreatment markers of good neurological outcomes were DWI-ASPECTS >2, intravenous thrombolysis and glycaemia ≤6.8 mmol/l.

Major histocompatibility complex-linked social signalling affects female fertility.

Genes of the major histocompatibility complex (MHC) have been shown to influence social signalling and mate preferences in many species, including humans. First observations suggest that MHC signalling may also affect female fertility. To test this hypothesis, we exposed 191 female horses (Equus caballus) to either an MHC-similar or an MHC-dissimilar stimulus male around the time of ovulation and conception. A within-subject experimental design controlled for non-MHC-linked male characteristics, and instrumental insemination with semen of other males (n = 106) controlled for potential confounding effects of semen or embryo characteristics. We found that females were more likely to become pregnant if exposed to an MHC-dissimilar than to an MHC-similar male, while overall genetic distance to the stimulus males (based on microsatellite markers on 20 chromosomes) had no effect. Our results demonstrate that early pregnancy failures can be due to maternal life-history decisions (cryptic female choice) influenced by MHC-linked social signalling.

Outcome after urgent microvascular revision of free DIEP, SIEA and SGAP flaps for autologous breast reconstruction.

INTRODUCTION: Microvascular complications after free flap breast reconstruction are devastating, and revision of a compromised breast reconstruction is very challenging. The aim of this study was to review the different characteristics of urgent microvascular revision in DIEP, SIEA and SGAP flaps and to evaluate the final outcome after revision. MATERIALS AND METHODS: A retrospective chart review was performed for all patients who underwent an autologous breast reconstruction with a DIEP, SIEA or SGAP flap at the University Hospitals of Leuven between August 1997 and December 2013. The number of revisions, time to revision, reason for revision, and outcome after microvascular free flap revision were analysed. RESULTS: A total of 1562 free flaps were evaluated during the study period, of which 4.42% required urgent exploration. DIEP flaps (3.38%) had a statistically significant lower revision rate than SIEA flaps (11.76%) and SGAP flaps (8.42%). Venous insufficiency was the main reason for revision of DIEP flaps (86.7%) and SGAP flaps (62.5%). SIEA flaps mostly failed because of an arterial problem (62.5%). SIEA flaps (62.5%) had a higher revision failure rate than DIEP flaps (37.8%) and SGAP flaps (12.5%). We found a statistically significant difference (p < 0.001) in the outcome of revision in DIEP flaps in correlation to the time to revision. Our overall flap failure rate was 1.79% (DIEP 1.28%; SIEA 7.35%; SGAP 1.05%). CONCLUSIONS: The DIEP flap remains the most reliable flap for microvascular breast reconstructions. SIEA flaps are only performed when no suitable perforator for a DIEP flap is present. Multiple revisions are no longer performed, as the outcome after more than one revision is very disappointing. The difference in reason for revision between the different flaps led to the introduction of some technical refinements.

[Suicide attempts by young adolescents: epidemiological characteristics of 517 15-year-old or younger adolescents admitted in French emergency departments]. / Tentatives de suicide: étude descriptive d'une cohorte de 517 adolescents de moins de 15 ans et 3 mois.

Adolescents who attempt suicide are a major concern. A growing body of literature seeks to explain this phenomenon and to identify its predictive factors, but relatively little information is available and children and adolescents under 15 years of age who present to general hospitals because of a suicide attempt. This study aimed to describe the demographic, social, medical, and psychological characteristics of a large sample of 517 French adolescents aged not more than 15 years 3 months. A second purpose was to measure observance of psychological care in a 1-year follow-up. Third, we aimed to document the reattempt rate during the follow-up in this population of young adolescents. Following the French official recommendations, a systematic 72-h hospitalization as well as a somatic, social, and psychological assessment was proposed to every suicide attempter after his or her admission to the emergency department. The adolescent was followed for 1 year after the suicide attempt, called the index episode. This follow-up was organized by two physicians, one of whom was not associated with the care of any of the patients. It consisted in seeking regular information as well as organization and/or optimization of the patient's psychological care, which was delivered in dedicated structures for adolescents, in outpatient care by a psychiatrist, or in an adolescent psychiatric inpatient care unit. In case of a repeated suicide attempt or persistence of alarming symptoms, this follow-up was prolonged for 1 more year. Patient data were compiled by experienced clinicians during initial assessment and alongside the 1-year follow-up through patient self-reports, but also through interviews with informants (family members, social professionals) and clinical sources (general practitioner, psychiatrist, etc.). The areas covered were the characteristics of the index episode, those of the population at the time of the index episode, as well as those of the 1-year follow-up including observance to the care and potential repetition of the suicide attempt. The mean age was 14 years with a minimum of 7 years 9 months. The vast majority of the population was female (86.1%), less than one-third lived with both parents, and 27% had academic problems. The most frequent means of suicide attempt was medication (83.9%), 92.6% of adolescents were hospitalized following the index episode, only 7.5% of them were admitted to adolescent psychiatric unit inpatient care following the initial care. Psychiatric evaluation was documented for 93.3% of the adolescents. Half (n=222) had at least one symptom of a psychiatric disorder. One-year follow-up data were available for 394 adolescents: 40 had not yet completed the year and 83 were lost to follow-up. Among the analyzable population of 391 adolescents, 35.3% were optimally observant of the care proposed and 21.4% did not observe treatment. Fifty-nine youths (15%) were referred to the hospital because of a repeated suicide attempt. Two of the patients who repeated the suicide attempt within the year had died. The findings from this study are informative with regard to prevention and intervention efforts with suicidal young and very young adolescents. First, repetition of the suicide attempt in young adolescents is not rare since nearly 15% of the cohort were repeaters within the year following the index episode. Nevertheless, intensive care and follow-up resulting in good attendance appeared to have a positive impact on the repetition of the suicide attempt.

A randomized, double-blind, placebo-controlled study of benfluorex in HIV-infected patients with insulin resistance or impaired glucose tolerance.

PURPOSE: We previously reported a beneficial effect of benfluorex (BFL) on oral glucose tolerance test (OGTT) and visceral fat mass in an open-label study conducted in 60 HIV-infected patients. The objective of this study was to assess whether administration of BFL compared to placebo (PBO) improves insulin resistance (IR) in HIV+ patients with HAART- induced lipodystrophy. METHOD: 22 HIV-infected patients with IR or impaired glucose tolerance were double-blind randomly assigned to receive BFL 3 tablets/day or PBO for 24 weeks. Efficacy assessments included OGTT, abdominal computed tomography, and the measurement of fasting lipids. RESULTS: Change of median insulin AUC was -53.0 microIU/mL (IQR, -126.0 to -12.7) in the BFL group vs. +33.6 microIU/mL (IQR, 7.0 to 115.6) (p = .01) in PBO group. Weight decreased significantly in the BFL group (-2 kg +/- 2.6; IQR, -6.8 to 2.0) compared to the PBO group (0.8 kg +/- 1.7; IQR, -2.0 to 0.5) (p = .02). No significant changes in visceral or subcutaneous fat mass and plasma lipid level were observed between the two groups. CONCLUSION: Added to antiretroviral therapy, a 6-month therapy with BFL improved insulin sensitivity but is not sufficient to reduce significantly visceral fat mass.

Aggressive cutaneous squamous cell carcinoma associated with prolonged voriconazole therapy in a renal transplant patient.

A 69-year-old man, with a history of end-stage renal disease due to polyarteritis nodosa, followed by invasive pulmonary aspergillosis secondary to cyclophosphamide and corticosteroids, received a renal transplant 2 years ago under prophylactic treatment with voriconazole. Because of the severity of the aspergillosis, it was decided to continue voriconazole for a prolonged period. Eighteen months after transplantation, the patient developed a severe facial phototoxic reaction. A few months later, he developed multiple actinic keratoses and a large, rapidly expanding, poorly differentiated squamous cell carcinoma (SCC) with perineural invasion and metastatic lymph nodes, necessitating radical surgery and radiotherapy. Voriconazole therapy has been suggested to be involved in the development of multi-focal invasive SCC when complicated by a phototoxic reaction. Therefore, an alternative antifungal prophylaxis regimen (for instance with posaconazole) should be considered when evaluating patients for solid organ transplantation who are at high risk for the development of cutaneous malignancies.

[Breast reconstruction after prolonged tissue expansion. About 247 cases]. / Reconstruction mammaire prothétique après expansion préalable prolongée. A propos de 247 cas.

SUBJECT: The purpose of this study was to analyse the complications and the aesthetic results in case of slow tissue expansion in prosthetic breast reconstruction. PATIENTS AND METHODS: We tracked 237 patients representing 247 mammary reconstructions operated between 1992 and 2004. These patients were distributed in two series, a series of 148 operated mammary reconstructions between 1992 and 2000 and a series of 99 reconstructions operated between 2001 and 2004. For every reconstruction, we analysed the progress of the expansion, the complications and the quality of the aesthetic results according to the radiotherapy and the type of implant used. RESULTS: The radiotherapy increases the risk of failure of the breast reconstruction and degrades the quality of the aesthetic results. Capsular contractures are rare and their frequency does not depend on the irradiation. Prosthesis infections and exposure are more frequent on irradiated ground. DISCUSSION: The tissue expansion in prosthetic breast reconstruction is a technique studied well in the literature, but few authors use a chronic expansion and compare the long-term results according to the radiotherapy. If our study confirms the noxious role of the radiotherapy as for the complications and for the aesthetic aspect of the results, it is not a question for us of an absolute contraindication. The weak rate of capsular contracture is attributable to the chronic character of the expansion, which allows the maturation of the capsule. The use of silicone gel implants decreases the deflations but does not improve the results. CONCLUSIONS: The radiotherapy increases the risks of failure of the tissulaire expansion and decreases the quality of the aesthetic results. The chronic character of the expansion allows to obtain a rate of capsular contracture weak, even on irradiated ground. The silicone gel implants make it possible to obtain a perennial result.

Abdominal aplasia cutis congenita: case report and review of the literature.

A case of bilateral abdominal aplasia cutis congenita without skull defect is reported and was treated successfully by a combination of allografts and growth factors delivered by allogenic cultured keratinocytes.

Target-controlled sedation-analgesia using propofol and remifentanil in women undergoing late termination of pregnancy.

Twenty-one women undergoing termination of pregnancy for severe fetal abnormality received remifentanil and propofol using a target-controlled infusion system and were studied prospectively. Target concentrations were initially set at 1 ng.mL(-1) for remifentanil and 0.8 microg.mL(-1) for propofol. Remifentanil concentration was adjusted to obtain visual analog scores <50 mm with preservation of ventilation. Visual analog scores assessed by the patients and physiologic data were recorded every 15 min until delivery. The median duration of administration was 150 min [10th-90th centiles: 42-282 min). Visual analog scores decreased within the first 5 min (P < 0.05) and remained under 50 mm for 91.7% of time. The median rate of infusion of remifentanil was 0.056 microg.kg(-1) min(-1) [10th-90th centiles: 0.037-0.15 ng.mL(-1)]. At delivery, the median target concentration was 2.2 ng.mL(-1) [10th-90th centiles: 1.25-4 ng.mL(-1)] for remifentanil and 0.8 microg.mL(-1) [10th-90th centiles: 0.32-1.12 microg.mL(-1)] for propofol. Remifentanil requirements were statistically correlated to gestational age, parity and duration of labor. No episodes of ventilatory depression, nausea, vomiting or pruritus were noted. Patients scored analgesia as excellent in 12 cases, good in 7 cases and moderate in 2 cases. Further studies are required to determine the place and the best regimen of remifentanil infusion for pain management in labor in those cases when epidural analgesia is contraindicated.

Declared hepatitis C screening strategies in blood recipients in French hospitals.

AIMS OF THE STUDY: In spite of official recommendations and measures in France, screening strategies of hepatitis C performed in the field of transfusion are not clearly known. The aim of this study is to describe the screening strategies before and after the current year of the transfusion in blood recipients in several French medical departments and hospitals. MATERIALS AND METHODS: A qualitative study using the key informant technique was carried out. A sample of 179 departments and 64 hospitals in charge of patients transfused with low or high-volumes of homologous blood products was constituted. The key informants were asked about the number of homologous blood products, the number of recipients transfused in the hospital, the volume of transfusion performed, the existence of a single defined screening strategy, the time of prescription of the biological tests (before or after transfusion), the tests performed on cryopreserved blood samples, and the indications of the transfusion. RESULTS: The main screening strategy was HCV serology (second or third generation of enzyme immunoassays) with transaminase assessments before and after transfusion in 14% of the declared screening strategies. Screening tests were more frequently prescribed after transfusion, in at least 64% of the declared screening strategies according to the volume of transfusion. HCV serology was the common test prescribed in 61 and 50% of the screening strategies for low and high-volume transfusion respectively. The screening strategies showed a large heterogeneity combining HCV serology, transaminase assessment, before or after transfusion. CONCLUSION: A great heterogeneity of screening strategies was found. The most frequent was HCV serology with transaminase assessment before and after transfusion. Recommendations on screening strategies are needed in order to limit practice heterogeneity. This study will help building a cost-efficacy model in order to guide public health decision making.

[Femoral shaft fractures in the elderly treated by intramedullary nailing]. / Fractures diaphysaires fémorales au-delà de 70 ans.

PURPOSE OF THE STUDY: Little work has been devoted to femoral shaft fractures in the elderly, contrasting with the data available for proximal neck or trochanteric fractures. The purpose of this study was to determine the epidemiological and clinical features of femoral shaft fractures in the elderly from a retrospective series of 58 patients who underwent locked intramedullary nailing procedures with Grosse and Kempf (GK) or long gamma (GL) nails. MATERIAL AND METHODS: The series included 38 women and 20 men, mean age 83.6 years, who suffered a fracture of the femoral diaphysis due to a fall at home (49 fractures), a traffic accident (8 fractures) or a high-energy fall (1 fracture). Prior to the fracture, 10 patients had homolateral osteoarthritis and two had a contralateral hip arthroplasty. Twenty-six patients were in very good health, 19 had a history of cardiovascular disease, 9 had diabetes and 12 suffered parkinsonian syndromes or dementia. The ASA score was I in 24, II in 23 and III in 11. The diaphyseal fracture was isolated in 31 cases and associated with trochanteric involvement in 27. The upper third of the femur was involved in 37 cases, the middle third in 7 and the lower third in 14. Generally there was a simple spiroid subtrochanteric fracture line (36 cases), or a torsion wedge with or without a proximal extension. Mean delay to surgery was 1.9 days. Subtrochanteric fractures with a proximal line were stabilized with a GL (34 nails) and diaphyseal fractures with a GK (24 nails). Mean duration of the procedure was 1.9 for GL and 2 hours for GK. In 22 cases (17 GL and 5 GK), a minimally invasive access was needed to achieve reduction or stabilization during reaming and insertion of complementary fixation (3 screw fixations, 7 cerclages). RESULTS: Six patients died before six months, 4 during the initial hospitalization. Twenty patients experienced general complications: 7 cases of phlebitis and 5 "end-of-life" syndromes. Infection occurred in 3 cases including one septic arthritis leading to a bedridden situation. A new fracture beyond the ends of the implant occurred in 2 others. The upright position was achieved within 31 days and total weight bearing within 69 days. Bone fusion was achieved at 4 months (mean). Six patients died between 6 and 12 months, giving a 20.6% mortality at 1 year. Clinical outcome at 12 months was available for 42 living patients: 21 were walking without assistance, 7 used a cane, 8 required crutches or another assistance device and 6 were bedridden. DISCUSSION: The general and functional prognosis of femoral shaft fractures in the elderly is the same as for proximal fractures. These diaphyseal fractures can be individualized due to their characteristic mechanical and anatomic features: composite fracture with a rotation element involving the distal portion of the trochanter and the proximal quarter of the diaphysis. Several types of ostheosynthesis have been proposed for fixation. Locked intramedullary nailing has been found to be effective despite the difficulty in reduction, especially for particularly proximal fractures. There is a risk of iterative fracture in the transition zones between the femoral component and the osteoporotic bone.

Expression and induction of CYP1A1/1A2, CYP2A6 and CYP3A4 in primary cultures of human hepatocytes: a 10-year follow-up.

1. The aims were to refine experimental conditions (using 76 human hepatocyte preparations) in terms of the selection of enzyme inducers and their optimal concentration, the treatment duration with inducers and the choice of specific cytochrome P450 isoform(s) probes to optimize the use of primary hepatocytes for predicting the potential induction by new chemical entities of cytochrome P450 isoforms in vivo in man. 2. In the absence of any inducer, basal cytochrome P450 isoform(s)-mediated activities decreased to 20% of their initial activity (end of the seeding period) by 72-96 h. In contrast, UGT-dependent enzyme activities remained at a constant level (+/- 20%) up to the fifth day of culture. 3. Beta-naphthoflavone, at an optimal concentration of 50 microM and after a 3-day treatment, specifically and potently induced 7-ethoxyresorufin (10.4 +/- 10.4-fold, n = 74) and phenacetin (6.6 +/- 6.4-fold, n = 60) O-deethylation processes, markers for CYP1A1 and CYP1A2 isoforms respectively. Only a 2-fold increase was noted following treatment with 2 mM phenobarbitone, whereas dexamethasone and rifampicin had no effect at all. 4. A 3-day treatment of human hepatocytes with 50 microM dexamethasone was associated with a major induction of both coumarin 7-hydroxylation (9.4 +/- 11.4-fold, n = 49) and nifedipine dehydrogenation (4.7 +/- 3.8-fold, n = 61), markers for CYP2A6 and CYP3A4 respectively. Phenobarbitone, however, exhibited a broad but moderate inducing effect on 7-ethoxyresorufin (2.2 +/- 1.5-fold, n = 55) and phenacetin (1.7 +/- 0.9-fold, n = 54) O-deethylation, coumarin 7-hydroxylation (3.9 +/- 9.2-fold, n = 50) and nifedipine dehydrogenation (2.1 +/- 2.0-fold, n = 47). 5. Km obtained for the different cytochrome P450 isoform substrates in untreated hepatocytes were in the same range of magnitude that those determined on human hepatic microsomal fractions. Enzyme induction processes were characterized by a large increase in apparent Vmax whereas apparent Km were not affected. 6. These studies demonstrate that human hepatocytes in primary culture can respond specifically and quantitatively to model inducers. This in vitro system offers a useful approach to study the regulation of human hepatic biotransformation activities and should facilitate the demand for a reproducible method for addressing cytochrome P450 induction.

[Fall in the seroprevalence of hepatitis A in French youth]. / Effondrement de la séroprévalence de l'hépatite virale A chez les jeunes français.

OBJECTIVE: An anti-hepatitis A virus seroprevalence survey was performed in 1997 in 1052 French army recruits (mean age: 21.2 years). To describe epidemiological trends, the current pattern was compared to previous results obtained by similar methods in 1985, 1990 and 1993. RESULTS: In 1997, overall anti-hepatitis A virus seroprevalence was 11.5%. The greatest risk factor of hepatitis A infection was related to travel in intermediate or highly endemic areas for hepatitis A virus: 46% of overseas residents (odds ratio = 10.3), 28% of recruits who had travelled in developing countries (odds ratio = 3.7) and 7.65% of French living in industrialised countries are anti-hepatitis A virus antibody positive. Moreover, seroprevalence was higher in subjects with a history of icteria (adjusted odds ratio = 3.5) and families with at least 3 children (adjusted odds ratio = 3). No association was found with drinking water, socioeconomic status such as baccalaureat degree, or parents profession. The seroepidemiological shift of hepatitis A, as assessed in three previous studies, shows a marked decrease of 20% in 12 years from 30.4% in 1985, to 21.3% in 1990, to 16.3% in 1993, and to 11.5% in 1997. The decrease in the prevalence of anti-hepatitis A virus was more marked in young adults who had never travelled in endemic countries (decrease of 20%) than those who had visited or lived in developing countries (decrease of 10%). CONCLUSION: Although France is not highly endemic for hepatitis A thanks to improved hygiene and housing conditions over the past 20 years, a pattern of intermediate endemicity was seen in French overseas areas in which the risk of outbreaks of hepatitis A was higher. The decrease in anti-hepatitis A virus seroprevalence in French youth can be used to draft a public health policy for hepatitis A control.

Role of cytochrome P-4502C9 in irbesartan oxidation by human liver microsomes.

The oxidative metabolism of irbesartan, a new nonpeptide angiotensin II receptor antagonist, was investigated on 12 human fully characterized hepatic microsomes and purified cytochrome P-450 (CYP) isoforms. After incubation of microsomes with irbesartan and NADPH, four main hydroxy metabolites were formed, as confirmed by liquid chromatography-mass spectrometry analysis. Irbesartan oxidation follows Michaelis-Menten kinetics, consistent with the involvement of a single CYP isoform in these hydroxylation processes. Only a low interindividual variability (2-fold difference) was observed in drug oxidation, even in preparations lacking CYP2D6. Km and Vmax for irbesartan oxidation were 54 +/- 6.5 microM and 0.62 +/- 0.18 nmol/min/mg, respectively. Irbesartan oxidation correlated (r2 = 0. 769) with tolbutamide (CYP2C9 substrate) 4-methyl-hydroxylation. Oxidation of irbesartan was markedly inhibited by sulfaphenazole (CYP2C9 inhibitor), but not by any of several other CYP inhibitors. In the same manner, both tolbutamide and warfarin (CYP2C9 substrates), were competitive-type inhibitors of irbesartan oxidation with Ki values of 500 and 30 microM, respectively. Moreover, irbesartan was a competitive-type inhibitor of tolbutamide 4-methylhydroxylation (Ki = 317 microM). Nifedipine also potentially decreased irbesartan oxidation, whereas neither ketoconazole and triacetyloleandomycin (CYP3A inhibitors), nor diltiazem and verapamil, (CYP3A4 substrates), exhibited an inhibitory effect. Additional studies demonstrated that nifedipine was an inhibitor of irbesartan (Ki = 20 microM) and tolbutamide oxidation processes, whereas irbesartan had no effect at all on nifedipine dehydrogenation. Enzyme kinetics suggest that nifedipine is a noncompetitive-type inhibitor of CYP2C9-mediated catalytic activities. Finally, only microsomes containing recombinant human liver CYP2C9 were capable of oxidizing irbesartan. These results provide evidence that CYP2C9 plays a major role in irbesartan oxidation.

The Use of Long-term Hepatocyte Cultures for Detecting Induction of Drug Metabolising Enzymes: The Current Status.

In this report, metabolically competent in vitro systems have been reviewed, in the context of drug metabolising enzyme induction. Based on the experience of the scientists involved, a thorough survey of the literature on metabolically competent long-term culture models was performed. Following this, a prevalidation proposal for the use of the collagen gel sandwich hepatocyte culture system for drug metabolising enzyme induction was designed, focusing on the induction of the cytochrome P450 enzymes as the principal enzymes of interest. The ultimate goal of this prevalidation proposal is to provide industry and academia with a metabolically competent in vitro alternative for long-term studies. In an initial phase, the prevalidation study will be limited to the investigation of induction. However, proposals for other long-term applications of these systems should be forwarded to the European Centre for the Validation of Alternative Methods for consideration. The prevalidation proposal deals with several issues, including: a) species; b) practical prevalidation methodology; c) enzyme inducers; and d) advantages of working with independent expert laboratories. Since it is preferable to include other alternative tests for drug metabolising enzyme induction, when such tests arise, it is recommended that they meet the same level of development as for the collagen gel sandwich long-term hepatocyte system. Those tests which do so should begin the prevalidation and validation process.

Correlation between oral drug absorption in humans, and apparent drug permeability in TC-7 cells, a human epithelial intestinal cell line: comparison with the parental Caco-2 cell line.

PURPOSE: To determine and compare the relationship between in vivo oral absorption in humans and the apparent permeability coefficients (Papp) obtained in vitro on two human intestinal epithelial cell lines, the parental Caco-2 and the TC-7 clone. METHODS: Both cell lines were grown for 5-35 days on tissue culture-treated inserts. Cell monolayers were analysed for their morphology by transmission electron micrography, and for their integrity with respect to transepithelial electrical resistance, mannitol and PEG-4000 transport, and cyclosporin efflux. Papp were determined for 20 compounds exhibiting large differences in chemical structure, molecular weight, transport mechanisms, and percentage of absorption in humans. RESULTS: The TC-7 clone exhibits morphological characteristics similar to those of the parental Caco-2 cell line, concerning apical brush border, microvilli, tight junctions and polarisation of the cell line. The TC-7 clone however appeared more homogenous in terms of cell size. Both cell lines achieved a similar monolayer integrity towards mannitol and PEG-4000. Monolayer integrity was achieved earlier for the TC-7 clone, mainly due to its shorter doubling time, i.e. 26 versus 30 hours for parental Caco-2 cells. When using cyclosporin A as a P-glycoprotein substrate, active efflux was lower in the TC-7 clone than in the parental Caco-2 cells. The Papp and mechanisms of transport (paracellular or transcellular routes, passive diffusion and active transport) were determined for 20 drugs. A relationship was established between the in vivo oral absorption in humans and Papp values, allowing to determine a threshold value for Papp of 2 10(-6) cm/sec, above for which a 100% oral absorption could be expected in humans. Both correlation curves obtained with the two cell types, were almost completely superimposable. These studies also confirmed that the dipeptide transporter is underexpressed in both cell lines. CONCLUSIONS: On the basis of morphological parameters, biochemical activity and drug transport characteristics, the TC-7 clone appeared to be a valuable alternative to the use of parental Caco-2 cells for drug absorption studies.