Elastic Properties of Few Nanometers Thick Polycrystalline MoS2 Membranes: A Nondestructive Study.

The performance gain-oriented nanostructurization has opened a new pathway for tuning mechanical features of solid matter vital for application and maintained performance. Simultaneously, the mechanical evaluation has been pushed down to dimensions way below 1 µm. To date, the most standard technique to study the mechanical properties of suspended 2D materials is based on nanoindentation experiments. In this work, by means of micro-Brillouin light scattering we determine the mechanical properties, that is, Young modulus and residual stress, of polycrystalline few nanometers thick MoS2 membranes in a simple, contact-less, nondestructive manner. The results show huge elastic softening compared to bulk MoS2, which is correlated with the sample morphology and the residual stress.

Nanoscale conductive pattern of the homoepitaxial AlGaN/GaN transistor.

The gallium nitride (GaN)-based buffer/barrier mode of growth and morphology, the transistor electrical response (25-310 °C) and the nanoscale pattern of a homoepitaxial AlGaN/GaN high electron mobility transistor (HEMT) have been investigated at the micro and nanoscale. The low channel sheet resistance and the enhanced heat dissipation allow a highly conductive HEMT transistor (Ids > 1 A mm(-1)) to be defined (0.5 A mm(-1) at 300 °C). The vertical breakdown voltage has been determined to be ∼850 V with the vertical drain-bulk (or gate-bulk) current following the hopping mechanism, with an activation energy of 350 meV. The conductive atomic force microscopy nanoscale current pattern does not unequivocally follow the molecular beam epitaxy AlGaN/GaN morphology but it suggests that the FS-GaN substrate presents a series of preferential conductive spots (conductive patches). Both the estimated patches density and the apparent random distribution appear to correlate with the edge-pit dislocations observed via cathodoluminescence. The sub-surface edge-pit dislocations originating in the FS-GaN substrate result in barrier height inhomogeneity within the HEMT Schottky gate producing a subthreshold current.

Nanoscale investigation of AlGaN/GaN-on-Si high electron mobility transistors.

AlGaN/GaN HEMTs are devices which are strongly influenced by surface properties such as donor states, roughness or any kind of inhomogeneity. The electron gas is only a few nanometers away from the surface and the transistor forward and reverse currents are considerably affected by any variation of surface property within the atomic scale. Consequently, we have used the technique known as conductive AFM (CAFM) to perform electrical characterization at the nanoscale. The AlGaN/GaN HEMT ohmic (drain and source) and Schottky (gate) contacts were investigated by the CAFM technique. The estimated area of these highly conductive pillars (each of them of approximately 20-50 nm radius) represents around 5% of the total contact area. Analogously, the reverse leakage of the gate Schottky contact at the nanoscale seems to correlate somehow with the topography of the narrow AlGaN barrier regions producing larger currents.

Turbulent Flow Over Large Roughness Elements: Effect of Frontal and Plan Solidity on Turbulence Statistics and Structure.

Wind-tunnel experiments were carried out on fully-rough boundary layers with large roughness ( δ / h ≈ 10 , where h is the height of the roughness elements and δ is the boundary-layer thickness). Twelve different surface conditions were created by using LEGO™ bricks of uniform height. Six cases are tested for a fixed plan solidity ( λ P ) with variations in frontal density ( λ F ), while the other six cases have varying λ P for fixed λ F . Particle image velocimetry and floating-element drag-balance measurements were performed. The current results complement those contained in Placidi and Ganapathisubramani (J Fluid Mech 782:541-566, 2015), extending the previous analysis to the turbulence statistics and spatial structure. Results indicate that mean velocity profiles in defect form agree with Townsend's similarity hypothesis with varying λ F , however, the agreement is worse for cases with varying λ P . The streamwise and wall-normal turbulent stresses, as well as the Reynolds shear stresses, show a lack of similarity across most examined cases. This suggests that the critical height of the roughness for which outer-layer similarity holds depends not only on the height of the roughness, but also on the local wall morphology. A new criterion based on shelter solidity, defined as the sheltered plan area per unit wall-parallel area, which is similar to the 'effective shelter area' in Raupach and Shaw (Boundary-Layer Meteorol 22:79-90, 1982), is found to capture the departure of the turbulence statistics from outer-layer similarity. Despite this lack of similarity reported in the turbulence statistics, proper orthogonal decomposition analysis, as well as two-point spatial correlations, show that some form of universal flow structure is present, as all cases exhibit virtually identical proper orthogonal decomposition mode shapes and correlation fields. Finally, reduced models based on proper orthogonal decomposition reveal that the small scales of the turbulence play a significant role in assessing outer-layer similarity.

Wind resource assessment in heterogeneous terrain.

High-resolution particle image velocimetry data obtained in rough-wall boundary layer experiments are re-analysed to examine the influence of surface roughness heterogeneities on wind resource. Two different types of heterogeneities are examined: (i) surfaces with repeating roughness units of the order of the boundary layer thickness (Placidi & Ganapathisubramani. 2015 J. Fluid Mech.782, 541-566. (doi:10.1017/jfm.2015.552)) and (ii) surfaces with streamwise-aligned elevated strips that mimic adjacent hills and valleys (Vanderwel & Ganapathisubramani. 2015 J. Fluid Mech.774, 1-12. (doi:10.1017/jfm.2015.228)). For the first case, the data show that the power extraction potential is highly dependent on the surface morphology with a variation of up to 20% in the available wind resource across the different surfaces examined. A strong correlation is shown to exist between the frontal and plan solidities of the rough surfaces and the equivalent wind speed, and hence the wind resource potential. These differences are also found in profiles of [Formula: see text] and [Formula: see text] (where U is the streamwise velocity), which act as proxies for thrust and power output. For the second case, the secondary flows that cause low- and high-momentum pathways when the spacing between adjacent hills is beyond a critical value result in significant variations in wind resource availability. Contour maps of [Formula: see text] and [Formula: see text] show a large difference in thrust and power potential (over 50%) between hills and valleys (at a fixed vertical height). These variations do not seem to be present when adjacent hills are close to each other (i.e. when the spacing is much less than the boundary layer thickness). The variance in thrust and power also appears to be significant in the presence of secondary flows. Finally, there are substantial differences in the dispersive and turbulent stresses across the terrain, which could lead to variable fatigue life depending on the placement of the turbines within such heterogeneous terrain. Overall, these results indicate the importance of accounting for heterogeneous terrain when siting individual turbines and wind farms.This article is part of the themed issue 'Wind energy in complex terrains'.

Involvement of human liver cytochrome P450 3A in vinblastine metabolism: drug interactions.

Vinblastine biotransformation was investigated by using a human liver microsomes library. The drug was converted into one major metabolite (M) upon incubation with the microsomes. A large interindividual variation in vinblastine metabolism was observed among the samples tested, with a 4.4 ratio between the lowest and the highest metabolic rates. The biotransformation of vinblastine followed Michaelis-Menten kinetics (Km = 6.82 +/- 0.27 microM and Vmax = 0.64 +/- 0.06 nmol/min/mg protein). The involvement of the cytochrome P450 3A subfamily in vinblastine metabolism was demonstrated by the following body of evidence: (a) the competitive inhibition of vinblastine biotransformation by cytochrome P450 3A specific probes with Ki values of 0.17, 22.5, 14.8, and 35.3 microM for ketoconazole, erythromycin, troleandomycin, and vindesine, respectively; (b) the immunoinhibition of vinblastine metabolism by polyclonal anti-cytochrome P450 3A antibodies; (c) the highly significant correlation between the level of cytochrome P450 3A determined by Western blots and vinblastine metabolism (r = 0.759, P < 0.001); (d) the highly significant correlation between erythromycin N-demethylase activity (mediated by cytochrome P450 3A) and vinblastine metabolism (r = 0.83, P < 0.001); (e) the significant correlation between the CYP3A4 mRNA level and vinblastine metabolism (r = 0.60, P < 0.1). Although vincristine and navelbine (members of the Vinca alkaloid family) also inhibit the metabolism of vinblastine, suggesting the involvement of the cytochrome subfamily in their respective metabolisms, other anticancer drugs currently associated with vinblastine in chemotherapy (etoposide, Adriamycin, lomustine, and teniposide) also interfere with vinblastine biotransformation. These metabolic drug interactions may alter the antitumor activity and/or toxicity of the drug during anticancer chemotherapy.

Disposition and metabolism of the angiogenic moderator O-(chloroacetyl-carbamoyl) fumagillol (TNP-470; AGM-1470) in human hepatocytes and tissue microsomes.

The biotransformation of O-(chloroacetyl-carbamoyl) fumagillol (TNP-470; AGM 1470), a potent in vitro inhibitor of angiogenesis, was investigated in primary cultured human hepatocytes and microsomal fractions of various human tissues. Exposure of human hepatocytes to 5 microM [3H]TNP-470 led to a rapid metabolism of unchanged drug to six metabolic derivatives within 30 min. The predominant extracellular metabolites were M-II and M-IV, attaining a maximum level of 3.23 +/- 0.34 and 0.88 +/- 0.10 microM, respectively. M-II leveled off, while M-IV rapidly declined to 0.06 +/- 0.05 microM by 3 h. TNP-470 was undetectable after 60 min. M-V and M-VI slowly reached maximal concentrations of 0.26 +/- 0.12 and 0.32 +/- 0.16 microM, respectively. M-I only reached a concentration of 0.18 +/- 0.07 microM at 60 min and leveled at 0.13 +/- 0.06 microM for the remaining time of the experiment. The intracellular profile was different, with M-III and M-V representing the major metabolites detected. Studies using human liver microsomes demonstrated that M-IV formation was associated with an esterase-like enzymatic cleavage of TNP-470 and that this metabolite was then further metabolized by microsomal epoxide hydrolase to M-II, as evidenced by inhibition of this metabolic step by cyclohexene oxide, a microsomal epoxide hydrolase inhibitor. Extrahepatic metabolism of TNP-470 was also demonstrated using different sites of human intestinal, stomach, and kidney microsomes, with metabolite M-IV as the principal derivative detected in these tissues. Hepatic microsomal samples from seven different donors demonstrated large interindividual variations in the formation of both M-II and M-IV. In summary, this study demonstrates a rapid and extensive metabolism of TNP-470 in human tissues. The data emphasize the need to evaluate the in vivo formation and extent of TNP-470 metabolites to adequately assess the pharmacodynamic effects of this novel anticancer drug with a novel mechanism of action.

Reduction of 3'-azido-3'-deoxythymidine to 3'-amino-3'-deoxythymidine in human liver microsomes and its relationship to cytochrome P450.

The formation of 3'-amino-3'-deoxythymidine (AMT) in patients receiving 3'-azido-3'-deoxythymidine (zidovudine) and the potential role of this metabolite in zidovudine-induced toxicity was recently demonstrated by our laboratory. This study evaluated the formation of AMT versus cytochrome P450 (P450) content, cytochrome B5 (B5) content and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase activity in human liver microsomes obtained from 24 different donors. Significant interindividual differences in total P450 content and P450 reductase activity were observed, whereas no variation was observed in B5 content. Of particular importance, metabolism of zidovudine to AMT varied widely and correlated with P450 content but not with B5 content or P450 reductase activity. The apparent values for the Michaelis-Menten constant and the maximum rate of metabolism of the reaction were 46.1 mmol/L and 3.5 nmol/min/mg microsomal protein. These large variations of AMT levels as a function of P450 suggest that major interindividual differences may be observed in the pharmacokinetics and formation of this metabolite that may affect the pharmacodynamic properties of zidovudine. Potential drug-drug interactions may occur with therapeutic agents that interact with or induce P450 (zidovudine).

Human liver microsomal cytochrome P450 3A isozymes mediated vindesine biotransformation. Metabolic drug interactions.

Vindesine biotransformation was investigated using a bank of human liver microsomes. The drug was converted into one major metabolite (M) upon incubation with the microsomes. Large interindividual variations were observed: vindesine biotransformation rates ranged from 1.2 to 12.9 pmol/min/mg protein. Vindesine metabolic processes followed Michaelis-Menten kinetics: Km = 24.7 +/- 9.4 microM, Vmax = 1.5 +/- 0.8 nmol/min/mg protein. The involvement of human cytochrome P450 3A isozymes in vindesine metabolism was demonstrated by: (1) competitive inhibition of vindesine biotransformation by compounds known to be specifically metabolized by human cytochrome P450 3A. Apparent Ki values were 3.6, 17.9 and 19.8 microM for quinidine, troleandomycin and erythromycin, respectively; (2) immunoinhibition of vindesine metabolism by polyclonal anti-P450 3A antibody; (3) significant correlation between immunoquantified P450 3A and vindesine biotransformation (r = 0.800, P < 0.001); and (4) significant correlation between erythromycin N-demethylase activity, which was supported by P450 3A in humans, and vindesine biotransformation (r = 0.853, P < 0.001). Other vinca alkaloids also exerted an inhibitory effect on vindesine biotransformation with apparent Ki values of 3.8, 10.6 and 19.2 microM for vinblastine, vincristine and navelbine, respectively, suggesting a possible involvement of the same cytochrome subfamily in their hepatic metabolism. Moreover, a number of anticancer drugs currently associated with the vinca alkaloids, such as teniposide, etoposide, doxorubicin, lomustine, folinic acid and mitoxantrone, significantly inhibited vindesine biotransformation.

Characterization of midazolam metabolism using human hepatic microsomal fractions and hepatocytes in suspension obtained by perfusing whole human livers.

Isolated human hepatocytes provide a useful model for studying xenobiotic metabolism. However, in vitro studies using human hepatocytes are scarce due to the limited availability of this material. A new methodology is described for obtaining hepatocytes from a whole adult human liver. This procedure is based on (i) the rapid and intense in situ washing step of the organ with Eurocollins then glucose supplemented HEPES buffer (10 mM, pH 7.4) at 4 degrees in order to both minimize the warm ischemic period and remove erythrocytes, and (ii) a perfusion of collagenase solution (0.05% in 10 mM HEPES buffer at 37 degrees) throughout the portal vein according to a recirculated model. All perfused buffers are oxygenized. Hepatocyte viability averaged 85% as determined by Trypan Blue dye exclusion. The ability of these hepatocytes to catalyze certain metabolic transformations such as Phase I and Phase II reactions has been particularly investigated using the benzodiazepine drug, midazolam, as a substance probe. Freshly isolated human hepatocytes in suspension retained the ability to metabolize midazolam to its different hydroxylated derivatives--mainly the 1-hydroxy-midazolam--which was further conjugated with glucuronic acid. For a better understanding of the cytochrome P-450 mediated reactions, we studied the metabolism of midazolam in microsomal fractions prepared from twelve human livers. It was concluded that human microsomes (i) exhibited a Type I binding spectrum upon midazolam addition (Ks = 3.3 microM) and (ii) intensively metabolized the drug to its different derivatives. Furthermore, and since we demonstrated that midazolam was predominantly transformed by a single cytochrome P-450 enzyme, we could attribute the large inter-individual variations in midazolam metabolism to differences in human liver cytochrome P-450 content.

Tissue-specific expression and methylation of the human CYP2E1 gene.

The level and number of CYP2E1 gene transcripts were investigated by northern blot analysis in various human adult tissues including liver, lung, placenta, skin and neurinoma. Three transcripts of 1.8, 2.6 and 4 Kb were expressed in a tissue-specific manner. The origin of the various transcripts was studied and showed that both 4 and 2.6 Kb mRNAs contained sequences from the 3' non-translated region of the gene and that the 4 Kb also contained region localized in the 5' non-translated region. Furthermore, it clearly appeared that a catalytically active CYP2E1 enzyme (as proved by NDMA demethylase activity) was only detected in tissues expressing the 1.8 Kb. The human CYP2E1 was also identified through immunohistochemical techniques. Finally, we observed a relation between the hypomethylation of the human CYP2E1 gene and the hypoexpression of the corresponding protein.

Metabolism of digoxin, digoxigenin digitoxosides and digoxigenin in human hepatocytes and liver microsomes.

In vitro metabolism of digoxin and its cleavage-related compounds was investigated using hepatocytes in primary culture and microsomal fractions both isolated from human livers. On these models, digoxin (DG3) and digoxigenin bisdigitoxoside (DG2) were not shown to be significantly metabolized in vitro in man. Therefore, it appeared that the stepwise cleavage of DG3 and DG2 sugars was not cytochrome P450 dependent. This enzymatic system probably plays a minor role in humans for this particular reaction. However, digoxigenin monodigitoxoside (DG1) and digoxigenin (DG0) which are known to be formed after intra-gastric hydrolysis of DG3, were extensively converted to polar compounds (mainly glucuronides). In addition, using human liver microsomes, a wide variability in UDP-glucuronyl transferase (UDPGT) activities responsible for DG1 glucuronidation was demonstrated. These results suggest that two main factors may contribute to the overall interindividual variability of digoxin biotransformation: 1), the individual intra-gastric pH which influences the sugar cleavage leading to DG1 and DG0; ii), a variability in the level of the hepatic UDPGT specific for digitalis compounds conjugation.

Identification of the human and animal hepatic cytochromes P450 involved in clonazepam metabolism.

This report characterizes the cytochrome P450 isozyme involved in clonazepam metabolism. This study was undertaken using a library of liver microsomal fractions prepared from untreated rabbits or those treated with drugs known to specifically induce various cytochrome P450 isozymes (ie P450 2B4 by phenobarbital, P450 1A1 and P450 1A2 by 3-methylcholanthrene and beta-naphthoflavone, P450 2E1 by acetone and ethyl alcohol, and P450 3A6 by erythromycin). Only microsomes obtained from phenobarbital-treated rabbits exhibited a type II binding spectrum upon addition of clonazepam (Ks(app) = 31.4 +/- 3.8 microM) and significantly metabolized clonazepam to 7-aminoclonazepam. Benzphetamine, which is a known substrate for P450 2B1 was also extensively metabolized by microsomes prepared from phenobarbital treated rabbits. This indicates that the same isozyme (P450 2B subfamily) was involved in the biotransformation of both substrates. Experiments performed on 14 human liver microsomal preparations showed a wide interindividual variability (from 1-4) and a good correlation (r = 0.70) between benzphetamine and clonazepam metabolism. Since P450 3A4 (nf25) was involved in benzphetamine metabolism, clonazepam was probably nitroreduced by the same isozyme. An oligonucleotide specific for the P450 3A4 gene subfamily was synthetized and used for hybridization on total RNA from human liver samples. Two transcripts of 2.2 and 3.0 kb were detected and the level of the 2.2 kb mRNA expression was significantly correlated (r = 0.61) with the intensity of clonazepam nitroreduction by the corresponding microsome batches.

Uptake and metabolism of vinca alkaloids by freshly isolated human hepatocytes in suspension.

A study was carried out to evaluate the uptake, release and metabolism of four currently used vinca alkaloids, including vinblastine, vincristine, vindesine and navelbine, using freshly isolated human hepatocytes in suspension. The drugs were rapidly taken up and intensely metabolised by the cells, giving a number of yet unidentified biotransformation products. Navelbine was the most rapidly and intensely accumulated drug followed by vinblastine, vindesine and vincristine. The extent of cell uptake appeared to parallel the lipophilicities of these compounds. Interestingly, we found a significant correlation between the mean uptake rates of the vinca alkaloids into the cells, which were 0.279, 0.343, 0.568 and 0.834 pmol/min/10(6) cells for vincristine, vindesine, vinblastine and navelbine, respectively, and the in vivo plasma clearances of the drugs (r = 0.9995, p < 0.001). This finding is of great importance as regards a better understanding of the structure-activity relationship among this class of antitumour drugs, as well as a reliable extrapolation of in vitro results to the in vivo situation.

A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco-toxicology.

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.

Human hepatic uptake of two vinca alkaloids: navelbine and vincristine.

A human liver plasma membrane model for the evaluation of the specific binding and transport processes of drugs presenting high hepatic clearance such as vinca alkaloids was developed. Uptake of the two structural antitumor analogs, navelbine (NVB) and vincristine (VCR), which exhibit wide variabilities in their respective pharmacokinetic parameters and antitumor spectra, was investigated. The high yield, the enzymatic profile and the retention of physiologic transport capacities, as demonstrated by taurocholate uptake, revealed that this membrane preparation was well suited for studies of hepatic drug transport systems. For both drugs two distinct processes were observed: mainly membrane binding and transport. NVB was found to bind to the membrane vesicles more intensively than VCR, but the transport processes were almost identical. However only NVB uptake seems to involve Na(+)-dependent processes. These significant differences may be related to the respective lipophilicity of the drugs. The more lipophilic molecule (NVB) presents the highest uptake, which is presumably at the origin of its greatest distribution volume in vivo.

Freshly isolated or cryopreserved human hepatocytes in primary culture: Influence of drug metabolism on hepatotoxicity.

The use of human hepatocytes in drug metabolism studies overcomes the difficulty in extrapolating from animal data. As drug metabolites are often involved in pharmacological activity and toxicity, it is essential to determine these compounds early in the development of a drug. We previously demonstrated that mitoxantrone metabolism exhibits a great interspecies variability: the two main metabolites in humans being dicarboxylic acid (the major metabolite) and monocarboxylic acid. In this paper we analyse their respective cytotoxicities with two endpoint tests: neutral red uptake (cell viability) and MTT reduction (cell metabolism) after a 24- or 48-hr exposure. Results demonstrated less or no toxicity of mono- and dicarboxylic acid derivatives compared with mitoxantrone. In vitro studies using human hepatocytes are scarce because of the limited availability of human livers. After developing a new method for cryopreservation of human hepatocytes, we evaluated them before and after cryopreservation using erythromycin as a test molecule. The cell viability test was more reproducible than the cell metabolism test after 38 hr of cell seeding.

Use of a human liver microsome bank in drug glucuronidation studies.

A bank of readily available, well characterized human hepatic microsomal fractions (29 samples from different livers) has been established. The enzymatic characteristics of each microsome sample were determined using three specific UDP-glucuronosyltransferase (UDPGT) substrates (p-nitrophenol for UDPGT(1), 4-hydroxybiphenyl for UDPGT(2)A and monodigitoxoside digitoxigenin (DT(1)) for UDPGT(7)). After characterization, the bank was used to study the phase II biotransformation processes of two drugs: an antiviral, zidovudine (AZT), and an intermediate metabolite of digoxin, monodigitoxoside digoxigenin (DG(1)). Results showed a wide interindividual variability for the glucuronidation rate of 4-hydroxybiphenyl, DT(1), AZT and DG(1). Surprisingly, this variability was lower for p-nitrophenol. No significant correlation was found between the various activities except between DG(1) and DT(1) UDPGT activities and 4-hydroxybiphenyl and AZT UDPGT activities. For AZT, further experiments showed that glucuronidation of this antiviral compound was inhibited by 4-hydroxybiphenyl and chloramphenicol but not by morphine, p-nitrophenol, acetaminophen, or bilirubin. Finally, the results strongly suggest the respective involvement of UDPGT(2)A and UDPGT(7) in the glucuronidation of AZT and DG(1).

A multicentre study of acute in vitro cytotoxicity in rat liver cells.

A multicentre validation study of the acute in vitro cytotoxicity of drugs involving six French laboratories from INSERM or pharmaceutical companies has been carried out. Thirty liquid or solid chemicals such as antibiotics, anticancer drugs and solvents were selected and incubated for 20 hr with normal rat hepatocytes and FaO hepatoma cells. Miniaturized and automated methods were defined for the evaluation of cytotoxic effects. Four endpoints were evaluated: the ratio of extracellular lactate dehydrogenase to total lactate dehydrogenase, total cellular protein content, reduction of a tetrazolium salt, and neutral red uptake. For each test IC(50) values were calculated. A good interlaboratory reproducibility was demonstrated. The neutral red assay was found to be the most sensitive and the least reproducible endpoint. More compounds were shown to be cytotoxic to hepatocytes than to hepatoma cells (18 v. 12). On the basis of the IC(50) values a few compounds were found to be much less cytotoxic than predicted from in vivo data, suggesting that a simple experimental protocol and non-specific cytotoxicity parameters are not sufficient to test certain drug families. However, such methods appear to provide a useful means of defining the concentration range of the drug that will be selected for further analysis using more specific tests.

In vivo and in vitro pharmacokinetics and metabolism of vincaalkaloids in rat. I. Vindesine (4-deacetyl-vinblastine 3-carboxyamide).

Vindesine (VDS) pharmacokinetics, including tissue distribution, metabolism and elimination, were investigated in rats using both in vivo and in vitro models. VDS was found to be intensively distributed in tissues after i.v. injection in rat. The most important drug accumulation site was the spleen (615.0 ng/g at 24 h). Liver and kidneys also retained VDS in significant amounts (respectively 170.1 +/- 11.0 ng/g and 145.0 +/- 17.0 ng/g at 24 h). Urine excretion of drug over 7 days was low (10.1 +/- 1.8% of total dose) and consisted mainly of unchanged drug (more than 85%). The major excretion route for VDS was the feces (69.6 +/- 2.5% of total dose) via the bile (50% of total dose excreted in 72 h). High performance liquid chromatography analysis (HPLC) of collected bile samples revealed the excretion of three VDS biotransformation products. These results were confirmed in vitro using freshly isolated rat hepatocytes in suspension. Rapid and high VDS uptake by liver cells, probably through a passive diffusion mechanism followed by a tight cellular binding, was demonstrated. Moreover, VDS was intensively converted, in vitro, into four metabolites which were rapidly excreted into the extracellular medium. In contrast, the intracellular medium contained almost exclusively unchanged drug, presumably fixed to tubulin proteins. Two anti-VDS monoclonal antibodies with different specificities were used to test metabolite immunoreactivities. The results suggested that some structural modifications occurred in the catharantine moiety of the molecule but that the VDS dimeric structure seemed well conserved after biotransformation.