The effects of combined antifolates on inhibition of growth of murine leukemia cells cultured in vitro

PURPOSE: Long-term patency of cryopreserved vascular grafts is determined by maintained cellular and tissue viability, which implies preservation of various biochemical, smooth muscle, and endothelial functions. Therefore, it was investigated whether the presence of fetal calf serum (FCS) in the cryomedium improves the postthaw contractile and endothelial function of human arteries. METHODS: Rings from human mesenteric (HMA) and left circumflex coronary arteries (HCA) obtained from organ donors were randomized into three groups and studied either unfrozen or after storage for 3 to 6 weeks at -196 degrees C while suspended in Krebs-Henseleit solution without or with 20% FCS as the vehicles and 1.8 mol/L dimethyl sulfoxide and 0.1 mol/L sucrose as cryoprotecting agents. The samples were slowly frozen to -70 degrees C and then stored in liquid nitrogen. Before use, the tissues were thawed within 3 minutes in a 40 degrees C water bath. RESULTS: After thawing the sensitivity to various agonists and maximal responses to the endothelium-independent relaxing agent sodium nitroprusside were unchanged. However, after cryopreservation of HMA was performed without and with FCS, maximal contractile responses to noradrenaline were significantly reduced to 10.1 +/- 0.7 gm and 9.9 +/- 0.9 gm compared with 13.3 +/- 0.6 gm in unfrozen HMA (mean +/- SEM, n = 15). After cryopreservation of HCA was performed without and with FCS, maximal contractile responses to prostaglandin F2 alpha (6.9 +/- 0.4 gm in unfrozen HCA) were significantly reduced to 4.3 +/- 0.3 gm and 3.8 +/- 0.2 gm (mean +/- SEM, n = 6). In both types of arteries cryopreservation also attenuated significantly the endothelium-dependent relaxant responses to bradykinin during U46619 (10 nmol/L)-induced tone. In HMA the maximal bradykinin-induced relaxation (85% +/- 4%) was significantly diminished to 29% +/- 7% and 38% +/- 9% after cryopreservation without and with FCS (mean +/- SEM, n = 6). In HCA maximal bradykinin-induced relaxation (88% +/- 4%) was significantly diminished to 26% +/- 10% and 36% +/- 11% after cryopreservation without and with FCS (mean +/- SEM, n = 6). This result was reflected by a marked endothelial denudation in all groups of cryopreserved arteries. Neither functional nor morphologic preservation of the endothelial cell lining was significantly improved by FCS supplementation of the cryomedium. CONCLUSIONS: Cryopreservation diminished contractile and endothelium-dependent relaxant responses of human arteries. The presence of FCS in the cryomedium did not modify these changes.     

Cryopreservation of rat and mouse hepatocytes. II. Assessment of metabolic capacity using testosterone metabolism

Cryopreservation of hepatocytes is widely used, but to validate the use of cryopreserved (CP) hepatocytes in metabolic studies, CP cells must compare favorably with fresh cell activities. We have assessed the metabolic capacity of fresh and CP rat and mouse hepatocytes in primary culture. Total cytochrome P450 (P450) contents and metabolism of testosterone were measured up to 72 hr in culture. At 0 hr, total P approximately 450 in CP rat hepatocytes was 102.5 +/- 32.8 pmol/10(6) cells, compared with fresh rat hepatocytes that had 148.2 +/- 75.7 pmol/10(6) cells. The P450 contents of mouse hepatocytes were also unaltered by cryopreservation (176.7 +/- 56.0 pmol/ 10(6) fresh cells; 196.4 +/- 59.9 pmol/10(6) CP cells). There were no significant differences in the total P450 contents of fresh and CP rat and mouse cell cultures with time over 72 hr in culture. The overall metabolism of testosterone was lower in CP suspensions than in freshly isolated hepatocytes. When CP hepatocyte suspensions were permeabilized (with digitonin) and incubated with NADPH and ATP, testosterone metabolism was significantly increased. Testosterone hydroxylase activities (16 alpha-, 6 beta-, 2 alpha-, and 7 alpha-hydroxylase) were equivalent in fresh and CP rat hepatocytes over 72 hr in culture. There was a marked and sustained loss of 6 beta-hydroxylase activity in CP mouse hepatocyte cultures, compared with fresh hepatocytes throughout 72 hr in culture (436.9 +/- 118.0 pmol/min/10(6) cells and 37.3 +/- 41.0 pmol/min/10(6) cells at 72 hr in fresh and CP mouse hepatocytes, respectively). The total metabolism of testosterone was, however, unaffected because 16 alpha-hydroxylase activity increased in CP mouse hepatocytes (475.4 +/- 80.8 pmol/min/10(6) CP cells, compared with 148.7 +/- 39.4 pmol/min/10(6) fresh cells).     

Xenobiotic metabolism in rat, dog, and human precision-cut liver slices, freshly isolated hepatocytes, and vitrified precision-cut liver slices

Human, rat, and dog phase I and phase II xenobiotic metabolism in precision-cut liver slices and freshly isolated hepatocytes was compared using a range of substrates. Carbamazepine (50 microM) and styrene (2 mM) were used as probes to study the maintenance of cytochrome P450 and epoxide hydrolase-mediated metabolism in male Sprague-Dawley rat, precision-cut liver slices and hepatocytes. Carbamazepine metabolism in both models resulted in the formation of the bioactive 10,11-epoxide (KM = 766 microM and Vmax = 2.5 pmol/min/mg protein in precision-cut slices). Epoxide formation was higher (2.4-fold) in hepatocytes than slices. Styrene was deactivated to styrene diol at a higher rate in hepatocytes (9.7-fold) than slices. The lower rate of metabolism in slices compared with hepatocytes confirms our previous observations using testosterone, 7-ethoxycoumarin, 1-chloro-2,4-dinitrobenzene and 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone in the rat. Testosterone 6 beta-hydroxylation in human liver slices was similar to cultured hepatocytes, but lower than in freshly isolated hepatocytes. 7-Ethoxycoumarin O-deethylation was higher in freshly isolated human hepatocytes, as was the ratio of glucuronide to 7-hydroxycoumarin. Testosterone hydroxylations, 7-ethoxycoumarin O-deethylation, and 1-chloro-2,4-dinitrobenzene conjugation were also lower in male beagle dog slices, compared with freshly isolated hepatocytes. Attempts at long-term preservation of dog liver slices using vitrification and storage for up to 9 days at -196 degrees C resulted in the retention of phase I and phase II metabolism, although conjugation was lower than in freshly prepared slices. Xenobiotic metabolism in short-term incubations is consistently lower in dog and rat precision-cut slices than in freshly isolated hepatocytes; whereas, in humans, this quantitative difference is partly hidden by the large interindividual variation.     

Controlled-rate versus uncontrolled-rate cryopreservation of peripheral blood progenitor cells: a prospective multicenter study. Group for Cryobiology and Biology of Bone Marrow Transplantation (CBTMO), Spain

BACKGROUND AND OBJECTIVE: Cryopreservation of hemopoietic progenitors for transplantation has been traditionally performed by the use of a controlled-rate freezer. Several groups have reported successful cryopreservation of progenitor cells at -80 degrees C without a controlled-rate freezer. In an attempt to elucidate whether both methods are equally efficient, we compared controlled-rate versus uncontrolled cryopreservation of peripheral blood progenitor cells (PBPC) in a prospective, multicenter study. DESIGN AND METHODS: Apheresis products from patients undergoing PBPC mobilization were split into two aliquots, and cryopreserved simultaneously by both methods, in autologous plasma plus 10% dimethylsulfoxide. Controlled-rate samples were placed into a programmable freezer with a cooling rate of 1-2 degrees C/min. Uncontrolled-rate samples were directly introduced into a -80 degrees C mechanical freezer. After thawing, cell counts, assays for viability, clonogenic cultures and CD34+ cell enumeration were performed. RESULTS: A total of 105 cases were included. No significant differences were found in viability (mean 88.8 +/- 13% in the controlled-rate group vs. 89.7 +/- 12% in the uncontrolled-rate group), nucleated cell loss (23.5 +/- 23% vs. 23 +/- 22%), mononuclear cell loss (19 +/- 23% vs. 19.1 +/- 22%), and loss of CD34+ cells (34.3 +/- 33% vs. 28.6 +/- 34%). On the other hand, recovery of granulomonocytic colony-forming units (CFU-GM), was significantly better with the controlled-rate technique, than with the non-controlled-rate method (104.3 +/- 95 vs. 86.5 +/- 80, respectively; p = 0.048). INTERPRETATION AND CONCLUSIONS: Our results indicate that both techniques are suitable for cryopreservation of PBPC, although a better recovery of committed progenitors is achieved by the controlled-rate method. Therefore, the use of controlled-rate freezer should probably be recommended.     

Sparing of mdx extraocular muscles from dystrophic pathology is not attributable to normalized concentration or distribution of neuronal nitric oxide synthase

Human T lymphocytes isolated from peripheral blood were cryopreserved at -196 degreesC for different periods of 3, 14, 21, 35, and 50 days. Viability and cytokine-producing activity of T cells were examined before and after cryopreservation. A high recovery (90 +/- 1%) of viable T cells was obtained at each frozen period, indicating that a 10% loss of cells was due to the freezing process rather than the duration of cryopreservation. There was no difference in cell cycle distribution between PHA-treated fresh and frozen lymphocytes. Resting human T cells produced little or no cytokine. After stimulation of fresh T cells with PHA, an apparent increase in cytokine production was noted in IL-2 (35.5 +/- 8.3 pg/ml), IL-6 (1280.4 +/- 64.7 pg/ml), tumor necrosis factor-alpha (874.3 +/- 71.7 pg/ml), interferon-gamma (58.9 +/- 2.2 pg/ml), and granulocyte macrophage-colony-stimulating factor (59.5 +/- 4.4 colonies/5 x 10(4) bone marrow cells). Compared with PHA-activated fresh T cells, all the above cytokines did not diminish in their levels in conditioned medium from PHA-treated frozen T cells thawed at each storage period, suggesting that cryopreservation could well retain the cytokine-producing activity of human T lymphocytes. In addition, our results also revealed that cryopreservation rendered T lymphocytes more responsive to PHA in IL-2 production than fresh T cells.     

Myenteric neurons with different projections have different dendritic tree patterns: a morphometric study in the pig ileum

Species differences in the biotransformation of the antiemetic tropisetron, a potent 5-hydroxytryptamine type 3 (5-HT3) receptor antagonist, were evident in liver slice incubates of human, rat and dog, and reflected the species differences observed in vivo with respect to the relative importance of individual pathways. The dominant biotransformation pathway of tropisetron (10 microM) in human liver slices was formation of 6-hydroxy-tropisetron, whereas in rat liver slices it was 5-hydroxy-tropisetron, and in dog liver slices N-oxide formation. Initial rates of tropisetron metabolite formation in the liver slices (8 mm in diameter, 200 +/- 25 microns thickness) of human (83 +/- 61 pmol/h/mg slice protein), rat (413 +/- 98 pmol/h/mg slice protein) and dog (426 +/- 38 pmol/h/mg slice protein) would predict less of a first-pass effect in humans compared to the rat or the dog. For human and rat, the prediction matched well with the species ranking of tropisetron bioavailability; however, for dog the in vitro data overestimated the apparent first-pass effect. The jejunum is not expected to contribute to the first-pass effect in humans, since human jejunum microsomes did not metabolize tropisetron. The major organ of excretion for tropisetron and its metabolites is the kidney, but the contribution of the kidney to the overall metabolism of tropisetron would be small. Species independent N-oxide formation (2-12 pmol/h/mg slice protein) was the major pathway in human, rat and dog kidney slices, and was comparable to N-oxide formation in the rat and human liver slices but was 1/10 the rate in dog liver slices. This study has demonstrated that the liver is the primary site of tropisetron biotransformation, and the usefulness of organ slices to characterize cross species differences in the dominant biotransformation pathways.     

Effect of albumin on the estimation, in vitro, of phenytoin Vmax and Km values: implications for clinical correlation

The effect of bovine serum albumin (BSA) on human liver metabolism, in vitro, of 14C-phenytoin (PHT) was studied. Michaelis Menten parameters were determined for the conversion of PHT to p-hydroxy phenytoin in seven different microsomal preparations with the addition of 0, 2, and 4% BSA. The unbound Km (Kmu) values were 30.8 +/- 18.6, 1.57 +/- 0.21 and 1.50 +/- 0.17 microM (mean +/- S.D.), respectively; however, there was excellent agreement among the Vmax values (29.1, 31.8 and 31.5 pmol/min/mg). With intact tissue slices, BSA (4%) added to incubations of PHT had a minimal effect on the Vmax values in two of the four livers studied and resulted in a mean Kmu value of 2.20 +/- 0.59 microM, although the Kmu in the absence of BSA was 6.64 +/- 3.17. In scaling-up to the whole body, Vmax values were 3.9 and 1.0 mg/kg/day for microsomes and slices, respectively, compared to 5.9 mg/kg/day, in vivo. The Kmu values determined in the presence of albumin in both microsomes and slices were similar to those based on in vivo human steady state data (Kmu = 2-3 microM), and the intersubject variation, in vitro, was decreased in the presence of BSA. These findings for phenytoin metabolism suggest that the addition of albumin to incubation media for slices or microsome experiments may yield Km estimates that are more representative of in vivo values.     

Escherichia coli and Lactobacillus plantarum responses to osmotic stress

Artificial insemination using cryopreserved semen is a common management tool of the contemporary livestock producer. However, cryopreservation is detrimental to sperm function and fertility, killing some 50% of the spermatozoa during the process. Prediction of cryopreservation damage from prefreeze samples remains elusive. Computer-automated sperm head morphometry was used in this study to determine the effects of cryopreservation on bovine sperm head morphometry. Semen was collected from 18 bulls and was divided. One portion was extended to 200 x 10(6) sperm/ml and a microscope slide was prepared, while the remaining portion was cryopreserved in a Triscitrate-yolk extender. After thawing, the cryopreserved samples were prepared on microscope slides. All slides were air dried and were stained with hematoxylin and rose bengal. The morphometric dimensions for length, width, width/length, area, and perimeter for a minimum of 200 sperm heads were analyzed from each slide by computer-aided sperm head morphometry analysis, and the mean measurements were recorded. Bull sperm heads were significantly (P < 0.01) smaller in cryopreserved spermatozoa than in the companion extended samples for length (8.56+/-0.07 vs. 8.63+/-0.08 microm), width (4.39+/-0.05 vs. 4.48+/-0.05 microm), area (28.42+/-0.07 vs. 29.14+/-0.08 microm), and perimeter (23.33+/-0.21 vs. 23.70+/-0.23 microm) for all bulls. Width/length was also different (0.513 vs. 0.519). In addition, differences (P < 0.05) were found within 14 of 18 bulls for at least four of the morphometric parameters. The percent change in measures after cryopreservation were correlated (P < or = 0.05) to the variability of the extended sample. Variations in sperm head measurements were lower (P < or = 0.05) in extended samples of the four bulls in which no changes occurred than in extended samples of the remaining 14 bulls. These data suggest that the variability in sperm head measurements of individual bulls, or ejaculates, may be an indicator of sperm cryosurvivability.     

Monooxygenation, cytochrome P450-mRNA expression and other functions in precision-cut rat liver slices

Precision-cut rat liver slices (KRUMDIECK slicer, slice thickness 200-250 microm) were incubated in rollers containing modified William's medium E at 37 degrees C for 2, 24 and 48 hrs. Protein, DNA, potassium and glutathione concentrations did not decrease during 48 hrs. Lactate dehydrogenase (LDH) leakage into the medium was relatively marked during the first 2 hrs of incubation, from the 2nd to the 48th hr LDH leakage was very low. The same is true of the release of thiobarbituric acid-reactive substances. Albumin synthesis and transport into the medium decreased to about 70% after 48 hrs. Cytochrome P450 (CYP)-dependent 7-ethoxycoumarin O-deethylation rate was relatively stable up to 48 hrs, whereas testosterone hydroxylation decreased significantly without alterations of the proportions of the 7 quantified hydroxylated metabolites. After exposure of the slices to beta-naphthoflavone for 6 hrs CYP1A1-mRNA expression, measured by competitive RT-PCR, was increased by a factor of at least 1000. Precision-cut liver slices are a useful tool for the study of various hepatic functions, drug metabolism and its induction in vitro.     

Metabolism of [ring-U-14C] agaritine by precision-cut rat, mouse and human liver and lung slices

Agaritine [(beta-N-[gamma-L(+)glutamyl]-4-hydroxymethylphenylhydrazine] is present in the common cultivated mushroom Agaricus bisporus and several agaritine derivatives have been shown to produce tumours in experimental animals. In this investigation the metabolism of [ring-U-14C]agaritine has been studied in precision-cut rat, mouse and human liver slices and in precision-cut rat and mouse lung slices. To confirm the functional viability of the tissue slice preparations, the metabolism of 7-ethoxycoumarin was also studied. Liver and lung slices from all species metabolized 50 microM 7-ethoxycoumarin to 7-hydroxycoumarin, which was conjugated with D-glucuronic acid and sulfate. Incubation of rat, mouse and human liver slices, and rat and mouse lung slices with 25 microM [14C]agaritine resulted in a time-dependent formation of metabolite(s), which bound covalently to tissue slice proteins. Agaritine metabolite covalent binding was greater in mouse liver than in rat and human liver slices and was greater in mouse lung than in rat lung slices. No correlation was observed between agaritine metabolite covalent binding and tissue slice gamma-glutamyltransferase activity. Additional studies with mouse liver slices showed that [14C]agaritine was also metabolized to a number of unknown polar metabolites. These results demonstrate that agaritine can be metabolized by enzymes present in mammalian liver and lung.     

Factors influencing sperm-zona pellucida binding in vitro using the intact zona model

The zona pellucida binding assay assesses the ability of spermatozoa to bind to the zona pellucida. The present study investigated the influence of: (i) prior oocyte exposure to spermatozoa on subsequent sperm-zona pellucida binding in vitro; and (ii) cryopreservation of oocytes. Only oocytes obtained from fertile donors were used and the binding capacity of non-inseminated, cryopreserved oocytes was compared with both inseminated/unfertilized, cryopreserved oocytes and inseminated/unfertilized, non-cryopreserved oocytes recovered from in-vitro fertilization cultures on sperm-zona pellucida binding using an intact zona model. There was no statistically significant difference in sperm-zona binding between non-inseminated, cryopreserved oocytes (range 9.6-23.2), inseminated/unfertilized, cryopreserved oocytes (range 15.0-16.0) and inseminated/ unfertilized, non-cryopreserved oocytes (range 3.3-23.0). The coefficient of variation for sperm binding to all oocyte groups was very large (range 37-121%). We conclude that neither prior exposure of human oocytes to human spermatozoa nor cryopreservation of human oocytes influences the subsequent binding of a different population of spermatozoa to the zona pellucida. However, large oocyte-to-oocyte variation of sperm-zona binding may diminish the usefulness of this assay in clinical practice and as a research tool.     

Clearance of N-nitrosodimethylamine and N-nitrosodiethylamine by the perfused rat liver. Relationship to the Km and Vmax for nitrosamine metabolism

The first-pass clearance of dietary N-nitrosodimethylamine (NDMA) by the liver is the most important factor in the pharmacokinetics of this carcinogen in the rat, but is less important in the pharmacokinetics of N-nitrosodiethylamine (NDEA). The reason for the difference in clearance of these two nitrosamines is not known. These experiments were carried out to see whether the general characteristics of the clearance of these two carcinogens in vivo could be reproduced in the perfused liver, and whether the clearance could be correlated with the Michaelis-Menten parameters Km and Vmax for their metabolism. If this could be done one would be able to predict the possible extent of first-pass clearance of nitrosamines in man from measurement of Km and Vmax for nitrosamine metabolism by the human liver. The Km (22 microM) and Vmax (10.2 and 13.4 nmol/g liver/min) for the metabolism of NDMA by slices from two human livers, the inhibition of that metabolism by ethanol (Ki 0.5 microM), and the rate of N-7 methylation of DNA when slices are incubated with NDMA, were measured. These results are similar to those reported previously with rat liver. The Km (27 microM) for the metabolism of NDEA by rat liver slices and the inhibition of that metabolism by ethanol (Ki 1 microM) were estimated from the rate of ethylation of the DNA of the slices. The clearance of both these nitrosamines by the perfused rat liver was measured, and the results appeared to parallel those in vivo with a striking difference between the clearance of NDMA and NDEA. The maximal rate of clearance of NDMA was 11.2 nmol/g liver/min and of NDEA 8.9 nmol/g liver/min, similar to the Vmax for metabolism of NDMA by liver slices and to the estimated maximal rate of liver metabolism of both nitrosamines in the living rat. However, although the Km for metabolism of these two nitrosamines by liver slices is similar (about 25 microM), the logarithmic mean sinusoidal concentration [see Bass and Keiding, Biochem Pharmacol 37: 1425-1431, 1988] giving half maximal clearance during perfusion (the equivalent to Km) was 2.3 microM for NDMA and 10.6 microM for NDEA. The almost 5-fold difference between these two values is the basis for the difference between the clearance of the two nitrosamines.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of cord blood CD34+ cells purified after cryopreservation

Many practical issues regarding processing blood samples for cord blood banking remain. After cryopreservation, a reduction in clonogenicity has been reported, although it is unknown whether this is associated with lower potential for long-term engraftment. CD34+ cell purification of cryopreserved cord blood (CB) may be important for the clinical application of in vitro expansion. We compared purity, yield, clonogenicity, and growth in long-term stromal-based culture of fresh and cryopreserved CD34+ purified cells (n = 12) using the miniMACS separation system. Mean purity of CD34+ cells was 93% when processed before and 73% when processed after cryopreservation. Fresh CD34+ cells had higher clonogenic potential than cryopreserved cells (45 vs 20%, p < 0.05) in CFU-Mix assays, indicating that progenitor cell loss during cryopreservation is due in part to reduced cloning efficiency of viable CD34+ cells. In long-term culture (LTC) on irradiated normal human bone marrow stroma (n = 7), CFU-GM production in the two groups was the same over 12 weeks, suggesting identical long-term culture-initiating cell (LTC-IC) numbers. We conclude that apparent clonogenic cell loss during cryopreservation is associated with relative sparing of the more primitive LTC-ICs. CFU-Mix assays may therefore underestimate the transplant potential of cryopreserved CB. Purification of CD34+ cells following cryopreservation gives sufficient purity for detailed evaluation of CD34+ cells and for stem cell expansion.     

Inhibitory monoclonal antibody to human cytochrome P450 2B6

The human cytochrome P450 2B6 metabolizes, among numerous other substrates, diazepam, 7-ethoxycoumarin, testosterone, and phenanthrene. A recombinant baculovirus containing the human 2B6 cDNA was constructed and used to express 2B6 in Sf9 insect cells. The 2B6 was present at 1.8 +/- 0.4% of the total cellular protein and was purified to a specific content of 13.3 nmol/mg protein. Mice were immunized with the purified 2B6, and a total of 811 hybridomas were obtained from the fusion of NS-1 myeloma cells and spleen cells of the immunized mice. Monoclonal antibodies (MAbs) from 24 of the hybrids exhibited immunobinding to 2B6 as determined by ELISA. One of the MAbs, 49-10-20, showed a strong immunoblotting activity and was highly inhibitory to 2B6 enzyme activity. MAb 49-10-20 inhibited cDNA-expressed 2B6-catalyzed metabolism of diazepam, phenanthrene, 7-ethoxycoumarin, and testosterone by 90-91%. MAb 49-10-20 showed extremely high specificity for 2B6 and did not bind to 17 other human and rodent P450s or inhibit the metabolism of phenanthrene catalyzed by human 1A2, 2A6, 2C8, 2C9, 2D6, 2E1, 3A4, and 3A5. MAb 49-10-20 was used to determine the contribution of 2B6 to the metabolism of phenanthrene and diazepam in human liver. In ten liver samples, MAb 49-10-20 inhibited phenanthrene metabolism variably by a wide range of 8-42% and diazepam demethylation by 1-23%. The degree of inhibition by the 2B6 specific MAb 49-10-20 defines the contribution of 2B6 to phenanthrene and diazepam metabolism in each human liver. This technique using inhibitory MAb 49-10-20 determines the contribution of 2B6 to the metabolism of its substrates in a human tissue containing multiple P450s. This study is a prototype for the use of specific and highly inhibitory MAbs to determine individual P450 function.     

The biotransformation of the ergot derivative CQA 206-291 in human, dog, and rat liver slice cultures and prediction of in vivo plasma clearance

Liver slice cultures from humans, dogs, and rats were used to investigate the biotransformation of the dopaminergic ergot agonist CQA 206-291 and to predict pharmacokinetic values for hepatic intrinsic clearance and plasma clearance. CQA 206-291 was extensively metabolized in the liver slice cultures and in vivo. The HPLC metabolite patterns from the liver slice cultures were similar for all three species, indicating the occurrence of the same metabolic pathways for CQA 206-291 biotransformation. The rate of formation of CQ 32-084, a pharmacologically active N-deethylated metabolite, exceeded that of metabolite d, a primary metabolite, by 1.4 fold in human liver slices, and by 1.7 fold in rat liver slices. In dog liver slice cultures, metabolite d formation exceeded CQ 32-084 formation by 1.3 fold and was formed at a statistically significantly greater rate (3 fold) than in either human or rat liver slices. The metabolism of ergots like CQA 206-291 by human fetal liver was also demonstrated in this study. However, the prominent metabolite from fetal and adult human liver microsomes was metabolite d with minor amounts of CQ 32-089 being formed. A major route of excretion for the metabolites of CQA 206-291 is the kidney, yet the kidney does not contribute to the metabolism of CQA 206-291. Kidney slices derived from humans, rats, and dogs did not metabolize CQA 206-291 within 24 hr. CQA 206-291 intrinsic clearance was derived from the half-life of parent drug disappearance in the liver slice and hepatocyte cultures, and from the ratio of Vmax/Km of human and rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Severe and complicated malaria. Report of six cases

The major obstacle for successful xenotransplantation of islets to large animals and human diabetics is the host rejection. To address the rejection problem, we studied the efficacy of UV-B irradiation, cryopreservation and immunosuppression on the in vivo functional time and immunogenicity of adult porcine islets (PI) in outbred CD1 mice. Exposure of PI to UV-B irradiation between 300-1800J/M2 did not affect the cellular viability as assessed by fluorescein diacetate or their daily insulin secretion in vitro. Fresh PI normalized the blood glucose (BG) of diabetic CD1 mice for 3.1+/-0.6 (n = 8, mean+/-SEM) days. Islets treated with 600J/M2 UV-B irradiation or cryopreservation had similar graft functional times to fresh islets upon transplantation in diabetic CD1 mice. Immunosuppression with cyclosporin A (CsA), antilymphocyte serum (ALS) and FK506 prolonged the functional time of fresh pig islets to 7.9+/-0.9 (n = 9), 6.2+/-1.3 (n = 5) and 24.2+/-10.4 (n = 12) days, respectively. However, additional pretransplant treatment with either UV-B irradiation or cryopreservation did not further increase the functional time of pig islets in mice immunosuppressed with CsA. Furthermore, there was no apparent difference in the frequency of appearance of cytotoxic antibodies and antibody titers in the recipients of UV-B irradiated or cryopreserved pig islet compared with non-treated islets. The UV-B irradiation and cryopreservation of PI before transplantation with the present protocols did not appear to have significant effect on the islet immunogenicity when assessed by in vivo survival duration and anti-donor antibody titer production.     

Application of a first-pass effect model to characterize the pharmacokinetic disposition of venlafaxine after oral administration to human subjects

Venlafaxine (VEN), a drug used in the treatment of depression, undergoes significant first-pass metabolism after oral dosing to O-desmethylvenlafaxine (ODV), a metabolite with comparable therapeutic activity to that of parent drug. The pharmacokinetic disposition of VEN was characterized using a "first-pass" model that incorporates a presystemic compartment (liver) to account for the first-pass metabolism of VEN to ODV. A series of differential equations were simultaneously fitted to plasma concentrations of parent and metabolite. A good fit of the model to observed data was demonstrated, generating estimates for the following parameters: ka (1.31 +/- 0.009 hr-1), VVEN (252 +/- 87.6 liters), CLint (65.8 +/- 39.7 liters/hr), RL (liver:plasma partition coefficient, 29.6 +/- 18. 3), VODV (181 +/- 84.1 liters), and CLODV (23.5 +/- 12.5 liters/hr). Parameter estimates correlated closely with those obtained through noncompartmental methods. These results indicate that the time-course disposition of a compound undergoing first-pass hepatic metabolism after oral dosing can be successfully modeled.     

Hypoglycemic effect of water extract of the root of Pandanus odorus RIDL

Testosterone and 7-ethoxycoumarin were used as substrates to quantify the maintenance of phase I and II enzymes in precision-cut rat liver slices in dynamic organ culture. Testosterone hydroxylations, 7-ethoxycoumarin O-deethylation, 7-hydroxycoumarin sulfate, and 7-hydroxycoumarin glucuronide formation were all maintained at initial levels in the slice incubation media after incubation for up to 4 hr. The activities of various cytochrome P450 isozymes, measured using the stereospecific and regiospecific hydroxylation of testosterone and the maintenance of phase I and II metabolism using 7-ethoxycoumarin, are therefore suggested to be stable over short-term incubations in a physiological buffer. The testosterone hydroxylation assay is also suggested as a versatile slice metabolic viability marker for various P450 activities over longer periods.     

Use of precision-cut rat liver slices for studies of xenobiotic metabolism and toxicity: comparison of the Krumdieck and Brendel tissue slicers

1. In this study we have compared freshly cut and cultured precision-cut rat liver slices produced by the Krumdieck and Brendel-Vitron tissue slicers. 2. No significant differences were observed in levels of protein, potassium, total glutathione (i.e. GSH and GSSG), reduced glutathione (GSH) and cytochrome P450 and activities of 7-ethoxyresorufin O-deethylase and 7-benzoxyresorufin O-debenzylase in freshly cut rat liver slices produced by the two tissue slicers. However, levels of oxidized glutathione (GSSG) were significantly greater in liver slices produced with the Brendel-Vitron tissue slicer. 3. Precision-cut rat liver slices produced with both tissue slicers were cultured for 0 (i.e. a 1-h preincubation), 24 and 72 h in a dynamic organ culture system in an atmosphere of either 95% 02/5% CO2 or 95% air/5% CO2. 4. Apart from small differences in glutathione levels in 0 and 24 h cultured liver slices, no significant differences were observed in the parameters measured between liver slices prepared with both tissue slicers and cultured in both gas phases. 5. With liver slices produced by both tissue slicers 50 microM sodium arsenite produced a greater induction of heat shock protein 70 levels in slices cultured for 24 h in a high oxygen than in an air atmosphere. 6. These results suggest that both tissue slicers can readily produce precision-cut liver slices for studies of xenobiotic metabolism and toxicity. However, the data suggest that for any given application of precision-cut tissue slices it is desirable to establish optimal culture conditions     

Identification and characterization of human cytochrome P450 isoforms interacting with pimozide

Using human liver microsomes (HLMs) and recombinant human cytochrome P450 (CYP450) isoforms, we identified the major route of pimozide metabolism, the CYP450 isoforms involved, and documented the inhibitory effect of pimozide on CYP450 isoforms. Pimozide was predominantly N-dealkylated to 1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one (DHPBI). The formation rate of DHPBI showed biphasic kinetics in HLMs, which suggests the participation of at least two activities. These were characterized as high-affinity (K(m1) and Vmax1) and low-affinity (K(m2) and Vmax2) components. The ratio of Vmax1 (14 pmol/min/mg protein)/K(m1) (0.73 microM) was 5.2 times higher than the ratio of Vmax2 (244 pmol/min/mg protein)/K(m2) (34 microM). K(m2) was 91 times higher than K(m1). The formation rate of DHPBI from 25 microM pimozide in nine human livers correlated significantly with the catalytic activity of CYP3A (Spearman r = 0.79, P = .028), but not with other isoforms. Potent inhibition of DHPBI formation from 10 microM pimozide was observed with ketoconazole (88%), troleandomycin (79%), furafylline (48%) and a combination of furafylline and ketoconazole (96%). Recombinant human CYP3A4 catalyzed DHPBI formation from 10 microM pimozide at the highest rate (V = 2.2 +/- 0.89 pmol/min/pmol P450) followed by CYP1A2 (V = 0.23 +/- 0.08 pmol/min/pmol P450), but other isoforms tested did not. The K(m) values derived with recombinant CYP3A4 and CYP1A2 were 5.7 microM and 36.1 microM, respectively. Pimozide itself was a potent inhibitor of CYP2D6 in HLMs when preincubated for 15 min (Ki = 0.75 +/- 0.98 microM) and a moderate inhibitor of CYP3A (Ki = 76.7 +/- 34.5 microM), with no significant effect on other isoforms tested. Our results suggest that pimozide metabolism is catalyzed mainly by CYP3A, but CYP1A2 also contributes. Pimozide metabolism is likely to be subject to interindividual variability in CYP3A and CYP1A2 expression and to drug interactions involving these isoforms. Pimozide itself may inhibit the metabolism of drugs that are substrates of CYP2D6.