Local extensive granulomatous inflammation of the neck region and lymphangitis caused by Lichtheimia corymbifera infection in a Japanese Black calf.

A 7-month-old female Japanese Black calf developed elongated, nodular mass measuring 30 × 16 cm extended from the retropharyngeal region to mid lateral neck region. Histological examination revealed granulomatous lymphangitis with non-septate fungal hyphae recognized throughout the lesions. Fungal culture, DNA sequencing and molecular phylogenetic tree analysis confirmed the sequence of Lichtheimia corymbifera. The lymphogenous route was speculated to be the main route of fungal spread leading to the characteristic nodular appearance of this case.

Control of mammary tumor differentiation by SKI-606 (bosutinib).

C-Src is infrequently mutated in human cancers but it mediates oncogenic signals of many activated growth factor receptors and thus remains a key target for cancer therapy. However, the broad function of Src in many cell types and processes requires evaluation of Src-targeted therapeutics within a normal developmental and immune-competent environment. In an effort to understand the appropriate clinical use of Src inhibitors, we tested an Src inhibitor, SKI-606 (bosutinib), in the MMTV-PyVmT transgenic mouse model of breast cancer. Tumor formation in this model is dependent on the presence of Src, but the necessity of Src kinase activity for tumor formation has not been determined. Furthermore, Src inhibitors have not been examined in an autochthonous tumor model that permits assessment of effects on different stages of tumor progression. Here we show that oral administration of SKI-606 inhibited the phosphorylation of Src in mammary tumors and caused a rapid decrease in the Ezh2 Polycomb group histone H3K27 methyltransferase and an increase in epithelial organization. SKI-606 prevented the appearance of palpable tumors in over 50% of the animals and stopped tumor growth in older animals with pre-existing tumors. These antitumor effects were accompanied by decreased cellular proliferation, altered tumor blood vessel organization and dramatically increased differentiation to lactational and epidermal cell fates. SKI-606 controls the development of mammary tumors by inducing differentiation.

Homozygous CDA*3 is a major cause of life-threatening toxicities in gemcitabine-treated Japanese cancer patients.

Among 242 Japanese pancreatic cancer patients, three patients (1.2%) encountered life-threatening toxicities, including myelosuppression, after gemcitabine-based chemotherapies. Two of them carried homozygous CDA*3 (CDA208G>A [Ala70Thr]), and showed extremely low plasma cytidine deaminase activity and gemcitabine clearance. Our results suggest that homozygous *3 is a major factor causing gemcitabine-mediated severe adverse reactions among the Japanese population.

Functional characterization of CYP3A4.16: catalytic activities toward midazolam and carbamazepine.

1. To assess the substrate-dependent effects of the low-activity allele of human CYP3A4, CYP3A4*16 (Thr185Ser), a recombinant wild-type (CYP3A4.1) or variant (CYP3A4.16) protein was co-expressed with human NADPH-P450 reductase in Sf21 insect cells using a baculovirus-insect cell system. 2. The holo-CYP3A4 protein level of CYP3A4.16 in insect microsomes was slightly higher than that of CYP3A4.1, while no difference in total (apo- and holo-) CYP3A4 protein levels was observed between them. 3. When midazolam was used as a substrate, K(m) and V(max) for 1'-hydroxylation in CYP3A4.16 were significantly higher and lower, respectively, than those in the wild-type, resulting in a 50% decrease in intrinsic clearance (V(max)/K(m)) of the variant. In contrast, intrinsic clearance for 4-hydroxylation of the variant was decreased by 30% due to a significant increase in K(m) without a difference in V(max). 4. Both the wild-type and variant exhibited sigmoidal kinetic profiles for carbamazepine 10,11-epoxide formation. When the modified two-site equation was applied for the analysis of kinetic parameters, K(m2) and V(max2) of CYP3A4.16 were approximately two times higher and lower than those of the wild-type, resulting in a 74% decrease in intrinsic clearance. 5. These results demonstrated that CYP3A4.16 shows the substrate-dependent altered kinetics compared with CYP3A4.1.

A new statistical screening approach for finding pharmacokinetics-related genes in genome-wide studies.

Biomedical researchers usually test the null hypothesis that there is no difference of the population mean of pharmacokinetics (PK) parameters between genotypes by the Kruskal-Wallis test. Although a monotone increasing pattern with a number of alleles is expected for PK-related genes, the Kruskal-Wallis test does not consider a monotonic response pattern. For detecting such patterns in clinical and toxicological trials, a maximum contrast method has been proposed. We show how that method can be used with pharmacogenomics data to a develop test of association. Further, using simulation studies, we compare the power of the modified maximum contrast method to those of the maximum contrast method and the Kruskal-Wallis test. On the basis of the results of those studies, we suggest rules of thumb for which statistics to use in a given situation. An application of all three methods to an actual genome-wide pharmacogenomics study illustrates the practical relevance of our discussion.

Functional characterization of two novel CYP2C19 variants (CYP2C19*18 and CYP2C19*19) found in a Japanese population.

Cytochrome P450 2C19 (CYP2C19) plays an important role in the metabolism of a wide range of therapeutic drugs and exhibits genetic polymorphism with interindividual differences in metabolic activity. We have previously described two CYP2C19 allelic variants, namely CYP2C19*18 and CYP2C19*19 with Arg329His/Ile331Val and Ser51Gly/Ile331Val substitutions, respectively. In order to investigate precisely the effect of amino acid substitutions on CYP2C19 function, CYP2C19 proteins of the wild-type (CYP2C19.1B having Ile331Val) and variants (CYP2C19.18 and CYP2C19.19) were heterologously expressed in yeast cells, and their S-mephenytoin 4'-hydroxylation activities were determined. The K(m) value of CYP2C19.19 for S-mephenytoin 4'-hydroxylation was significantly higher (3.0-fold) than that of CYP2C19.1B. Although no significant differences in V(max) values on the basis of microsomal and functional CYP protein levels were observed between CYP2C19.1B and CYP2C19.19, the V(max)/K(m) values of CYP2C19.19 were significantly reduced to 29-47% of CYP2C19.1B. By contrast, the K(m), V(max) or V(max)/K(m) values of CYP2C19.18 were similar to those of CYP2C19.1B. These results suggest that Ser51Gly substitution in CYP2C19.19 decreases the affinity toward S-mephenytoin of CYP2C19 enzyme, and imply that the genetic polymorphism of CYP2C19*19 also causes variations in the clinical response to drugs metabolized by CYP2C19.

Ingestion of heat-treated Lactobacillus rhamnosus GG prevents development of atopic dermatitis in NC/Nga mice.

BACKGROUND: Continuous oral administration of live Lactobacillus rhamnosus GG (L. GG) to pregnant subjects with atopic dermatitis and their children, suppressed the frequency of atopic dermatitis. The details of mechanisms and immune systems involved in this suppressive effect, however, remain speculative. OBJECTIVE: We sought to clarify suppressive mechanisms of L. GG on atopic dermatitis by using NC/Nga mice, a model of human atopic dermatitis. METHODS: Maternal mice and infant mice were fed with food containing or not containing heat-treated L. GG during pregnancy and breastfeeding, and after weaning. RESULTS: Control NC/Nga mice raised under an air-uncontrolled condition spontaneously manifested typical skin lesions very similar to those in patients with atopic dermatitis. On the other hand, administration of food containing heat-treated L. GG inhibited the onset and development of atopic skin lesions, accompanied by smaller numbers of mast cells and eosinophils in the affected skin sites. Mice fed with L. GG showed a significant increase in plasma IL-10 levels compared with control mice, while there was no significant difference in the proportion of splenic CD4(+)CD25(+) regulatory T cells between mice fed with L. GG and control mice. The IL-10 mRNA expression was enhanced in both Peyer's patches and mesenteric lymph nodes in mice fed with L. GG. CONCLUSION: These findings suggest that some components of heat-treated L. GG may have an ability to delay the onset and suppress the development of atopic dermatitis, probably through a strong induction of IL-10 in intestinal lymphoid organs and systemic levels.

Genetic variations and haplotype structures of the ABCB1 gene in a Japanese population: an expanded haplotype block covering the distal promoter region, and associated ethnic differences.

As functional ABCB1 haplotypes were recently reported in the promoter region of the gene, we resequenced the ABCB1 distal promoter region, along with other regions (the enhancer and proximal promoter regions, and all 28 exons), in a total of 533 Japanese subjects. Linkage disequilibrium (LD) analysis based on 92 genetic variations revealed 4 LD blocks with the same make up as previously described (Blocks -1, 1, 2 and 3), except that Block 1 was expanded to include the distal promoter region, and that a new linkage between polymorphisms -1,789G>A in the distal promoter region and IVS5 + 123A>G in intron 5 was identified. We re-assigned Block 1 haplotypes, and added novel haplotypes to the other 3 blocks. The reported promoter haplotypes were further classified into several types according to tagging variations within Block 1 coding or intronic regions. Our current data reconfirm the haplotype profiles of the other three blocks, add more detailed information on functionally-important haplotypes in Block 1 and 2 in the Japanese population, and identified differences in haplotype profiles between ethnic groups. Our updated analysis of ABCB1 haplotype blocks will assist pharmacogenetic and disease-association studies carried out using Asian subjects.

Postnatal expression profile of OBCAM implies its involvement in visual cortex development and plasticity.

This study examined the expression of a neuron-specific cell adhesion molecule, OBCAM (opioid-binding cell adhesion molecule), at both the mRNA and protein levels in the cat primary visual cortex at various postnatal ages, using cDNA array analysis and immunocytochemistry. Results obtained using both methods showed that the expression level of OBCAM was high in young and low in older and adult visual cortex. OBCAM-immunoreactivities were associated predominantly with perikarya and dendrites of pyramidal neurons, and OBCAM-immunopositive neurons were present in all cortical layers. Immunostaining of OBCAM in adult visual cortex showed a reduced number of immunopositive neurons and neurites and relatively lower staining intensities as compared with younger animals. In addition, the number of OBCAM-immunopositive neurons was significantly higher in the visual cortex of 4-month-old animals dark-reared from birth than those in age-matched normally reared animals. These results suggest that OBCAM may play an important role in visual cortex development and plasticity.

Haplotype structures of the UGT1A gene complex in a Japanese population.

Genetic polymorphisms of UDP-glucuronosyltransferases (UGTs) are involved in individual and ethnic differences in drug metabolism. To reveal co-occurrence of the UGT1A polymorphisms, we first analyzed haplotype structures of the entire UGT1A gene complex using the polymorphisms from 196 Japanese subjects. Based on strong linkage disequilibrium between UGT1A8 and 1A10, among 1A9, 1A7, and 1A6, and between 1A3 and 1A1, the complex was divided into five blocks, Block 8/10, Block 9/6, Block 4, Block 3/1, and Block C, and the haplotypes for each block were subsequently determined/inferred. Second, using pyrosequencing or direct sequencing, additional 105 subjects were genotyped for 41 functionally tagged polymorphisms. The data from 301 subjects confirmed the robustness of block partitioning, but several linkages among the haplotypes with functional changes were found across the blocks. Thus, important haplotypes and their linkages were identified among the UGT1A gene blocks (and segments), which should be considered in pharmacogenetic studies.

FT-IR study of the interlamellar water confined in glycolipid nanotube walls.

The local hydrogen-bonding environment of water confined in glycolipid nanotubes (LNTs) was investigated by Fourier transform infrared (FT-IR) spectroscopy. Using X-ray diffraction (XRD), we estimated the thickness of an interlamellar water layer, which was confined between the bilayer membranes constructing the walls of the LNTs, to be 1.3 +/- 0.3 nm. FT-IR spectroscopic measurement of the confined water showed an obvious reduction in IR absorption in both the low-energy (around 3000 cm(-1)) and high-energy regions (around 3600 cm(-1)) of the OH stretching band as compared to bulk water. The reduction around 3000 cm(-1) indicated a decrease in the relative proportion of the water molecules with a long-range network structure due to a geometrical restriction. This agrees with the results obtained for other multilamellar systems. On the other hand, the remarkable reduction around 3600 cm(-1), which was not observed in the other systems, indicated the absence of weakly hydrogen-bonded water aggregates due to the effect of sugar headgroups.

Genetic polymorphisms and haplotypes of the human cardiac sodium channel alpha subunit gene (SCN5A) in Japanese and their association with arrhythmia.

Genetic variations in cardiac ion channels have been implicated not only as the causes of inherited arrhythmic syndromes, but also as genetic risk factors for some acquired arrhythmias. To elucidate the potential roles of genetic polymorphisms of the alpha subunit of the voltage-gated sodium channel type V (SCN5A) in cardiac rhythm disturbance, the entire SCN5A coding exons and their flanking introns were sequenced in 166 Japanese arrhythmic patients and 232 healthy controls. We detected 69 genetic variations, including 54 novel ones. Out of the 12 novel nonsynonymous single nucleotide polymorphisms (SNPs), p.Leu1988Arg was found at a frequency of 0.015. The other 11 SNPs were rare (0.001), with 6 found in arrhythmic patients and 5 in healthy controls. The frequency of a novel intronic SNP, c.703+130G>A, was significantly higher in the patients than in the controls, suggesting this SNP is associated with an unknown risk factor for arrhythmia. Following linkage disequilibrium analysis, the haplotype structure of SCN5A was inferred using high-frequency SNPs. The frequency of the haplotype harbouring both p.Leu1988Arg and the common SNP p.His558Arg (haplotype GG) was significantly lower in the patients than in the controls. This finding suggests that this haplotype (GG) might have been positively selected in the controls because of its protective effect against arrhythmias. This study provides fundamental information necessary to elucidate the effect of genetic variations in SCN5A on channel function and cardiac rhythm in Japanese, and probably in the Asian population.

Non-synonymous single nucleotide alterations in the microsomal epoxide hydrolase gene and their functional effects.

1. By sequencing genomic DNA from 72 established cell lines derived from Japanese individuals, we detected 25 single nucleotide alterations in the microsomal epoxide hydrolase (EPHX1) gene. Of them, five were exonic alterations resulting in amino acid alterations (77C>G, T26S; 128G>C, R43T; 337T>C, Y113H; 416A>G, H139R; 823A>G, T275A). The T26S, R43T, Y113H and H139R substitutions were found at relatively high frequencies and seemed to be polymorphic, and T26S and T275A were novel. 2. To examine the effects of these amino acid alterations on EPHX1 function, EPHX1 cDNA constructs of wild-type and five variants were transfected into COS-1 cells, and their hydrolytic activities for cis-stilbene oxide were determined in vitro. Although all of the transfectants expressed EPHX1 mRNA and protein at similar levels, the variant H139R protein was expressed at a significantly higher level (128% of the wild-type). K(m) values were not significantly different between the wild-type and variants. 3. Increase (140%) in the enzymatic activity (V(max)) of the variant H139R was accompanied by the increased EPHX1 protein level without any significant change in the intrinsic EPHX1 activity. On the other hand, the variant R43T showed increased values for V(max) and clearance (V(max)/K(m)) (around 130%) both on a microsomal protein basis and on a EPHX1 protein basis. 4. These results suggest that R43T as well as H139R increase epoxide hydrolase activity.

Gene expression patterns during enhanced periods of visual cortex plasticity.

During a critical period in its postnatal development the mammalian visual cortex displays susceptibility to experience-dependent alterations of neuronal response properties. Plasticity represents an integrated set of developmental processes controlled by a transcriptional hierarchy that coordinates the action of many genes. To illuminate the expression of these critical genes, we examined gene expression patterns of 18371 non-redundant cDNAs in the visual cortex of cats at birth, at eye opening, at the peak of the critical period of eye dominance plasticity and in the adult cat using filter-based cDNA arrays and software-based hierarchical cluster analysis. We identified a small set of genes that were selectively expressed during the peak of the critical period for plasticity. We further examined the patterns of expression of these genes by analyzing the gene expression pattern of dark-reared chronologically older animals that are known to retain this ocular dominance plasticity beyond the chronologically defined critical period. This additional cluster assessment allowed us to separate age-related changes in the patterns of gene expression from plasticity-related changes, thus identifying a subset of genes that we define as plasticity candidate genes. Those plasticity candidate genes that have previously characterized functions include participants in second messenger systems, in cell adhesion, in transmitter recycling and cytokines, among others. Comparison of cDNA array quantitation with reverse transcription-polymerase chain reaction showed almost identical expression profiles for three genes that we examined. The expression pattern of one identified gene, opioid binding cell adhesion molecule, from the cDNA array analysis, is also in agreement with immunocytochemical results. We conclude that the approach of high-density cDNA array hybridization can be used as a useful tool for examining a complex phenomenon of developmental plasticity since it is amenable to multiple developmental stage gene expression comparisons.

Gene expression profiling of Ca2+-atpase inhibitor DTBHQ and antigen-stimulated RBL-2H3 mast cells.

OBJECTIVE AND DESIGN: Ca2+ signaling is critical for mast cell activation by antigen stimulation, and we previously described that the signaling can be mimicked by Ca2+-ATPase inhibitors. We therefore investigated the effect of the Ca2+-ATPase inhibitor and antigen stimulation on the gene expression profiles of RBL-2H3 mast cells. MATERIAL: A Ca2+-ATPase inhibitor, 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ), an antigen (dinitrophenylated BSA), a high-density oligonucleotide microarray (Affymetrix GeneChip) technique, and a well-characterized rat mast cell line RBL-2H3 were used. TREATMENT: RBL-2H3 cells were activated for 3 h with 10 microM DTBHQ, which increases cytosolic Ca2+ concentration, or 10 microg/ml antigen, which cross-links IgE receptors, and the mRNA expression profiles (8,799 genes) were analyzed with GeneChip arrays (n = 3). METHODS: Expression levels were measured by GeneChip, and the differences were tested by Welch's t-test and P-values less than 0.05 were considered statistically significant. Values are expressed as means +/- SEM. RESULTS: The genes, including MCP-1, GADD45, Relaxin H1, CSF-1, c-jun-oncogene, Pyk-2, NKR-P2 and CREM, were significantly up-regulated by both DTBHQ and antigen stimuli, whereas the genes including interleukin (IL)-3, IL-4, IL-9, IL-13, GADD153, butyrate response factor, and Fas ligand, were up-regulated by DTBHQ alone. On the other hand, the expression of several genes, including GATA-1, were down-regulated by DTBHQ stimulation. CONCLUSIONS: These results suggest 1) that DTBHQ seems to induce proinflammatory responses by stimulating the production of several cytokines through the expression of several transcription factors, 2) that the changes in gene expression profile induced by DTBHQ and by IgE receptor cross-linking in mast cells were almost the same, but many more stress-inducible genes like GADD 153 were up-regulated by the former.

Human liver UDP-glucuronosyltransferase isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin.

1. The human liver UDP-glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), have been studied using microsomes from human liver and insect cells expressing human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B15). 2. The glucuronidation of SN-38 was catalysed by UGT1A1, UGT1A3, UGT1A6 and UGT1A9 as well as by liver microsomes. Among these UGT isoforms, UGT1A1 showed the highest activity of SN-38 glucuronidation at both low (1 microM) and high (200 microM) substrate concentrations. The ranking in order of activity at low and high substrate concentrations was UGT1A1 > UGT1A9 > UGT1A6 > UGT1A3 and UGT1A1 > UGT1A3 > UGT1A6 > or = UGT1A9, respectively. 3. The enzyme kinetics of SN-38 glucuronidation were examined by means of Lineweaver-Burk analysis. The activity of the glucuronidation in liver microsomes exhibits a monophasic kinetic pattern, with an apparent Km and Vmax of 35.9 microM and 134 pmol min(-1) mg(-1) protein, respectively. The UGT isoforms involved in SN-38 glucuronidation could be classified into two types: low-Km types such as UGT1A1 and UGT1A9, and high-Km types such as UGT1A3 and UGT1A6, in terms of affinity toward substrate. UGT1A1 had the highest Vmax followed by UGT1A3. Vmax of UGT1A6 and UGT1A9 were approximately 1/9 to 1/12 of that of UGT1A1. 4. The activity of SN-38 glucuronidation by liver microsomes and UGT1A1 was effectively inhibited by bilirubin. Planar and bulky phenols substantially inhibited the SN-38 glucuronidation activity of liver microsomes and UMT1A9, and/or UGT1A6. Although cholic acid derivatives strongly inhibited the activity of SN-38 glucuronidation by UGT1A3, the inhibition profile did not parallel that in liver microsomes. 5. These results demonstrate that at least four UGT1A isoforms are responsible for SN-38 glucuronidation in human livers, and suggest that the role and contribution of each differ substantially.

A new metabolite of irinotecan in which formation is mediated by human hepatic cytochrome P-450 3A4.

Irinotecan (CPT-11) is an anticancer prodrug. It is converted by carboxylesterase to yield an active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), which acts as a topoisomerase I inhibitor. Several oxidative metabolites of CPT-11 have been identified in humans, including 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin (APC) and 7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecin (NPC), generated by cytochrome P-450 3A4 (CYP3A4). Other minor metabolites in which metabolic pathways and biologic activities have not been identified also exist. To further investigate the metabolism of CPT-11 in human liver, we analyzed metabolites of CPT-11 in human hepatic microsomes using a high-performance liquid chromatography/mass spectrometry (HPLC/MS) system and detected a new metabolite that was the major one produced in the microsomal system. HPLC-tandem mass spectrometry (HPLC/MS/MS) analysis indicated that this compound was an oxidation product formed by the loss of two hydrogen atoms from the terminal piperidine ring. Kinetic analyses indicated that a single enzyme generated the metabolite, and we have identified this enzyme in two in vitro systems. The formation of the new metabolite was significantly inhibited by SKF525A, ketoconazole, and an anti-CYP3A4 antibody and catalyzed specifically by CYP3A4 expressed in insect microsomes. A significant correlation was observed between the generation of this metabolite and the CYP3A4 content in individual human hepatic microsomes. These findings indicate that this newly detected metabolite is a CYP3A4-generated product that may be produced in hepatic microsomes of patients treated with CPT-11.

Apparent low frequency of sequence variability within the proximal promoter region of the cytochrome P450 (CYP) 3A5 gene in established cell lines from Japanese individuals.

The members of the cytochrome P450 (CYP) 3A subfamily play an important role in the metabolism of more than 50% of the drugs metabolized by CYPs. Among the CYP3A members, CYP3A5 is known to exhibit polymorphic expression within the human liver. We hypothesized that a single nucleotide polymorphism (SNP) in the 5'-regulatory region of the CYP3A5 gene might be the cause of CYP3A5 polymorphic expression. Due to the existence of "CYP3AP1," a highly homologous sequence to the CYP3A5 gene, it was necessary to make specific primers to the CYP3A5 gene. In the present study, we designed a series of oligonucleotide primers for sequencing the proximal promoter region of the CYP3A5 gene in order to search for the putative regulatory single nucleotide polymorphism. We examined 86 established cell lines derived from Japanese individuals as a representation of the Japanese population. However, no SNP was detected in the promoter region of the CYP3A5 gene isolated from the cell lines used, suggesting other causal factors for the observed polymorphism of CYP3A5-dependent drug metabolism.

High-performance liquid chromatographic assay for glucuronidation activity of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in human liver microsomes.

A simple and sensitive assay for glucuronidation activity of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in human liver microsomes by high-performance liquid chromatography (HPLC) with fluorescence detection is reported. The method was validated for the determination of SN-38 glucuronide (SN-38G) with respect to specificity, linearity, recovery, stability, precision, accuracy, and limits of detection and quantitation. There was no interference from matrix and non-enzymatic reactions. The calibration curve for SN-38G was linear from 5 to 500 nM. Average recoveries ranged from 98 to 100% in spiked human liver microsome samples, and the SN-38G was stable at 4 degrees C for at least 72 h. The newly developed method was found to be more sensitive and selective than previous methods using thin layer chromatography and HPLC. The limit of quantitation for SN-38G was 5 nM (2.5 pmol/assay). The intra- and inter-day precision and accuracy were less than 7 and 4%, respectively. The intra- and inter-day precision of enzyme assay for UDP-glucuronosyltransferase (UGT) activity toward SN-38 in human liver microsomes was less than 4%. With this improved sensitivity, the kinetics of SN-38 glucuronidation in human liver microsomes could be determined more precisely. Therefore, this method is applicable to in vitro study on the side effects and drug interactions of CPT-11 using small amounts of biological sample.