Department of Biopharmaceutics, School of Pharmaceutical Sciences, Central South University, Changsha, Hunan 410013, PR China.
This study describes an high-performance liquid chromatographic (HPLC)-UV method for the simultaneous determination of 6beta-hydroxycortisol (6beta-OHF) and cortisol (F) in human urine and plasma. The within-day relative standard deviation of the three concentrations for both analytes was less than 6.2%. Accuracy determined at three concentrations ranged between 95 and 107%. The extraction recoveries were 64.1+/-4.3 and 88.1+/-2.4% at three concentrations for 6beta-OHF and F in urine, respectively. The extraction recoveries were 88.7+/-1.4% at three concentrations for F in plasma. This is the first HPLC method that can simultaneously determine 6beta-OHF and F in human urine and plasma and is suitable for routine assessment of the CYP3A activity expressed as 6beta-hydroxylation clearance.
Mesh-terms: Adult; Chromatography, High Pressure Liquid :: methods; Circadian Rhythm; Cytochrome P-450 CYP3A :: genetics; Female; Humans; Hydrocortisone :: analogs & derivatives; Hydrocortisone :: analysis; Hydrocortisone :: blood; Hydrocortisone :: urine; Male; Phenotype; Reproducibility of Results; Research Support, Non-U.S. Gov't; Sensitivity and Specificity; Spectrophotometry, Ultraviolet;
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Qian Gong,
Zhe-Yi Hu,
Zhi-Zhuang Huang,
Li-Qing Wang,
Wen-Fang Liu,
Xin Guo,
Wei Cao,
Ting Wang,
Ze-Neng Cheng
Objective To determine the pharmacokinetic interaction between cefalor and bromhexine in healthy Chinese volunteers. Methods Twelve subjects received a cefaclor (CEF) treatment, a bromhexine (BHX) treatment, and a co-treatment of CEF and BHX with a 3*3 Latin square design. The wash-out time between periods was 14 days. The plasma and urine drug concentrations of CEF and BHX were detected by HPLC-UV and LC/MS, respectively. Results All the 12 volunteers completed the study. There were no significant differences in AUC( -t) and C(max) of CEF in logarithm between the single administration group of CEF and the co-administration group of CEF with BHX. Two one sided t-test showed that CEF was bioequivalent in the 2 groups. There were no significant differences in t(max), MRT, t(1/2), and Clr between the 2 groups. V(d)/F was significantly lower in the single CEF group than in the co-administration group of CEF and BHX. There were no significant differences of AUC( -t) and C(max) of BHX in logarithm between the single administration group of BHX and the co-administration group of BHX with CEF. Two one sided t-test showed that BHX was bioequivalent in the 2 groups. There were no significant differences in t(max), MRT, t(1/2), V(d)/F, and Clr between the 2 groups. Conclusion There is no significant pharmacokinetic parameter change in the drug absorption, metabolism, and excretion, but V(d)/F of CEF significant increases in the co-administration of CEF with BHX. The co-administration of CEF and BHX has no adverse drug interaction. The increase of V(d)/F may be a favorable drug interaction, which may be the mechanism of the synergistic effect of the 2 drugs.
School of Pharmaceutical Sciences, Central South University, Changsha 410013, China.
OBJECTIVE: To explore the inhibitive effects of 1,3,8-trihydroxy-5-methoxyxanthone (TMX) on cytochrome P450s (CYP450s) in human liver microsomes. METHODS: Probe drugs were incubated with and without adding TMX to determine the changes of enzyme activities. The concentration ratio of metabolites to probe drugs was used to present enzyme activities. Concentrations of the probe drugs and their metabolites in the incubated mixture were detected by high performance liquid chromatography. RESULTS: The variations (mean, 95%CI) of the activities of CYP1A2, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 were 2.95 x 10(-3)(2.03 x 10(-3), 3.88 x 10(-3)), 3.14 x 10(-2)(1.87 x 10(-2), 4.42 x 10(-2)), 2.27 x 10(-3)(-1.4 x 10(-2),1.81 x 10(-2)), 7.72 x 10(-2)(- .83 x 10(-2), .2374), and - .2548 (-2.9802, 2.4707), respectively. The activities of CYP1A2 and CYP2C9 were significantly reduced in the present of TMX. CONCLUSION: TMX (10 micromol/L) has significant inhibitive effect on the activities of CYP1A2 and CYP2C9, but no significant inhibitive effect on the activities of CYP2C19, CYP2E1 and CYP3A4.
Department of Biopharmaceutics, School of Pharmaceutical Sciences, Central South University, Changsha 410013, PR China.
An HPLC-UV method was developed and validated for the determination of AKF-PD in whole blood of rat. Phenacetin was chosen as the internal standard, and the separation was achieved on a C(18) column with methanol and .02M phosphate buffer (pH 3.2) as mobile phase. The obtained calibration graphs were linear (r= .9999, n=9) in the range of .203-52.0mugml(-1). The low limit of quantitation was .203mugml(-1). This method can be used to study the pharmacokinetics of AKF-PD in rat.
Xiaoping Chen,
Liqing Wang,
Lianteng Zhi,
Gangqiao Zhou,
Haijian Wang,
Xiumei Zhang,
Bingtao Hao,
Yunping Zhu,
Zeneng Cheng,
Fuchu He
Department of Genomics and Proteomics, Beijing Institute of Radiation Medicine, Beijing, China.
OBJECTIVE: This study was designed to better understand genetic variation in the cytochrome P450 (CYP) gene CYP1A2 and its impact on CYP1A2 activity in Chinese subjects. METHODS: CYP1A2 genetic polymorphisms were screened by direct sequencing in 27 selected Chinese subjects. Plasma 1,7-dimethylxanthine/caffeine ratios 5 hours after a 100-mg caffeine administration, used as an index of CYP1A2 in vivo activity, were determined in 422 healthy subjects. Five single-nucleotide polymorphism markers, including G-860A (CYP1A2*1C), T-3594G, G-3113A, A-163C (CYP1A2*1F), and C5347T (CYP1A2*1B), were selected and genotyped by either polymerase chain reaction-restriction fragment length polymorphism or direct sequencing. RESULTS: Thirteen polymorphisms and 2 linkage disequilibrium blocks with a boundary around -2467 were identified at this locus. The allele frequency for -3860A,-3594G,-3113A,-163C, and 5347T was .21, .15, .10, .36, and .14, respectively, in the CYP1A2-phenotyped cohort. A significant difference in CYP1A2 activity was observed among genotypes of polymorphism G-3113A (P =.038), and CYP1A2 activity in subjects carrying the AA genotype was lower than that in those carrying the GA (P =.096) and GG genotypes (P =.036):- .45 +/- .05 (mean +/- SD),- .32 +/- .16, and - .29 +/- .16, respectively. Further analysis based on haplotype pairs found a 1.92-fold variation (95% confidence interval, 1.13-2.71) in mean CYP1A2 activity between haplotype pairs 13 and 15, and the difference was significant (- .19 +/- .15 versus - .45 +/- .05, P =.016). As compared with haplotype pair 10, haplotype pairs 9 and 15 and most haplotype pairs heterozygous for the haplotype with an A allele at -3113, including pairs 5, 8, and 12, also showed significantly lower CYP1A2 activity (P =.015,.048,.008,.024, and .014 for pairs 5, 8, 9, 12, and 15, respectively). In addition, haplotype pairs 5, 9, and 12 also showed significantly lower CYP1A2 activity than pair 13 (P =.034,.020, and .037 for pairs 5, 9, and 12, respectively). CONCLUSIONS: The G-3113A polymorphism is associated with decreased CYP1A2 activity, haplotype pairs 10 and 13 are responsible for high CYP1A2 activity, and haplotype pairs 5, 8, 9, 12, and 15 are responsible for low CYP1A2 activity in Chinese subjects.
Forensic Department, Xi'an Jiaotong University School of Medicine, 76# West Yanta Road, Xi'an 710061, P.R. China; The Key Laboratory of Health Ministry for Forensic Sciences, 76# West Yanta Road, Xi'an 710061, P.R. China.
Mitogen-activated protein kinases (MAPK) can be activated by opioids such as morphine via opioid receptor, and their activations have been observed in synaptic plasticity, learning, memory and addiction. Long-term exposure to morphine may induce physical dependence, manifested as somatic withdrawal symptoms such as diarrhea, body weight loss, jumping and headshaking, when drug is deprived. Though morphine dependence and withdrawal have been extensively studied, their molecular mechanisms have not been fully elucidated. In the present study, the physical dependence on morphine was developed in mice by an intermittent, escalating procedure of morphine injections, and was measured by the body weight loss and the behavioral signs (jumping and headshaking). We found that the mice with chronic morphine administration experienced dramatic body weight loss, compared with the saline-treated controls. Naloxone-precipitated withdrawal led to more body weight loss, compared with spontaneous withdrawal. Naloxone-precipitated withdrawal mice showed significantly aggravated morphine-withdrawal symptoms (including jumping and heading shaking), compared with spontaneous withdrawal mice. MAPK pathway activities in the frontal association cortex (FrA), accumbens nucleus (Acb) and caudate putamen (CPu) were examined to probe into molecular mechanism for morphine dependence and withdrawal. Compared with saline-treated mice,morphine-dependent mice and spontaneous withdrawal mice, naloxone-precipitated withdrawal mice showed a significantly increased ERK phosphorylation in FrA and Acb, but not in CPu. However, the activities of other protein kinases in the MAPK pathway, including p38 and JNK, showed no changes in FrA, Acb and CPu of the mice during the chronic morphine dependence and withdrawal phases. These results suggest that the ERK phosphorylation in FrA and Acb may be associated with naloxone-precipitated withdrawal syndrome.
Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, PA 19104.
BACKGROUND & AIMS:: Foxp3+ T regulatory cells (Tregs) help prevent autoimmunity; decreases in their numbers or functions could promote inflammatory bowel disease. Like other cells, Foxp3+ Tregs express histone/protein deacetylases (HDACs), which regulate chromatin remodeling and gene expression. We investigated whether disruption of HDAC9 activity in Tregs affects the pathogenesis of colitis in mice. METHODS:: We tested the effects of various HDAC inhibitors (HDACi) on development and progression of colitis (induced in my by injections of TsA, SAHA, or MS275) in mice. Tregs and non-Treg cells isolated from HDAC9-/- mice were transferred to immunodeficient mice and the effects were compared with those of cells transferred from wild-type mice; HDAC9 contributions to the functions of Tregs were determined during development and progression of colitis. RESULTS:: Pan-HDACi, but not class I-specific HDACi, increased the functions of Foxp3+ Tregs, prevented colitis, and reduced established colitis in mice, indicating the role of class II HDACs in controlling Treg function. The abilities of pan-HDACi to prevent/reduce colitis were associated with increased numbers of Foxp3+ Tregs and their suppressive functions. Colitis was associated with increased local expression of HDAC9; HDAC9-/- mice were resistant to the development of colitis. HDAC9-/- Tregs expressed increased levels of the heat-shock protein (HSP)70, compared with controls. Immunoprecipitation experiments indicated a interaction between HSP70 and Foxp3. Inhibition of HSP70 reduced the suppressive functions of HDAC9-/- Tregs; Tregs that over-expressed HSP70 had increased suppressive functions. CONCLUSIONS:: Strategies to decrease HDAC9 expression or function in Tregs or to increase expression of HSP70 might be used to treat colitis and other autoimmune disorders.
Division of Transplant Immunology, Children's Hospital of Philadelphia, Philadelphia 19104, USA.
Classical zinc-dependent histone deacetylases (HDACs) catalyse the removal of acetyl groups from histone tails and also from many non-histone proteins, including the transcription factor FOXP3, a key regulator of the development and function of regulatory T cells. Many HDAC inhibitors are in cancer clinical trials, but a subset of HDAC inhibitors has important anti-inflammatory or immunosuppressive effects that might be of therapeutic benefit in immuno-inflammatory disorders or post-transplantation. At least some of these effects result from the ability of HDAC inhibitors to enhance the production and suppressive functions of FOXP3(+) regulatory T cells. Understanding which HDACs contribute to the regulation of the functions of regulatory T cells may further stimulate the development of new class- or subclass-specific HDAC inhibitors with applications beyond oncology.
Department of Anatomy and Embryology, School of Basic Medical Sciences, Peking University, Beijing.
The localization of an axon growth inhibitory molecule Nogo and its receptor (NgR) was investigated in the mouse spinal cord during prenatal development of the commissural pathway. Using the antibody N18, intense signal for Nogo was localized largely on radial glia processes that are immunoreactive to RC2 antibody during the major period of commissural axon growth, and was gradually reduced towards the end of gestation. The glial processes ramified extensively in the ventral funiculus and resided within the interfascicular space between the longitudinally projecting axons. Axonal localization of Nogo was observed on the pre-midline segment of commissural axons and on axons in the dorsal and ventral funiculi, but only at the earliest stage of pathway development. Nogo signals were initially weak on the glial processes during the period of axon crossing in the floor plate but was elevated when the decussation is finished. NgR was expressed on the commissural axons; the expression pattern is spatially regulated, being low in the premidline and midline courses but is up-regulated when the axons leave the floor plate. These expression patterns raise the possibilities that the glial specific form of Nogo may be involved in the guidance of commissural axons by i) preventing recrossing of axons across the midline through an upregulation of axonal NgR; ii) partitioning axons in the ventral funiculus into longitudinal fascicles.
Zhenlin Hu,
Lei Qiu,
Zhenyu Xiao,
Jing Wang,
Qi Yu,
Jianzhong Li,
Hao Feng,
Cheng Guo,
Junping Zhang
Department of Biochemical Pharmacy, School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
Esculentoside A (EsA), a saponin isolated from the root of Phytolacca esculenta, has been reported to exert anti-inflammatory effects in several animal models of acute and chronic inflammation by inhibiting the production and activity of pro-inflammatory cytokines in macrophages and epithelial cells. However, little is known about its modulation on T cells. In the present study, we further investigated its potential in treatment of autoimmune disease and its modulation on T cells, using an experimental autoimmune model established through immunizing mice with campylobacter jejuni strain CJ-S(131) in Freund s complete adjuvant. Our results demonstrated that EsA administration markedly alleviated the inflammatory injury in liver and kidney of model mice, decreased the anti-CD3/CD28-stimulated proliferation of splenocytes and lymph node cells, and reduced the percentage of CD3+, CD4+, and CD8+ lymphocytes in peripheral blood. Furthermore, we demonstrated that EsA induced apoptosis in ConA-activated thymocytes but not in non-activated thymocytes. Gene expression analysis revealed that EsA up-regulated the expression of a group of pro-apoptotic genes more profoundly in Con A-activated thymocytes than in non-activated thymocytes. EsA-affected pro-apoptotic genes included those involved in Fas induction, p53 activation, redox metabolism, calcium- and glucocorticoid-induced apoptosis signals, suggesting that EsA may modulate multiple apoptotic signal pathways in activated T cells. Taken together, our findings suggest that EsA may be useful for treatment of autoimmune disease through modulation on T cell-mediated adaptive immunity.
Key Laboratory of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao, P. R. China. shushzhang@126.com
An MCE electrochemical enzyme immunoassay protocol for the determination of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) was reported. Two antigens (Ag), CEA and AFP, were incubated simultaneously with an excess amount of horseradish peroxidase-labeled antibody (Ab(*)). The free Ab(*) and the Ab(*)-Ag complex produced in the solution were first separated through a postcolumn reaction and then traced by the enzyme substrate o-aminophenol. The 3-aminophenoxazine produced in enzyme reaction was detected with downstream amperometric detection. The separations were performed at a separation voltage of +1.4 kV and were completed in less than 60 s. The better analytical performance and distinct miniaturization/portability for MCE at less assay time and sample volume consumption was achieved. The detection limit of CEA and AFP was calculated to be .25 and .13 ng/mL, respectively. Therefore, MCE could be used as a sensitive and new tool in separation science and offered considerable promise in biological sample analysis or quick clinical diagnosis.
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Research Institute of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Central South University, Changsha 410013, China.
AbstractAim:Currently, there is considerable debate as to which method is more accurate for measuring the activity of CYP3A in vivo: cortisol 6beta-hydroxylation clearance (Cl(m(6beta))) or the urinary ratio of 6beta-OHF to F (6beta-OHF/F). Furthermore, the value of measuring endogenous levels of cortisol over a 24 h period (AUC(F)) needs to be confirmed. The aim of the present study was to determine which method was most effective at measuring changes in the in vivo activity of CYP3A: AUC(F), Cl(m(6beta)), or 6beta-OHF/F.Methods:A two phase, cross-over design was adopted in this study. A total of 24 subjects (12 males and 12 females) were randomly assigned to one of two groups: the test group subjects were given 250 mg clarithromycin tablets twice a day for a period of 4 d, whereas the control group received a placebo twice daily for a similar period. On d 5 of the study, the last dose of either clarithromycin or placebo was supplemented with an oral dose of 7.5 mg midazolam (MDZ); blood and urine samples were then collected at various times. All samples collected at the same sampling times on d 4 were used to evaluate the effects of MDZ administration on cortisol levels and metabolism. The ratio of 1-hydroxymidazolam (1-OHMDZ) concentration to MDZ concentration at 1 h (MR) was taken as a measure of the in vivo CYP3A activity. AUC(F), Cl(m(6beta)), and 6beta-OHF/F were also used as biomarkers for CYP3A activity.Results:No correlations were found (either before or after inhibition) between CYP3A activity and any of the following measures: AUC(F), Cl(m(6beta)), or 6beta-OHF/F (r< .4, P> .05). After 4 d of clarithromycin administration, CYP3A activity (MR) decreased by 75%(P= .000), whereas AUC(F) increased by 19%(P= .040), and Cl(m(6beta)) and 6beta-OHF/F decreased by 54.2%(P= .000) and 50%(P= .003), respectively. No significant changes in AUC(F)(P= .178), or in the amount of urinary 6beta-OHF (P= .169), or in F (P= .391) were found over a 24 h time period, either with or without MDZ administration.Conclusion:Although Cl(m(6beta)) and 6beta-OHF/F can reflect the decline in CYP3A activity, the impression they provide is neither accurate nor complete. AUC(F) is completely ineffective for evaluating variations in CYP3A activity. MDZ administration had no evident effects on either cortisol metabolism or excretion over a period of 24 h.Acta Pharmacologica Sinica advance online publication, 24 August 2009; doi: 10.1038/aps.2009.116.
Dipartimento di Sanità pubblica Patologia comparata e Igiene veterinaria, Facoltà di Medicina Veterinaria, Università di Padova, Viale dell’Università 16, 35020 Legnaro, Padova, Italy.
Dexamethasone (DXM) is often illegally used as a growth promoter. To identify indirect biomarkers of illicit treatments, the urinary ratio between 6beta-hydroxycortisol (6beta-OHF) and cortisol (F) was measured in urines obtained from bulls experimentally treated per os and intramuscularly (im) with different DXM dosages. Dexamethasone, given per os at low doses elicited an early and lasting significant reduction of 6beta-OHF/F. No significant variations were seen in urines from bulls given DXM intramuscularly. These results suggest 6beta-OHF/F as a rapid, non-invasive, screening test for oral, low-dose, long-term corticosteroid treatment in cattle. Further studies are required to go deep inside the biochemical and molecular events underlying such an effect.
Department of Animal Physiology, University of Bayreuth, Bayreuth, Germany.
Daiichi Asubio Pharmaceuticals Inc, Rochelle Park, NJ.
Ya-Chi Chen,
S Karl Gotzkowsky,
Anne N Nafziger,
Robert W Kulawy,
Mario L Rocci Jr,
Joseph S Bertino Jr,
Angela D M Kashuba
University of North Carolina, School of Pharmacy, Chapel Hill, NC 27599, USA.
AIMS: A non-invasive proposed method for measuring CYP3A activity is the urinary 6beta-hydroxycortisol:cortisol ratio. This ratio has been used as an indicator of CYP3A induction and inhibition, with mixed results. This investigation evaluated the relationship between a validated, biomarker, intravenous midazolam clearance and the urinary cortisol ratio under constitutive conditions and with the influence of a moderate CYP3A inhibitor. METHODS: This was a sequential, cross-over study design. Intravenous midazolam .025 mg kg(-1) was administered to 10 male and 10 female subjects once every 14 days for 4 months. Fluvoxamine 150 mg day(-1) was given to all subjects during the last two visits. Total body clearance of midazolam and urinary 6beta-hydroxycortisol:cortisol molar ratio were used as biomarkers of hepatic CYP3A activity. RESULTS: No significant correlations were found between these two markers (r(2)< .5, P > .05). Larger interindividual and intra-individual variability in CYP3A activity was observed in 6beta-hydroxycortisol:cortisol ratios compared with midazolam clearances. With fluvoxamine therapy, midazolam clearance values decreased approximately 1.5-fold and cortisol ratios decreased approximately 1.9-fold. CONCLUSIONS: The high intra-individual variability of the urinary cortisol ratio, compared with midazolam, makes this a suboptimal CYP3A phenotyping tool.
Department of Pharmacology, Charles University in Prague, Faculty of Medicine in Hradec Kralove, PO Box 38, Simkova 870, 500 38 Hradec Kralove, Czech Republic; micuda@lfhk.cuni.cz.
The 24-hour urinary excretion of 6-beta-hydroxycortisol (6beta-OHC) and the urinary ratio of 6beta-hydroxycortisol/cortisol (6beta-OHC/UFC) have been proposed as noninvasive probes for human cytochrome P450 3A4 isoform (CYP3A4). In this study, we evaluated within- and between-day variability of 6beta-OHC excretion and 6beta-OHC/UFC ratio in nine Caucasian men with cardiac disease. Each study participant was asked to collect 24-hour urine specimens during four consecutive days in five standardized time intervals. Concentrations of UFC and 6beta-OHC were determined by immunoassay and high-performance liquid chromatographic (HPLC) method, respectively. The HPLC method was accurate and precise, as indicated by a recovery rate of 96.5-103.3% and less than 5.2% and 6.3% of coefficient of variation for within-run and between-run assay, respectively. In patients, diurnal variations in UFC and 6beta-OHC excretion were parallel. Consequently, 6 beta-OHC/UFC ratio remained stable during the day. Both, 6 beta-OHC excretion and 6 beta-OHC/UFC ratio showed significant relationship between 24-hour value and values measured in respective collection periods with best correlations obtained from night interval (22.00-06.00, r = .86- .91). These results indicated that urinary 6 beta-OHC excretion and 6 beta-OHC/UFC ratio measured in overnight/morning urine could precisely reflect 24-hour values even in severely ill patients. In addition, a simple and sensitive HPLC method was described for determination of 6 beta-OHC in urine.
Ya-Chi Chen,
S Karl Gotzkowsky,
Anne N Nafziger,
Robert W Kulawy,
Mario L Rocci Jr,
Joseph S Bertino Jr,
Angela D M Kashuba
University of North Carolina, School of Pharmacy, Chapel Hill, NC, USA.
Aims A non-invasive proposed method for measuring CYP3A activity is the urinary 6beta-hydroxycortisol:cortisol ratio. This ratio has been used as an indicator of CYP3A induction and inhibition, with mixed results. This investigation evaluated the relationship between a validated, biomarker, intravenous midazolam clearance and the urinary cortisol ratio under constitutive conditions and with the influence of a moderate CYP3A inhibitor. Methods This was a sequential, cross-over study design. Intravenous midazolam .025 mg kg(-1) was administered to 10 male and 10 female subjects once every 14 days for 4 months. Fluvoxamine 150 mg day(-1) was given to all subjects during the last two visits. Total body clearance of midazolam and urinary 6beta-hydroxycortisol:cortisol molar ratio were used as biomarkers of hepatic CYP3A activity. Results No significant correlations were found between these two markers (r(2)< .5, P > .05). Larger interindividual and intra-individual variability in CYP3A activity was observed in 6beta-hydroxycortisol:cortisol ratios compared with midazolam clearances. With fluvoxamine therapy, midazolam clearance values decreased approximately 1.5-fold and cortisol ratios decreased approximately 1.9-fold. Conclusions The high intra-individual variability of the urinary cortisol ratio, compared with midazolam, makes this a suboptimal CYP3A phenotyping tool.
Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, UT-160 012, India, kris_kohli2003@yahoo.com.
OBJECTIVES: To phenotype 200 healthy North Indians for cytochrome P450 3A (CYP3A) activity by measuring urinary ratio of 6beta-OH-cortisol/cortisol (6beta-OH-CS/CS) and to genotype the subjects demonstrating low and high CYP3A activity for the presence of CYP3A4*1B,*2,*4,*5,*6 and *10 alleles. METHODS: Morning spot urine samples were collected from 200 healthy North Indians. CS and 6beta-OH-CS were extracted and quantified by HPLC. Genotyping was performed by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). RESULTS: Urinary 6beta-OH-CS/CS ratio demonstrated a mean of 52. +/- 46 (1.1-290). North Indians demonstrated unimodal distribution with respect to urinary 6beta-OH-CS/CS ratio. On the basis of phenotypes, the subjects were divided into three groups demonstrating low (n=50), intermediate (n=100) and high (n=50) CYP3A activity. These groups demonstrated 6beta-OH-CS/CS ratio of 13.4 +/- 5.2 (1.1-21. ), 40 +/- 11.9 (21.2-63.2) and 114 +/- 51. (66-290), respectively. One hundred subjects, 50 in the low and 50 in the high activity group, were genotyped for CYP3A4*1B,*2,*4,*5,*6 and *10. Only 2 heterozygotes with genotype CYP3A4*1/*1B were found in the high CYP3A activity group. CYP3A4*2,*4,*5,*6 and *10 were not found in the subjects studied. CONCLUSION: This is the first investigation establishing CYP3A phenotypes and demonstrating the absence of common CYP3A4 genotypes in North Indians.
Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, UT-160 012, India, kris_kohli2003@yahoo.com.
OBJECTIVES: To phenotype 200 healthy North Indians for cytochrome P450 3A (CYP3A) activity by measuring urinary ratio of 6beta-OH-cortisol/cortisol (6beta-OH-CS/CS) and to genotype the subjects demonstrating low and high CYP3A activity for the presence of CYP3A4*1B,*2,*4,*5,*6 and *10 alleles. METHODS: Morning spot urine samples were collected from 200 healthy North Indians. CS and 6beta-OH-CS were extracted and quantified by HPLC. Genotyping was performed by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). RESULTS: Urinary 6beta-OH-CS/CS ratio demonstrated a mean of 52. +/- 46 (1.1-290). North Indians demonstrated unimodal distribution with respect to urinary 6beta-OH-CS/CS ratio. On the basis of phenotypes, the subjects were divided into three groups demonstrating low (n=50), intermediate (n=100) and high (n=50) CYP3A activity. These groups demonstrated 6beta-OH-CS/CS ratio of 13.4 +/- 5.2 (1.1-21. ), 40 +/- 11.9 (21.2-63.2) and 114 +/- 51. (66-290), respectively. One hundred subjects, 50 in the low and 50 in the high activity group, were genotyped for CYP3A4*1B,*2,*4,*5,*6 and *10. Only 2 heterozygotes with genotype CYP3A4*1/*1B were found in the high CYP3A activity group. CYP3A4*2,*4,*5,*6 and *10 were not found in the subjects studied. CONCLUSION: This is the first investigation establishing CYP3A phenotypes and demonstrating the absence of common CYP3A4 genotypes in North Indians.
Department of Pharmaceutical Chemistry, School of Pharmacy, Medical University of Silesia, Katowice, Poland. jbafeltowska@slam.katowice.pl
BACKGROUND: The aim of this study was to examine the pharmacokinetics of fluconazole in the CSF of children with hydrocephalus during CNS infection treatment after intravenous and/or intraventricular drug administration. Direct fluconazole administration into the ventricular CSF of patients to treat serious CNS infections is an aggressive therapy, and data on the pharmacokinetics of fluconazole in CSF are limited. METHODS: A method of fluconazole quantification in CSF by solid-phase extraction (SPE)-high-performance liquid chromatography (HPLC) was developed to conduct pharmacokinetic studies. The population of patients included 2 children with hydrocephalus. Fluconazole was administered intravenously at average multiple doses of 12.5 mg/kg/24 h and intraventricularly at doses of 4, 5 and 7.5 mg/24 h, and 7.5 and 10 mg/12 h. The CSF samples were taken 2-24 h after administration of fluconazole. The concentrations of fluconazole in CSF specimens were assessed, and after pharmacokinetic studies the fluconazole dosage was modified. RESULTS: The method of fluconazole determination in CSF using the SPE-HPLC method is specific, precise and accurate. After intravenous fluconazole administration, the concentration of this antifungal drug was not detected in the ventricular CSF. The pharmacokinetic parameters determined after intraventricular fluconazole administration were: steady-state peak CSF fluconazole concentration (19.54 +/- 5.63 mg/l); trough CSF fluconazole concentration ( . - .3 mg/l); elimination rate constant ( .4654 +/- .2097 h(-1)), and half-life (1.84 +/- .93 h). CONCLUSIONS: The authors developed a method to determine fluconazole in CSF by SPE-HPLC. After intravenous fluconazole administration, the drug was not detected in the examined CSF samples. The intraventricular multidose pharmacokinetic data suggest the necessity of fluconazole monitoring in children with hydrocephalus during the treatment of shunt infection.
