Lean body weight dosing avoids excessive systemic exposure to proton pump inhibitors for children with obesity.

BACKGROUND: Children with obesity are more likely to suffer gastroesophageal reflux disease, requiring acid-suppression therapy with proton pump inhibitors (PPIs) and no guidelines regarding dosing. OBJECTIVE: To prospectively evaluate lean-body-weight-based (LBW) dosing of the PPI pantoprazole for children with and without obesity. METHODS: Methods: Sixty-two children (6-17 years) received a one-time oral dose of liquid pantoprazole (1.2 mg kg-1 LBW). Plasma pantoprazole concentrations were measured at 10 time points over 8 h and pharmacokinetic (PK) profiles generated using non-compartmental techniques, in order to compare PK parameters of interest between children with and without obesity, while accounting for CYP2C19 genotype. RESULTS: Adjusted for milligram-per-kilogram total body weight (TBW) pantoprazole received, apparent drug clearance (CL/F) was reduced 50% in children with vs. without obesity (p=0.03). LBW-based dosing compensated for this reduction in CL/F (p = 0.15). CONCLUSION: To achieve comparable systemic PPI exposures for children with and without obesity, we recommend using LBW, rather than TBW-based dosing for pantoprazole.

Plasma ghrelin and liquid gastric emptying in children with functional dyspepsia consistent with post-prandial distress syndrome.

BACKGROUND: Adult studies indicate a role for ghrelin in functional dyspepsia (FD) mediated through ghrelin's effect on gastric emptying (GE). This study examines the relationship between ghrelin, liquid GE, and pain in children with FD. METHODS: Thirteen FD patients reporting symptoms consistent with post-prandial distress syndrome (PDS) and 17 healthy controls were enrolled. All participants received a liquid meal containing (13) C-sodium acetate. Pain severity, liquid GE utilizing exhaled (13) CO2 from the sodium acetate breath tests (ABT), plasma acyl ghrelin (AG), and des-acyl ghrelin concentrations were measured at specific intervals over 240 min following ingestion. KEY RESULTS: FD-PDS patients demonstrated lower mean baseline AG (14.8 ± 9.7 vs 27.2 ± 14.0 fmol/mL; p = 0.013), AG Cmax (24.6 ± 8.2 vs 40.5 ± 16.8 fmol/mL; p = 0.007), and AG flux (18.2 ± 7.8 vs 32.7 ± 17.3 fmol/mL; p = 0.015) than controls. The time to reach maximum exhaled (13) CO2 concentration (T max ) was longer in FD patients than controls (47.5 ± 18.5 vs 35.8 ± 11.8 min; p = 0.046). Significant relationships between ghrelin analyte ratios and ABT parameters were largely confined to control participants. CONCLUSIONS & INFERENCES: FD-PDS in children is associated with lower fasting and maximum AG concentrations, and dampened AG flux. These data suggest a possible role for altered ghrelin physiology in the pathogenesis of PDS.

Effects of valproic acid on organic acid metabolism in children: a metabolic profiling study.

Young children are at increased risk for valproic acid (VPA) hepatotoxicity. Urinary organic acid profiles, as a surrogate of mitochondrial function, were obtained in children 1.9 to 17.3 years of age (n = 52) who were undergoing treatment with VPA for seizure disorders. Age-matched patients receiving treatment with carbamazepine (CBZ; n = 50) and healthy children not undergoing treatment (n = 22) served as controls. Age-related changes in organic acid profiles were observed in all three groups. Although the untreated and CBZ control groups were indistinguishable from each other with respect to the principal-component analysis (PCA) score plots of the subjects, a distinct boundary was apparent between the VPA and each of the control groups. Interindividual variability was observed in the VPA-induced alterations in endogenous pathways corresponding to branched-chain amino acid metabolism and oxidative stress. The data suggest that more detailed metabolomic analysis may provide novel insights into biological mechanisms and predictive biomarkers for children at highest risk for serious toxicity.

Characterization of delayed liquid gastric emptying in children by the (13)C-acetate breath test.

The (13)C-acetate breath test represents a potential alternative to conventional scintigraphy to measure liquid gastric emptying (GE). The purpose of this study was to compare the (13)C-acetate breath test to gastric scintigraphy in children with functional dyspepsia. Simultaneous assessment of GE was performed in 28 children (9-17 years of age) using a liquid test meal that was double labeled with (13)C-acetate and (99 m)Technetium. (13)CO(2) versus time profiles were fit using traditional pharmacokinetic analyses. For each subject, GE half-life [Formula: see text] determined by scintigraphy was plotted against parameters determined from the (13)C-acetate breath test. Linear regression was used to explore the associations between the tests. Complete (13)CO(2) versus time profiles were available for 25 subjects. There was no association between the scintigraphy GE T½ and the(13)CO(2) half-exhalation time. However, significant associations were observed between the gastric half-emptying time as determined by scintigraphy and two of the breath test parameters: the enrichment of (13)CO(2) present in breath samples at 60 min (DOB(60)) (r = -0.52, p = 0.01) and the area under the curve from 0 to 60 min (AUC(0-60 min)) (r = -0.54; p < 0.01). The (13)C-acetate breath test has the potential to serve as a rapid, technically simple and inexpensive means to assess liquid GE in children with functional dyspepsia and possibly serve as a pharmacodynamic surrogate in studies of prokinetic drugs in children.

The CYP2D6 activity score: translating genotype information into a qualitative measure of phenotype.

Inferring CYP2D6 phenotype from genotype is increasingly challenging, considering the growing number of alleles and their range of activity. This complexity poses a challenge in translational research where genotyping is being considered as a tool to personalize drug therapy. To simplify genotype interpretation and improve phenotype prediction, we evaluated the utility of an "activity score" (AS) system. Over 25 CYP2D6 allelic variants were genotyped in 672 subjects of primarily Caucasian and African-American heritage. The ability of genotype and AS to accurately predict phenotype using the CYP2D6 probe substrate dextromethorphan was evaluated using linear regression and clustering methods. Phenotype prediction, given as a probability for each AS group, was most accurate if ethnicity was considered; among subjects with genotypes containing a CYP2D6*2 allele, CYP2D6 activity was significantly slower in African Americans compared to Caucasians. The AS tool warrants further prospective evaluation for CYP2D6 substrates and in additional ethnic populations.

Identification and characterization of CYP2D6*56B, an allele associated with the poor metabolizer phenotype.

A 5-year-old African-American girl presented with a CYP2D6*4xN/*10 genotype that was discordant with her poor metabolizer phenotype determined with the probe drug dextromethorphan. Both phenotype and genotype were confirmed in repeat assessments, suggesting that the CYP2D6*10 allele carried a novel debilitating sequence variation(s). The rationale for this study was to resolve the discordance and to describe the novel non-functional allelic variant of CYP2D6 and its frequency in populations of different ethnic backgrounds.

Ontogeny of dextromethorphan O- and N-demethylation in the first year of life.

The exponential increase in the number of drugs used to treat infant and childhood illnesses necessitates an understanding of the ontogeny of drug biotransformation for the development of safe and effective therapies. Healthy infants received an oral dose (0.3 mg/kg) of dextromethorphan (DM) at 0.5, 1, 2, 4, 6, and 12 months of age. DM and its major metabolites were measured in urine. CYP2D6 genotype was determined by polymerase chain reaction-restriction fragment length polymorphism. Genotyping data indicated a strong correlation between CYP2D6 genotype and DM O-demethylation (beta=-0.638; 95% CI: -0.745, -0.532; P<0.001). CYP2D6 activity was detectable and concordant with genotype by 2 weeks of age, showed no relationship with gestational age, and did not change with post natal age up to 1 year. In contrast, DM N-demethylation developed significantly more slowly over the first year of life. Genotype and the temporal acquisition of drug biotransformation are critical determinants of a drug response in infants.

Identification and characterization of novel sequence variations in the cytochrome P4502D6 (CYP2D6) gene in African Americans.

Cytochrome P4502D6 (CYP2D6) genotyping reliably predicts poor metabolizer phenotype in Caucasians, but is less accurate in African Americans. To evaluate discordance we have observed in phenotype to genotype correlation studies, select African American subjects were chosen for complete resequencing of the CYP2D6 gene including 4.2 kb of the CYP2D7-2D6 intergenic region. Comparisons were made to a CYP2D6(*)1 reference sequence revealing novel SNPs in the upstream, coding and intervening sequences. These sequence variations, defining four functional alleles (CYP2D6(*)41B, (*)45A and B and (*)46), were characterized for their ability to influence splice site strength, transcription level or catalytic protein activity. Furthermore, their frequency was determined in a population of 251 African Americans. A -692(TGTG) deletion (CYP2D6(*)45B) did not significantly decrease gene expression, nor could any other upstream SNP explain a genotype-discordant case. CYP2D6(*)45 and (*)46 have a combined frequency of 4% and can be identified by a common SNP. Carriers are predicted to exhibit an extensive or intermediate CYP2D6 phenotype.

Cytochrome P450 Involvement in the biotransformation of cisapride and racemic norcisapride in vitro: differential activity of individual human CYP3A isoforms.

Identification of the human cytochrome P450 (P450) enzymes involved in the metabolism of cisapride and racemic norcisapride [(+/-)-norcisapride] was investigated at 0.1 and 1 microM, concentrations that span the mean plasma C(max) for cisapride. Formation of norcisapride (Nor), 3-fluoro-4-hydroxycisapride (3F), and 4-fluoro-2-hydroxycisapride (4F) from cisapride and an uncharacterized metabolite (UNK) from (+/-)-norcisapride in human liver microsomes (HLMs) were consistent with Michaelis-Menten kinetics for a single enzyme (K(m), 6.0, 14.3, 13.9, and 107 microM; V(max), 1350, 696, 568, and 25 pmol/mg of protein, respectively). HLMs converted cisapride to Nor at rates that were at least 3 orders of magnitude greater than those observed for (+/-)-norcisapride conversion to UNK. The sample-to-sample variation in the rates of Nor, 3F, 4F, and UNK formation correlated strongly (r(2) > 0.796) with CYP3A4/5 activity in a panel of HLMs (n = 7) and was markedly reduced by ketoconazole, a potent CYP3A inhibitor. Ketoconazole virtually eliminated (+/-)-norcisapride conversion to UNK (94 +/- 0.5%). Studies with 10 cDNA-expressed enzymes revealed that CYP3A4 catalyzed the formation of Nor and 4F at rates >100 times those of non-CYP3A enzymes and >100- and 50-fold higher than CYP3A5 and CYP3A7, respectively. CYP3A4 was the only P450 capable of UNK formation. Therefore, CYP3A4 is the principal P450 enzyme responsible for the conversion of cisapride to Nor, 3F, and 4F and of (+/-)-norcisapride to UNK. Compared with cisapride, factors related to CYP3A4-mediated (+/-)-norcisapride metabolism (e.g., ontogeny of drug-metabolizing enzymes, inhibition, and induction) should be clinically unimportant due to the apparent lack of dependence on cytochromes P450 for elimination.

Effects of musk xylene and musk ketone on rat hepatic cytochrome P450 enzymes.

The purpose of the present work was to characterize the effect of musk xylene (MX) and musk ketone (MK) treatment on rat hepatic cytochrome P450 enzymes. Male F344 rats were dosed orally with MX (10, 50 or 200 mg/kg) or MK (20, 100 or 200 mg/kg) for 7 days, after which CYP1A, 2B and 3A enzyme activities and protein levels were determined. MX treatment resulted in a two- to four-fold increase in the activity of CYP1A, 2B and 3A enzymes. For CYP1A and 3A, these changes were consistent with small increases in immunoreactive proteins. However, for CYP2B, despite only a three-fold increase in enzyme activity, protein levels were increased nearly 50-fold relative to control. This induction occurred by transcriptional activation of the CYP2B1 gene as evidenced by increased steady state CYP2B1 mRNA levels. In contrast to MX, MK treatment increased CYP2B activity, protein and mRNA levels. However MK treatment also increased CYP1A enzyme activity nearly 30-fold higher than control rats, a profile that was markedly different from MX, and very different from its effects in mice (Stuard, S.B., Caudill, D., Lehman-Mc-Keeman, L.D., 1997. Characterization of the effects of musk ketone on mouse cytochrome P450 enzymes. Fund. Appl. Toxicol. 40, 264-271). These results indicate that in rats, MX is an inducer of CYP2B enzymes, but these enzymes are not functionally active. In contrast, MK also induces CYP2B enzymes, with no concurrent inactivation. MK also exhibits a unique pattern of cytochrome P450 induction by increasing both CYP1A and CYP2B in rats.

Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity.

The stability of cytochrome P450 enzymes, cytochrome b5, and NADPH-cytochrome c reductase was examined in (A) human liver samples frozen in liquid nitrogen and stored at -80 degrees C, (B) human liver microsomes suspended in 250 mM sucrose and stored at -80 degrees C, and (C) human liver microsomes subjected to as many as 10 cycles of thawing and freezing. In study A, microsomes from five human livers were prepared from fresh (unfrozen) tissue and from tissue that was stored frozen at -80 degrees C for 1, 2, 4, or 6 months. The apparent concentration of cytochromes P450 and b5 and the activity of NADPH-cytochrome c reductase decreased 20-40% as a result of freezing the liver, regardless of whether the liver was stored for 1 or 6 months. Similar decreases were observed in the activities of cytochrome P450 enzymes belonging to several gene families, namely CYP1A2 (7-ethoxyresorufin O-dealkylation and caffeine N3-demethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (tolbutamide methylhydroxylation), CYP2C19 (S-mephenytoin 4'- hydroxylation), CYP2D6 (dextromethorphan O-de-methylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4solidus5 (testosterone 6beta-hydroxylation), and CYP4A9solidus11 (lauric acid 12-hydroxylation). Freezing human liver did not convert cytochrome P450 to its inactive form, cytochrome P420, but it increased the contamination of liver microsomes with hemoglobin or other heme-containing proteins, which resulted in a uniform decrease in the specific activity of cytochromes P450 and b5 and in the specific activity of all P450 enzymes. In study B, the concentration of cytochromes P450 and b5, the activity of NADPH-cytochrome c reductase, and the activity of individual cytochrome P450 enzymes were determined in 10 samples of human liver microsomes stored at -80 degrees C for approximately 0, 1, or 2 years. The sample-to-sample variation in the concentration and activity of cytochrome P450, cytochrome b5, and NADPH-cytochrome c reductase was nominally affected by long-term storage of human liver microsomes at -80 degrees C, indicating there was no differential loss of cytochrome P450 activity, cytochrome b5 concentration, or NADPH-cytochrome c reductase activity. In study C, microsomes from a pool of human livers were subjected to 1, 2, 3, 5, 7, or 10 cycles of freezing at -80 degrees C followed by thawing at room temperature. Freezing/thawing liver microsomes for up to 10 cycles did not convert cytochrome P450 to P420, nor did it cause significant loss of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5, or CYP4A9/11 activity. Overall, these results suggest that our current methods for storing and processing human liver are well suited to preserving microsomal P450 enzyme activity.

Identification of the human P450 enzymes involved in lansoprazole metabolism.

The aim of this study was to identify which human P450 enzymes are involved in the metabolism of lansoprazole. In the presence of NADPH and oxygen, human liver microsomes converted lansoprazole to lansoprazole sulfide, lansoprazole sulfone and 5-hydroxylansoprazole. Formation of lansoprazole sulfide occurred nonenzymatically. The formation of lansoprazole sulfone appeared to be catalyzed by a single, low-affinity enzyme (apparent Km approximately 100 microM). In contrast, lansoprazole 5-hydroxylation appeared to be catalyzed by two kinetically distinct enzymes (apparent Km approximately 100 microM and approximately 15 microM). When human liver microsomes (n = 16) were incubated with 100 microM lansoprazole, both the 5-hydroxylation and sulfoxidation of lansoprazole appeared to be catalyzed by CYP3A4/5 (based on correlation analyses). Antibodies against rat CYP3A enzymes inhibited the rate of both 5-hydroxylation (approximately 55%) and sulfoxidation (approximately 70%) and cDNA-expressed CYP3A4 catalyzed both the 5-hydroxylation and sulfoxidation of lansoprazole (apparent Km approximately 100 microM). However, at the pharmacologically relevant substrate concentration of 1 microM, lansoprazole sulfoxidation was still highly correlated with CYP3A4/5 activity (r2 = .905), but lansoprazole 5-hydroxylation appeared to be catalyzed by CYP2C19 (r2 = .875) rather than CYP3A4/5 (r2 = .113). Antibodies and chemical inhibitors of CYP2C enzymes preferentially inhibited the 5-hydroxylation of lansoprazole, whereas lansoprazole sulfoxidation was preferentially inhibited by antibodies and chemical inhibitors of CYP3A4/5. The cDNA expressed enzymes CYP2C8, CYP2C9 and CYP2C19 catalyzed varying rates of lansoprazole 5-hydroxylation at a substrate concentration of 50 microM, but only CYPC19 catalyzed this reaction at 1 microM. These results suggest that at pharmacologically relevant concentrations, the 5-hydroxylation of lansoprazole is primarily catalyzed by CYP2C19, whereas the sulfoxidation of lansoprazole is primarily catalyzed by CYP3A4/5. It is possible that individuals lacking CYP2C19 will be poor metabolizers of lansoprazole.

Non-methylene-interrupted and omega4 dienoic fatty acids of the white shrimp Penaeus setiferus.

The total lipid fatty acids from the white shrimp Penaeus setiferus were found to contain several unusual dienoic fatty acid species. These included two methylene-interrupted species: delta 11, 14-C18:2 (18:2omega 4) and delta 13, 16-C20:2 (20:2omega4). Also found were several non-menthylene-interrupted dienoic fatty acids including delta 7, 11 and delta 7, 13-C20:2, delta 7, 13-C21:2, delta 7, 13, delta 7, 15, delta 9, 13, delta 9, 15,, and delta 7, 17-C22:2. Many minor C20:2 non-mentylene-interrupted dienes were found but could not be unequivocally characterized.

trans-6-Hexadecenoic acid in the spadefish Chaetodipterus faber.

Fatty acid methyl esters were prepared from spadefish (Chaetodipterus faber) liver oil and were analyzed by open tublar gas chromatography. The unusual fatty acid trans-6-hexadecenoic acid was identified as a major monoenoic component by reductive ozonolysis and by mass spectrometry of the di-trimethylsilyl ether. Very minor amounts of 7-methyl-7-hexadecenoic acid were found.