Excretion Rates Of Metabolites Research Articles

Effects of the Thiol Methyltransferase Inhibitor (±)-2,3-Dichloro-α-Methylbenzylamine (DCMB) on the Pharmacokinetics and Metabolism of Vicagrel in Rats.

P2Y12 receptor inhibitors are commonly used in clinical antiplatelet therapy, typically alongside other medications. Vicagrel, a promising P2Y12 receptor inhibitor, has submitted a new drug marketing application to the United States Food and Drug Administration. Its primary metabolites and some metabolic pathways are identical to those of clopidogrel. The aim of this study was to investigate the effects of the thiol methyltransferase inhibitor (±)-2,3-dichloro-α-methylbenzylamine (DCMB) on the metabolism and pharmacokinetics of vicagrel. In vitro incubation with human and rat liver microsomes revealed that DCMB significantly inhibited the methylation of vicagrel's thiol metabolite M15-1. Rats were orally administered 6 mg/kg [14C]vicagrel (100 μCi/kg) 1 hour after peritoneal injection with or without DCMB (80 mg/kg). Compared with the control group, the plasma of DCMB-pretreated rats exhibited maximum plasma concentration (C max) decrease and time to reach C max (T max) delay for all vicagrel-related substances, the methylation product of the thiol metabolite (M9-2), and the derivatization product of the active thiol metabolite (MP-M15-2). However, no significant changes in area under the curve (AUC) or half-life (t 1/2) were observed. DCMB had negligible effect on the total radiological recovery of vicagrel within 72 hours, although the rate of vicagrel excretion slowed down within 48 hours. DCMB had a negligible impact on the metabolic pathway of vicagrel. Overall, the present study found that DCMB did not significantly affect the total exposure, metabolic pathways, metabolite profiles, or total excretion rates of vicagrel-related metabolites in rats, but led to C max decrease, T max delay, and slower excretion rate within 48 hours. SIGNIFICANCE STATEMENT: This study used liquid chromatography-tandem mass spectrometry combined with radiolabeling technology to investigate the effects of the thiol methyltransferase inhibitor (±)-2,3-dichloro-α-methylbenzylamine on the absorption, metabolism, and excretion of vicagrel in rats. This work helps to better understand the in vivo metabolism of active thiol metabolites of P2Y12 inhibitors such as clopidogrel, vicagrel, etc.

Pharmacokinetic and safety profiles of mesalazine enema in healthy Chinese subjects: A single- and multiple-dose study.

Mesalazine is a well-established treatment for ulcerative colitis by oral or topical administration. However, the pharmacokinetic (PK) and safety profiles of mesalazine administered by an enema has not been clarified in Chinese population. We conducted an open-label study to assess the PK and safety profiles of mesalazine in 11 healthy Chinese subjects after receiving mesalazine enema (1 g/100 mL) once daily for 7 consecutive days. Blood and urine samples were collected for assay of mesalazine and N-acetyl mesalazine by liquid chromatography-tandem mass spectrometry. The PK and safety data were summarized using descriptive statistics. The mean (standard deviation) maximum plasma concentration (Cmax), area under plasma drug concentration-time curve from time 0 to the last measurable plasma concentration time point (AUC0-t) and elimination half-life (t1/2) of mesalazine were 1007.64 (369.00) ng/mL, 9608.59 (3533.08) h·ng/mL and 3.33 (1.99) h, respectively after the first dose administration. In multiple-dose study, the estimated accumulation factor of mesalazine was 1.09. The cumulative urinary excretion rate of parent and major metabolite of mesalazine was 27.77%. After the last doe administration, 2.21% of the administered dose was excreted as mesalazine and 24.47% as N-acetyl mesalazine in urine within 24 h. Overall, 9 adverse events (AEs) were reported in 4 of the 11 subjects (36.4%), including oral ulcer, toothache, upper respiratory tract infection (1 each) and laboratory abnormalities (6 cases). All AEs were mild and recovered spontaneously without treatment, and were not considered as related to mesalazine. Mesalazine enema (1 g/100 mL) was safe and well tolerated in healthy Chinese subjects. These findings support further clinical trials in Chinese patients. Trial registration: This trial was registered to Chinese Clinical Trial Registry (ChiCTR) at https://www.chictr.org.cn (registration number: ChiCTR2300073148).

Excretion of three alkaloids from Simiao Pills in urine, feces, and bile between normal and type 2 diabetic rats

This study investigated the differences in excretion kinetics of three alkaloids and their four metabolites from Simiao Pills in normal and type 2 diabetic rats. The diabetes model was established in rats by injection of streptozotocin, and the alkaloids in urine, feces, and bile of normal and diabetic rats were detected by LC-MS/MS to explore the effect of diabetes on alkaloid excretion of Simiao Pills. The results showed that 72 h after intragastric administration of the extract of Simiao Pills, feces were the main excretion route of alkaloids from Simiao Pills. The total excretion rates of magnoflorine and berberine in normal rats were 4.87% and 56.54%, which decreased to 2.35% and 35.53% in diabetic rats, which had statistical significance(P<0.05). The total excretion rates of phellodendrine, magnoflorine, and berberine in the urine of diabetic rats decreased significantly, which were 53.57%, 60.84%, and 52.78% of those in normal rats, respectively. After 12 h of intragastric administration, the excretion rate of berberine in the bile of diabetic rats increased significantly, which was 253.33% of that of normal rats. In the condition of diabetes, the excretion rate of berberine metabolite, thalifendine significantly decreased in urine and feces, but significantly increased in bile. The total excretion rates of jateorrhizine and palmatine in the urine increased significantly, and t_(1/2) and K_e changed significantly. The results showed that diabetes affected the in vivo process of alkaloids from Simiao Pills, reducing their excretion in the form of prototype drug, affecting the biotransformation of berberine, and ultimately increasing the exposure of alkaloids in vivo, which would be conducive to the hypoglycemic effect of alkaloids. This study provides references for the clinical application and drug development of Simiao Pills in diabetes.

The Metabolite Profile of Radioisotope-Labeled Biotin in Rats Indicates that Rat Biotin Metabolism is Similar to that in Humans

The egg white-fed rat is the most commonly used animal model of human biotin deficiency. We sought to determine whether the urinary excretion rates of biotin and biotin metabolites in rats are similar to those reported in humans. D-(Carbonyl-14C)biotin was injected intraperitoneally at physiologic doses in 6- to 10-wk-old rats; HPLC and radiometric flow detection were used to specifically identify and quantify biotin and metabolites in urine. Substantial amounts of bisnorbiotin and biotin sulfoxide, the two principal human metabolites, were detected. The excretion rates of biotin and metabolites were strikingly dependent on the dose of biotin. When the dose of [14C]biotin was 30% of the daily dietary intake (a physiologic dose), 50% of the administered 14C was excreted within 24 h; more than half of the excretion was the unchanged vitamin. After d 1, [14C]bisnorbiotin was the major form excreted. For the cumulative 5-d excretion, [14C]biotin accounted for 46 ± 9%, [14C]bisnorbiotin accounted for 47 ± 11%, and [14C]biotin sulfoxide accounted for 8 ± 4% of the total of biotin, bisnorbiotin, and biotin sulfoxide recovered radioactivity (mean ± 1 SD, n = 6). These proportions are similar to those reported in humans: biotin = 52 ± 12%, bisnorbiotin = 35 ± 9%, and biotin sulfoxide = 13 ± 4% of total biotin plus metabolites (mean ± 1 SD, n = 10). Thus, these, studies confirm the earlier identification of bisnorbiotin and biotin sulfoxide as the two principal biotin metabolites and provide evidence that the rat is an appropriate model for human biotin metabolism.

Role of glucocorticoid metabolism in childhood obesity-associated hypertension.

ObjectiveChildhood obesity is associated with alterations in hypothalamus–pituitary–adrenal axis activity. We tested the hypothesis that multiple alterations in the metabolism of glucocorticoids are required for the development of hypertension in children who become overweight.MethodsSpot urine for targeted gas chromatography-mass spectrometry steroid metabolome analysis was collected from (1) overweight/hypertensive children (n = 38), (2) overweight/non-hypertensive children (n = 83), and (3) non-overweight/non-hypertensive children (n = 56).ResultsThe mean (± s.d.) age of participants was 10.4 ± 3.4 years, and 53% of them were male. Group 1 and group 2 had higher excretion rates of cortisol and corticosterone metabolites than group 3 (869 (interquartile range: 631–1352) vs 839 (609–1123) vs 608 (439–834) μg/mmol creatinine × m2 body surface area, P < 0.01, for the sum of cortisol metabolites), and group 1 had a higher excretion rate of naive cortisol than group 3. Furthermore, groups differed in cortisol metabolism, in particular in the activities of 11β-hydroxysteroid dehydrogenases, as assessed from the ratio of cortisol:cortisone metabolites (group 2 < group 3), 5α-reductase (group 1 > group 2 or 3), and CYP3A4 activity (group 1 < group 2 or 3).DiscussionThe sequence of events leading to obesity-associated hypertension in children may involve an increase in the production of glucocorticoids, downregulation of 11β-hydroxysteroid dehydrogenase type 1 activity, and upregulation of 5α-reductase activity, along with a decrease in CYP3A4 activity and an increase in bioavailable cortisol.

No Effect of Permanent Tattoos on Local Sweat Concentration or Excretion Rate of Urea Nitrogen, Cortisol, Glucose, or Lactate During Exercise

Permanent tattooing is common on areas of the skin typically sampled for sweat diagnostics, yet its potential to alter local sweat concentration and excretion rate (ER) of metabolites and other compounds is unknown. The purpose of this study was to investigate the influence of tattooed skin (TAT) on local sweat concentration and ER of urea nitrogen (UN), cortisol (CORT), glucose (GLU) and lactate (LAC) when compared to contralateral non‐tattooed skin (NT) during a group fitness exercise session. Moderately trained participants (males n=8, females n=8, 34±8 y) with at least one permanent tattoo (age of tattoo 7±5 y) on the upper body (arm, scapula, or thorax regions) underwent a ~60 minute outdoor (~27±3°C, 63±4% RH, 26±3°C WBGT) group fitness exercise session. Sweat was collected with absorbent patches (3MTMTegaderm+PAD) placed on the TAT and on NT. Patches were monitored during exercise and removed upon moderate saturation. Local sweat rate (LSR) was calculated from sweat mass over patch surface area (11.9 cm2) and exercise duration (56±2 min). Excretion rates (ER) were calculated as the product of analyte concentration and local SR. Sweat was analyzed by ELISA for UN (Invitrogen, ThermoFisher Scientific), CORT (Invitrogen, ThermoFisher Scientific), GLU (Caymen Chemical Co.) and LAC (LSBio). To evaluate differences between TAT and NT skin, paired t‐tests or Wilcoxon signed rank tests were used. Significance level was set at p&lt;0.05. Data are presented as mean±SD or median±IQR for normally and non‐normally distributed data respectively. There were no differences in LSR for TAT vs. NT conditions (1.1±0.7 vs 1.2±0.6 mg/cm2/min; p=0.93). TAT and NT skin had similar concentrations for UN (19.2±6.0 vs 20.7±6.1 mg/dL; p=0.28), CORT (2.4±1.3 vs 2.5±1.4 ng/mL; p=0.41), GLU (0.22±0.3 vs 0.24±0.2 mg/dL; p=0.31), and LAC (5.8±6.3 vs 5.9±6.6 mM; p=0.83). ERs were also similar for all analytes: UN (252±150 vs 270±136 ng/cm2/min; p=0.34), CORT (1.8±3.4 vs 2.1±3.4 pg/cm2/min; p=0.46), GLU (2.3±1.6 vs 2.3±2.4 ng/cm2/min; p=0.40) and LAC (6.4±10.9 vs 7.1±10.1 ng/cm2/min; p&gt;0.999). In conclusion, TAT does not appear to affect the LSR, concentration, or ER of select analytes during ~60 min of fitness exercise.

Exploring the Temporal Relation between Body Mass Index and Corticosteroid Metabolite Excretion in Childhood.

Childhood obesity is associated with alterations in hypothalamus–pituitary–adrenal (HPA) axis activity. However, it is unknown whether these alterations are a cause or a consequence of obesity. This study aimed to explore the temporal relationship between cortisol production and metabolism, and body mass index (BMI). This prospective follow-up study included 218 children (of whom 50% were male), born between 1995 and 1996, who were assessed at the ages of 9, 12 and 17 years. Morning urine samples were collected for assessment of cortisol metabolites by gas chromatography-tandem mass spectrometry, enabling the calculation of cortisol metabolite excretion rate and cortisol metabolic pathways. A cross-lagged regression model was used to determine whether BMI at various ages during childhood predicted later cortisol production and metabolism parameters, or vice versa. The cross-lagged regression coefficients showed that BMI positively predicted cortisol metabolite excretion (p = 0.03), and not vice versa (p = 0.33). In addition, BMI predicted the later balance of 11β-hydroxysteroid dehydrogenase (HSD) activities (p = 0.07), and not vice versa (p = 0.55). Finally, cytochrome P450 3A4 activity positively predicted later BMI (p = 0.01). Our study suggests that changes in BMI across the normal range predict alterations in HPA axis activity. Therefore, the alterations in HPA axis activity as observed in earlier studies among children with obesity may be a consequence rather than a cause of increased BMI.

Sexual dimorphism in cortisol metabolism throughout pubertal development: a longitudinal study

ObjectiveSex differences in disease susceptibility might be explained by sexual dimorphism in hypothalamic-pituitary-adrenal axis activity, which has been postulated to emerge during puberty. However, studies conducted thus far lacked an assessment of Tanner pubertal stage. This study aimed to assess the contribution of pubertal development to sexual dimorphism in cortisol production and metabolism.MethodsParticipants (n = 218) were enrolled from a population-based Netherlands Twin Register. At the ages of 9, 12 and 17 years, Tanner pubertal stage was assessed and early morning urine samples were collected. Cortisol metabolites were measured with GC-MS/MS and ratios were calculated, representing cortisol metabolism enzyme activities, such as A-ring reductases, 11β-HSDs and CYP3A4. Cortisol production and metabolism parameters were compared between sexes for pre-pubertal (Tanner stage 1), early pubertal (Tanner stage 2–3) and late-pubertal (Tanner stage 4–5) stages.ResultsCortisol metabolite excretion rate decreased with pubertal maturation in both sexes, but did not significantly differ between sexes at any pubertal stage, although in girls a considerable decrease was observed between early and late-pubertal stage (P < 0.001). A-ring reductase activity was similar between sexes at pre- and early pubertal stages and was lower in girls than in boys at late-pubertal stage. Activities of 11β-HSDs were similar between sexes at pre-pubertal stage and favored cortisone in girls at early and late-pubertal stages. Cytochrome P450 3A4 activity did not differ between sexes.ConclusionsPrepubertally, sexes were similar in cortisol parameters. During puberty, as compared to boys, in girls the activities of A-ring reductases declined and the balance between 11β-HSDs progressively favored cortisone. In addition, girls showed a considerable decrease in cortisol metabolite excretion rate between early and late-pubertal stages. Our findings suggest that the sexual dimorphism in cortisol may either be explained by rising concentrations of sex steroids or by puberty-induced changes in body composition.

Long-Term Stability of Cortisol Production and Metabolism Throughout Adolescence: Longitudinal Twin Study.

Life-course experiences have been postulated to program hypothalamus-pituitary-adrenal (HPA) axis activity, suggesting that HPA axis activity is, at least partially, stable over time. Yet, there is paucity of data on the long-term stability of cortisol production and metabolism. We performed a prospective follow-up study in twins recruited from a nationwide register to estimate the stability of cortisol production and metabolism over time, and the contribution of genetic and environmental factors to this stability. In total, 218 healthy mono- and dizygotic twins were included. At the ages of 9, 12 and 17 years, morning urine samples were collected for assessment (by gas chromatography-tandem mass spectrometry) of cortisol metabolites, enabling the calculation of cortisol metabolite excretion rate and cortisol metabolism activity. Our results showed a low stability for both cortisol metabolite excretion rate (with correlations <.20) and cortisol metabolism activity indices (with correlations of .25 to .46 between 9 and 12 years, -.02 to .15 between 12 and 17 years and .09 to .28 between 9 and 17 years). Because of the low stability over time, genetic and environmental contributions to this stability were difficult to assess, although it seemed to be mostly determined by genetic factors. The low stability in both cortisol production and metabolism between ages 9 and 17 years reflects the dynamic nature of the HPA axis.

Quantitative targeted GC-MS-based urinary steroid metabolome analysis for treatment monitoring of adolescents and young adults with autoimmune primary adrenal insufficiency

PurposePrimary adrenal insufficiency (PAI) is a rare and life-threatening disease. A recent Endocrine Society guideline argued against hormonal monitoring of glucocorticoid replacement. However, about 50% of adolescents and young adults (AYAs) with chronic diseases are non-adherent to their treatment regimens. Therefore, suitable hormonal monitoring of glucocorticoid replacement would be highly desirable in AYAs with PAI. We investigated whether quantitative targeted gas chromatography-mass spectrometry urinary steroid metabolome analysis would be suitable for monitoring glucocorticoid replacement in AYAs with autoimmune PAI. MethodRetrospective analysis of 21 urinary steroid profiles of four AYAs aged 15.6 ± 2.0 years with autoimmune PAI on hydrocortisone and fludrocortisone treatment. 24-hr cortisol metabolite excretion rates (CMERs) were calculated using the sum of major seven urinary cortisol metabolites. CMERs were transformed into z-scores according to reference values of healthy age- and sex matched subjects. ResultsThree patients showed good treatment adherence (17 of 21 samples). Mean CMER of these samples was 7.4 ± 1.8 mg/m2/d, corresponding to a z-score of 1.8 ± 1.1. CMER reflected 59.7 ± 14.5% of prescribed hydrocortisone dosages. A forth patient displayed clinical symptoms of PAI during treatment. CMER was only 0.3 mg/m2 (−3.4 z), reflecting only 3.1% of prescribed hydrocortisone dosage, compatible with lack of treatment adherence. Thereafter, the parents supervised the intake of tablets and treatment adherence improved. ConclusionQuantitative targeted GCMS steroid metabolome analysis could support monitoring of glucocorticoid replacement treatment in patients with PAI.

Treatment of Hypertension with Stable Analogue of 14, 15‐Epoxyeicosatrienoic Acid (EET‐A) in Spontaneously Hypertensive Rats

IntroductionHypertension and concomitant complications, such as blood circulation disorders, stroke, heart attack or kidney failure, became one of the leading causes of death in the world. Recent reports indicate on epoxyeicosatrienoic acids (EETs) as significant factors in blood pressure regulation. These cytochrome P‐450 dependent metabolites of arachidonic acid, exhibit vasodilator and natriuretic activity. It has been shown that EETs facilitate the production of vasodilatory NO, protect the endothelium, exhibit antioxidant properties and might inhibit inflammation. All of the above‐mentioned features should contribute to the reduction of blood pressure and adverse effect of hypertension, hence we postulate the usefulness of EETs as novel drugs for the prevention and/or treatment of hypertension. In the present study the potential hypotensive efficiency of a stable analogue of 14,15‐EET: disodium (S)‐2‐(13‐(3‐entyl)ureido)‐tridec‐8(Z)‐enamido) succinate, called EET‐A, in genetic model of hypertension (spontaneously hypertensive rat, SHR) was evaluated.MethodsMale SHR (n = 6) in the established stage of hypertension (16 weeks) were treated for four weeks with EET‐A dissolved in drinking water (10 mg/kg/day); aged‐matched control group received water only (n = 6). Systolic blood pressure (SBP) was measured by telemetry technique every three days. Once a week observations in metabolic cages (food and water intake, feces, diuresis) were performed; urine and blood samples were collected for further analysis. Nitric oxide metabolites and vascular endothelial growth factor (VEGF) content in urine was measured with commercial ELISA kit.ResultsPreliminary results showed that EET‐A did not impact the blood pressure of adult SHR. We did not observe any changes in diuresis, water and food intake. Also sodium excretion did not change after EET‐A treatment, however, surprisingly, it significantly increased in the control group (EET‐A day 0: 0.5 ± 0.1 vs day 28: 0.5 ± 0.1, NS; control group day 0: 0.4 ± 0.2 vs day 28: 1.0 ± 0.1#* mmol/24h, *p&lt;0.05 within the control group; # values on day 28th between groups, p&lt;0.05). The excretion rates of NO metabolites (nitrates/nitrites; NOx) and VEGF‐A increased significantly in the EET‐A treated group (NOx day 0: 0.99 ± 0.18 vs day 28: 1.56 ± 0.13 μmol/24h, p&lt;0.05; VEGF‐A day 0: 3.2 ± 0.7 vs day 28: 9.5 ± 2.0 ng/24h, p&lt;0.05).SummaryAnalysis of the obtained data suggests that EET‐A in the applied dose does not lower blood pressure. Further studies are necessary to investigate if, despite the lack of blood pressure decrease, EET‐A improved kidney function.Support or Funding InformationNational Science Centre 2017/26/M/NZ5/00367This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Absorption and metabolism of isothiocyanates formed from broccoli glucosinolates: effects of BMI and daily consumption in a randomised clinical trial.

Sulphoraphane originates from glucoraphanin in broccoli and is associated with anti-cancer effects. A preclinical study suggested that daily consumption of broccoli may increase the production of sulphoraphane and sulphoraphane metabolites available for absorption. The objective of this study was to determine whether daily broccoli consumption alters the absorption and metabolism of isothiocyanates derived from broccoli glucosinolates. We conducted a randomised cross-over human study (n 18) balanced for BMI and glutathione S-transferase μ 1 (GSTM1) genotype in which subjects consumed a control diet with no broccoli (NB) for 16 d or the same diet with 200 g of cooked broccoli and 20 g of raw daikon radish daily for 15 d (daily broccoli, DB) and 100 g of broccoli and 10 g of daikon radish on day 16. On day 17, all subjects consumed a meal of 200 g of broccoli and 20 g of daikon radish. Plasma and urine were collected for 24 h and analysed for sulphoraphane and metabolites of sulphoraphane and erucin by triple quadrupole tandem MS. For subjects with BMI >26 kg/m2 (median), plasma AUC and urinary excretion rates of total metabolites were higher on the NB diet than on the DB diet, whereas for subjects with BMI <26 kg/m2, plasma AUC and urinary excretion rates were higher on the DB diet than on the NB diet. Daily consumption of broccoli interacted with BMI but not GSTM1 genotype to affect plasma concentrations and urinary excretion of glucosinolate-derived compounds believed to confer protection against cancer. This trial was registered as NCT02346812.

Identification and Quantification of Volatile Ramson-Derived Metabolites in Humans.

Ramson (Allium ursinum) is known for its typical garlic-like aroma. Both ramson and garlic belong to the genus allium which is characterized by a high content of sulfurous compounds. However, in contrast to garlic, ramson is in general not associated with an unpleasant breath following consumption. While there is data available regarding the metabolism of volatile garlic constituents in the human body, the metabolism of ramson was not yet addressed. To elucidate if ramson has an impact on the body odor, this study aimed at identifying volatile ramson-derived metabolites in human milk and urine. Therefore, milk and urine samples were gathered before and after ramson consumption, and were analyzed sensorially by a trained human sensory panel as well as chemo-analytically applying gas chromatography-mass spectrometry/olfactometry (GC-MS/O). Sensory evaluation revealed a garlic-/cabbage like odor in milk samples obtained after ramson consumption, demonstrating that ramson consumption affected the milk aroma. Analyzes by means of GC-MS/O further confirmed excretion of three ramson-derived metabolites in milk and urine samples collected after ramson consumption, namely allyl methyl sulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO2). Of these metabolites only AMS had a garlic-/cabbage-like odor, while the other two were odorless. These metabolites were subsequently quantified using stable isotope dilution assays. Nine urine sets, each comprising eight urine samples, and nine milk sets, each comprising four samples, were analyzed. In case of the urine sets a time interval of about 24 h was monitored, in case of the milk sets a time interval of up to 9 h. Despite the fact that all samples contained the same metabolites there were relevant differences found between individual subjects, especially with regard to the temporal rate of metabolite excretion. Generally, the maxima of metabolite excretion were observed in milk sets within 3 h after ramson consumption. In urine the highest AMS and AMSO amounts were observed within 2 h whereas the maximum concentration of AMSO2 was reached about 2 to 4 h after ramson ingestion. This study suggests that ramson constituents are heavily metabolized in the human body.

Absorption and Metabolism of Isothiocyanates Formed from Broccoli Glucosinolates: Effects of Daily Consumption

Sulforaphane is derived from glucoraphanin, the predominant glucosinolate in broccoli, and is associated with anti‐cancer effects. A preclinical study suggested that daily consumption of broccoli may increase the production of sulforaphane and sulforaphane metabolites available for absorption. To test this result in humans, we designed a randomized crossover study (n=18) during which subjects consumed a Brassica‐free control diet for 16 days or the same diet with 200 g of broccoli and 20 g of fresh daikon radish for 16 days. On day 17, all subjects consumed 200 g of broccoli with 20 g of daikon radish. Plasma and urine were collected over 24 h and analyzed for sulforaphane and metabolites of sulforaphane and erucin by tandem mass spectrometry. Females and subjects with the GSTM1+ genotype had higher concentrations of total metabolites in plasma when they had been consuming the control diet prior to day 17. Subjects with BMI&gt;26 (median) had higher total plasma metabolites following the control diet compared to the daily broccoli diet, whereas subjects with BMI&lt;26 had higher levels following the broccoli diet compared to the control diet. The urinary excretion rate of total metabolites was higher following the control diet compared to the broccoli diet for subjects with BMI&gt;26. These results indicate that multiple factors affect the plasma concentrations of compounds believed to confer protection against cancer.Support or Funding InformationSupported by the National Cancer Institute and the U.S. Department of Agriculture.

Abstract B14: Hsp90-inhibitor drug conjugate STA-12-8666 demonstrates complete tumor regression in preclinical models of pediatric sarcoma

Abstract Long-term survival in patients with metastatic relapsed or recurrent Ewing sarcoma (ES) and rhabdomyosarcoma (RMS) is dismal. Encouraging responses to irinotecan, a topoisomerase 1 (Top1) inhibitor, have been seen in these patients; however, limitations in irinotecan bioavailability, including low conversion rate to the active metabolite (SN-38) and high excretion rate of the inactive form, hinder efficacy. HSP90 is widely expressed in cancer cells, and HSP90 inhibitors (HSP90i) have favorable pharmacokinetics for anticancer use, as they remain in tumors for longer periods of time and at higher steady-state levels compared to normal tissue. This property makes them ideal intracellular delivery vehicles for chemotherapeutic drugs, allowing for high tumor exposure and low systemic toxicity. STA-12-8666 (Synta Pharmaceuticals) is an HSP90i drug conjugate (HDC) consisting of a weak HSP90i attached to SN-38 through a cleavable chemical linker. The purpose of this study was to test this HDC in xenograft models of pediatric sarcoma and to investigate its mechanism of action. To test therapeutic efficacy of this HDC, female SCID mice underwent orthotopic injection of ES or RMS cells from established cell lines or PDX tissue. When tumors reached a desired size, mice were randomized and then treated weekly with HDC, vehicle, ganetespib (a highly potent HSP90i), high dose irinotecan, protracted dose irinotecan, or irinotecan plus ganetespib. Tumors were measured twice per week, and mice were weighed weekly to determine drug tolerability. Tumors were harvested at midpoints and at study endpoint for biology studies. Activity of pharmacodynamic (PD) markers was investigated in tumor tissue. In xenograft models of ES and RMS, treatment with HDC produced superior antitumor efficacy compared to the other arms. When initial treatment began in mice with palpable tumors (between 100 and 500 mm3 (ES) or 50 and 90 mm3 (RMS)), all tumors underwent complete regression after 2 doses of HDC, with total tumor eradication in all ES mice and several RMS mice. In the RMS group, 6/8 mice relapsed by 23 weeks, and all 6 of those responded to retreatment with HDC. When initial treatment was delayed until tumors reached between 800 mm3 and 1000 mm3, complete regressions were again achieved in ES after 2 doses and RMS after 4 doses. Compared with high dose weekly irinotecan, which also induced tumor regression, mice treated with HDC had longer and more persistent remissions. A dose response effect was seen in HDC with cures noted in mice with ES at the 100- and 150-mg/kg doses and longest remissions noted in RMS in the 150-mg/kg group. Tolerability of the HDC was excellent with no toxicity-related deaths or weight loss in any treated mice. Studies using PDX models are ongoing and will be reported. Activity of γH2AX in tumor samples was explored as a PD marker of Top1 inhibitor activity. Mice bearing ES were treated with a single dose of vehicle, irinotecan, or HDC and one mouse per group was sacrificed at serial intervals between 6 hours and 10 days post-treatment. Expression of γH2AX in irinotecan mice began to wane between days 1 and 3, whereas in HDC mice, it was still detectable at day 7, suggesting that HDC results in more persistent inhibition of topoisomerase 1 compared to irinotecan. To look at the potential role of HSP90 inhibition in this HDC, HSP70 activity was investigated as a marker of HSP90 inhibition in samples from the dose finding experiment. In ES, no HSP70 was detected in samples from mice treated with irinotecan or HDC, suggesting the primary mechanism of action is via SN-38. In RMS, HSP70 was slightly induced in mice treated with higher doses of the HDC, perhaps contributing to the higher relapse rate in this model. Preclinical data suggest that this HDC may be a promising anticancer agent for ES and RMS patients. Citation Format: Christine M. Heske, Arnulfo Mendoza, Choh Yeung, David A. Proia, Len Neckers, Lee J. Helman. Hsp90-inhibitor drug conjugate STA-12-8666 demonstrates complete tumor regression in preclinical models of pediatric sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B14.

Phenotypic, metabolic, and molecular genetic characterization of six patients with congenital adrenal hyperplasia caused by novel mutations in the CYP11B1 gene

Congenital adrenal hyperplasia (CAH) is an autosomal recessive inherited disorder of steroidogenesis. Steroid 11β-hydroxylase deficiency (11β-OHD) due to mutations in the CYP11B1 gene is the second most common form of CAH.In this study, 6 patients suffering from CAH were diagnosed with 11β-OHD using urinary GC–MS steroid metabolomics analysis. The molecular basis of the disorder was investigated by molecular genetic analysis of the CYP11B1 gene, functional characterization of splicing and missense mutations, and analysis of the missense mutations in a computer model of CYP11B1.All patients presented with abnormal clinical signs of hyperandrogenism. Their urinary steroid metabolomes were characterized by excessive excretion rates of metabolites of 11-deoxycortisol as well as metabolites of 11-deoxycorticosterone, and allowed definite diagnosis. Patient 1 carries compound heterozygous mutations consisting of a novel nonsense mutation p.Q102X (c.304C>T) in exon 2 and the known missense mutation p.T318R (c.953C>G) in exon 5. Two siblings (patient 2 and 3) were compound heterozygous carriers of a known splicing mutation c.1200+1G>A in intron 7 and a known missense mutation p.R448H (c.1343G>A) in exon 8. Minigene experiments demonstrated that the c.1200+1G>A mutation caused abnormal pre-mRNA splicing (intron retention). Two further siblings (patient 4 and 5) were compound heterozygous carriers of a novel missense mutation p.R332G (c.994C>G) in exon 6 and the known missense mutation p.R448H (c.1343G>A) in exon 8. A CYP11B1 activity study in COS-1 cells showed that only 11% of the enzyme activity remained in the variant p.R332G. Patient 6 carried a so far not described homozygous deletion g.2470_5320del of 2850bp corresponding to a loss of the CYP11B1 exons 3–8. The breakpoints of the deletion are embedded into two typical 6 base pair repeats (GCTTCT) upstream and downstream of the gene.Experiments analyzing the influence of mutations on splicing and on enzyme function were applied as complementary procedures to genotyping and provided a rational basis for understanding the clinical phenotype of CAH.

Human microsomal cyttrochrome P450-mediated reduction of oxysophocarpine, an active and highly toxic constituent derived from Sophora flavescens species, and its intestinal absorption and metabolism in rat

Oxysophocarpine (OSC), an active and toxic quinolizidine alkaloid, is highly valued in Sophora flavescens Ait. and Subprostrate sophora Root. OSC is used to treat inflammation and hepatitis for thousands of years in China. This study aims to investigate the CYP450-mediated reduction responsible for metabolizing OSC and to evaluate the absorption and metabolism of OSC in rat in situ. Four metabolites were identified, with sophocarpine (SC) as the major metabolite. SC formation was rapid in human and rat liver microsomes (HLMs and RLMs, respectively). The reduction rates in the liver are two fold higher than in the intestine, both in humans and rats. In HLMs, inhibitors of CYP2C9, 3A4/5, 2D6, and 2B6 had strong inhibitory effects on SC formation. Meanwhile, inhibitors of CYP3A and CYP2D6 had significant inhibition on SC formation in RLMs. Human recombinant CYP3A4/5, 2B6, 2D6, and 2C9 contributed significantly to SC production. The permeability in rat intestine and the excretion rates of metabolites were highest in the duodenum (p<0.05), and the absorbed amount of OSC in duodenum and jejunum was concentration-dependent. The metabolism could be significantly decreased by CYP3A inhibitor ketoconazole. In conclusion, the liver was the main organ responsible for OSC metabolism. First-pass metabolism via CYP3A4/5, 2B6, 2D6, and 2C9 may be the main reason for the poor OSC bioavailability.

Urine Excretion of Caffeine and Select Caffeine Metabolites Is Common in the US Population and Associated with Caffeine Intake1–4

Background: Caffeine is a widely consumed psychoactive stimulant and is of epidemiologic interest. Major sources of caffeine are challenging to standardize, and the use of biomarkers is proposed as an alternative means of assessing intake.Objective: We described urine caffeine and caffeine metabolite concentrations (n = 2466) and excretion rates (n = 2261) in the US population ≥6 y by age, sex, race-ethnicity, and caffeine intake (from foods, beverages, and dietary supplements).Methods: We measured caffeine and 14 of its metabolites in spot urine samples from the cross-sectional NHANES 2009–2010 by use of LC-tandem mass spectrometry.Results: Caffeine and its metabolites were detectable in the urine of most persons, generally at concentrations ≥1 μmol/L. Median concentrations (95% CI) ranged from 0.560 (0.497, 0.620) μmol/L to 58.6 (48.6, 67.2) μmol/L; median excretion rates from 0.423 (0.385, 0.468) nmol/min to 46.0 (40.7, 50.2) nmol/min. Urine concentrations and excretion rates for 9 analytes (caffeine, theophylline, paraxanthine, 1-methylxanthine, 1-methyluric acid, 1,3-dimethyluric acid, 1,7-dimethyluric acid, 1,3,7-trimethyluric acid, and 5-acetylamino-6-amino-3-methyluracil) had moderate correlations with caffeine intake (Spearman ρ = 0.55–0.68, P < 0.0001); the remaining analytes had low correlations (ρ = 0.15–0.33, P < 0.0001). We observed larger differences in geometric mean concentrations and excretion rates between the highest vs. lowest quartiles of caffeine intake for these 9 compounds than the rest. Consistent with dietary caffeine intake, we observed that urine concentrations and excretion rates for most compounds were significantly (P < 0.05) higher in men than women, non-Hispanic whites than Hispanics and non-Hispanic blacks, and highest in persons aged 40–59 y.Conclusion: Excretion of caffeine and its metabolites in urine is common in the US population. According to the observed associations between spot urine concentrations or excretion rates with caffeine intake, several of these compounds show promise as potential biomarkers of caffeine intake.

Reduction in Cortisol Inactivation is Part of the Adrenal Stress Response to Cardiac and Noncardiac Pediatric Surgery: A Prospective Study Using Gas Chromatography-Mass Spectrometry Analysis

We examined the hypothesis that major cardiac surgery triggers a more intense adrenal stress response than less intensive noncardiac surgery, which then alters cortisol inactivation. Urinary excretion rates of glucocorticoid metabolites were determined before and after surgery using gas chromatography-mass spectrometry in 29 children undergoing scheduled major cardiac surgery and 17 control children undergoing conventional noncardiac surgery in a prospective observational study. Excretion rates of glucocorticoid metabolites were summed and corrected for creatinine excretion to calculate cortisol production rates (mg/mmol creatinine/m(2) body surface area). Precursor/product ratios from individual metabolites were calculated to characterize cortisol inactivation (11β-hydroxysteroid dehydrogenase). Postoperatively, median cortisol production rates increased in both groups ( from 2.7 to 9.3; controls: from 2.7 to 5.8; p<0.001) with no significant difference between groups (p=0.12). Ratios of cortisol to cortisone metabolites, indicating the overall activity of 11β-hydroxysteroid dehydrogenase, increased postoperatively in both groups (p<0.001). In conclusion, surgery resulted in a distinct postoperative increase in cortisol production. In contrast to our hypothesis, children undergoing major cardiac surgery did not show an increased adrenal stress response compared to children undergoing conventional surgery. Furthermore, the reduction in cortisol inactivation appears to be an essential part of the stress response to pediatric surgery in general.

Metabolism, Distribution, and Excretion of Deoxynivalenol with Combined Techniques of Radiotracing, High-Performance Liquid Chromatography Ion Trap Time-of-Flight Mass Spectrometry, and Online Radiometric Detection

Dispositions of deoxynivalenol (DON) in rats and chickens were investigated, using a radiotracer method coupled with a novel γ-accurate radioisotope counting (γ-ARC) radio-high-performance liquid chromatography ion trap time-of-flight tandem mass spectrometry (radio-HPLC-IT-TOF-MS/MS) system. 3β-(3)H-DON was chemically synthesized and orally administrated to both sexes of rats and chickens as single or multiple doses. The results showed that DON was widely distributed and quickly eliminated in all tissues. The highest concentration was found in the gastrointestinal tract at 6 h post-administration. Substantially lower levels were detected in the kidney, liver, heart, lung, spleen, and brain. Three new metabolites were identified tentatively as 10-deoxynivalenol-sulfonate, 10-deepoxy-deoxynivalenol (DOM-1)-sulfonate, and deoxynivalenol-3α-sulfate. Deoxynivalenol-3α-sulfate was a major metabolite in chickens, while the major forms in rats were DOM-1 and DON. Additionally, a higher excretion rate in urine was observed in female rats than in male rats. The differences in metabolite profiles and excretion rates, which suggested diverse ways to detoxify, may relate to the different tolerances in different genders or species.