Total Urinary Recovery Research Articles

Gastrointestinal permeability and inflammatory status of preweaning dairy calves in response to decreasing the ratio of n-6 to n-3 fatty acid in milk replacer

The ratio of n-6 to n-3 fatty acid (FA) is between 2 and 10 times higher in milk replacer (MR) than in whole milk, which may promote inflammation and compromise the integrity of the intestinal epithelium. To evaluate how decreasing the n-6:n-3 FA ratio of MR affects gastrointestinal (GIT) permeability and inflammatory status, 30 dairy calves (2.8 ± 1.06 d of age; mean ± standard deviation) were randomly assigned to be fed an MR with an n-6:n-3 FA ratio of 40:1 (CON; 29.3% crude fat of DM; n = 15) or 6.5:1 (n-3; 29.1% crude fat of DM; n = 15). Calves were fed 7.0 L/d in 2 meals. Calves were weighed and fecal consistency was analyzed weekly. On d 22, calves were administered Cr-EDTA, lactulose, and d-mannitol to assess GIT permeability. Blood and total urine were sequentially collected for 6 and 24 h, respectively, and analyzed for marker content. Whole blood collected 4 h after the meal was subjected to an ex vivo lipopolysaccharide (LPS) challenge to evaluate cytokine secretion from blood cells. Calves were euthanized on d 25 for collection of intestinal tissue samples. Tissue samples were processed to assess FA composition by gas chromatography, histomorphology by bright-field microscopy, and gene expression of tight junction proteins, lipid metabolism enzymes, and immune molecules by real-time quantitative PCR. Data were analyzed using PROC GLIMMIX in SAS (version 9.4, SAS Institute Inc.). Growth performance and fecal consistency were unaffected. Calves fed MR with a lower ratio of n-6 to n-3 FA had 2-fold higher n-3 FA contents and 2-fold lower ratios of n-6 to n-3 FA in proximal jejunum and ileum tissues. Total urinary recovery (0-24 h relative to marker administration) and plasma concentrations of the markers were unaffected. Expression of TJP1 tended to be higher in proximal jejunum tissue and lower in ileum tissue of n-3 calves. The expression of TLR4 and TNFA tended to be higher and CD14 was higher in ileum tissue of n-3 calves. Plasma concentrations of interleukin-4 were decreased in response to the ex vivo LPS challenge in n-3 calves. Histomorphology and GIT permeability were largely unaffected by treatment. Furthermore, the inclusion of linseed and algae oil may promote inflammation, as suggested by greater concentrations of the acute-phase proteins haptoglobin and serum amyloid A postprandially, demonstrating that fat sources should be evaluated for their suitability for MR formulations. Understanding how MR composition affects dairy calf health may improve nutritional strategies on farm.

Nitrate and nitrite absorption, recycling and retention in tissues of sheep

Dietary nitrate is of increasing interest both for the pharmacological effects of its metabolites as well as its capacity to inhibit methanogenesis in the gut. A sequence of three experiments was conducted to investigate the absorption, metabolism and excretion of nitrate and nitrite through the gastrointestinal tract (GIT) of sheep, and to determine the fate of nitrate and nitrite in body fluids, tissues and faeces after intravenous dosing with 15N-labeled potassium nitrate (K15NO3) and with 15N-labeled sodium nitrite (Na15NO2). In Experiment 1, twelve female Merino sheep were assigned to one of two dietary treatments and adapted to the experimental diet over two weeks. Six sheep were fed a control diet of wheaten chaff mixture (600 g wheaten chaff plus 200 g wheat grain, CON) and six sheep were fed the CON diet with the inclusion of 18 g nitrate/kg DM (Nitrate). After acclimation to the diets, all sheep received a single intravenous dose of K15NO3 and were placed in metabolic cages for daily collection of total faeces and urine over 6 days. Experiment 2 studied movement of an intravenous dose of 15N in body fluids and tissues. Two sheep not adapted to dietary nitrate were dosed intravenously with K15NO3 or Na15NO2 and body fluids and tissue samples were collected 60 min after dosing. Finally, Experiment 3 was conducted to identify and quantify the major sites of nitrate and nitrite transfer within the body, focusing on absorption, partitioning and secretion into the GIT of anaesthetised sheep. A single dose of sodium nitrate (NaNO3) or sodium nitrite (NaNO2) was introduced into the rumen or abomasum or small intestine, and changes in nitrate and nitrite concentrations in other pools, including plasma, urine and saliva, were determined. Results from Experiment 1 showed that urinary recovery of 15N dose in urea after 46 h and total urinary recovery of 15N 141 h after dosing were greater in sheep fed the Nitrate diet relative to CON (P < 0.05). Recoveries of 15N in tissues indicated that nitrate and nitrite principally accumulated in the skin and muscle of sheep (Experiment 2). Finally, Experiment 3 indicated that nitrate and nitrite were rapidly absorbed from the rumen, abomasum and small intestine into the bloodstream. Nitrite was oxidized in plasma and the resultant nitrate was concentrated and recycled via saliva. Appearance of 15N in urinary urea confirmed the passage of plasma nitrate to the digestive tract, via saliva or transruminal flow to be reduced by gut biota to ammonia.

Abstract 1079: Pharmacokinetics and biodistribution of LNA-i-miR-221 in NOD.SCID mice and Cynomolgus monkeys

Abstract miRNAs therapeutics is based on the use of oligonucleotide engineered to efficiently replace or sequester endogenous miRNAs. miR-221 is upregulated in several tumors, including multiple myeloma (MM), where it promotes cancer cell proliferation mainly via targeting the negative regulators of G1/S cell cycle progression p27 and/or p57. We previously demonstrated that direct miR-221 inhibition by the use of an original LNA inhibitor, LNA-i-miR-221, induces significant anti-MM activity and upregulates canonical targets in vitro and in vivo.To assess pharmacokinetics (PK)/pharmacodynamics of LNA-i-miR-221, we set-up, and report here, novel assays for oligo quantification in plasma, urine and tissues of NOD.SCID mice and Cynomolgus monkeys. We investigated the LNA-i-miR-221 PKs by UHPLC-MS/MS, following solid-phase extraction, in plasma and urine, and tissue uptake by in-situ hybridization (ISH). In particular, PK profile was calculated by UHPLC-MS/MS after single dose i.p injection of LNA-i-miR-221 (25 mg/kg) in mouse and the Tmax was identified at 1.5 hours with Cmax of 1110 ng/mL. Three hours after bolus injection, the plasma concentration was about 60% reduced and the molecule was not detectable at later time-points. The plasma exposure of the oligo showed an AUClast of 2330 h*ng/mL. Similarly, in monkeys the LNA-i-miR-221 plasma concentration following a single i.v. bolus injection (8.75 mg/kg) peaked at a Tmax of ½ hour, with a Cmax of 23600 ng/mL. The plasma exposure of the oligo showed an AUClast of 49300h*ng/mL, C0 of 61400 ng/mL and the t1/2 was 12.8 hours. Urine PK evaluation was performed in monkey on samples collected from 2 up to 48 hours. The excreted LNA-i-miR-221 was quantified equal to 0.68 mg with a total urinary recovery of approximately 2.68% of the amount injected (25,375 mg). Specifically, the urinary concentration/time-points plot shows the excretion of 9394,5 ng/mL (1.63% of recovery) in the interval of 2-6 hours. In addition, murine tissues analyzed by ISH revealed strong signals and long-lasting accumulation of the oligo in mice vital organs together with upregulation of the main canonical miR-221 target p27, as assessed by IHC. In addition, ISH analysis excluded that the LNA-i-miR-221 crosses the blood-brain barrier. All together PK results demonstrated that LNA-i-miR-221 has a short serum half-life with a rapid tissue uptake and minimum urinary excretion of the intact molecule, with a very long tissue half-life and biological activity. Moreover, we report preliminary toxicity analysis in non-human primates. No changes in the monkeys’ behavior, body weight or food consumption were observed in treated animals.In conclusion our results suggest that LNA-i-miR-221 is a promising anti-MM agent associated with a favorable PK, long-lasting tissue bioavailability and good safety profile in mice and in monkeys, providing the rationale for translation of this novel miR-221 inhibitor in early clinical investigation Citation Format: Maria Teresa Di Martino, Maria Eugenia Gallo Cantafio, Boye S. Nielsen, Mariamena Arbitrio, Annamaria Gullà, Chiara Mignogna, Massimo Breda, Niels M. Frandsen, Pierosandro Tagliaferri, Pierfrancesco Tassone. Pharmacokinetics and biodistribution of LNA-i-miR-221 in NOD.SCID mice and Cynomolgus monkeys. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1079.

The influence of age and weaning on permeability of the gastrointestinal tract in Holstein bull calves

Fourteen Holstein bull calves were used in a randomized complete block design to investigate the effect of calf age and weaning on permeability of the gastrointestinal tract (GIT). Calves were randomly assigned to 1 of 2 treatments: (1) a weaning protocol that was initiated on d 35; WN; n=7), or (2) a control treatment where calves were not weaned (CON; n=7). Calves were bottle-fed milk replacer (150g/L), in 3 equal portions/d targeting 15% of their body weight (BW) in liquid milk intake [approximately 21.1g/kg of BW/d, dry matter (DM) basis]. On d 35, the amount of milk replacer offered to WN calves was reduced to 7.5% of BW for 7 d before calves were weaned on d 42. On d 14, 28, and 42, calves were orally dosed with 500mL of Cr-EDTA (179mM Cr-EDTA solution) and housed in a metabolism crate to enable total urine collection and determination of total urinary Cr recovery as an indicator of total-tract permeability. On d 44, calves were killed and tissues from the rumen, omasum, duodenum, jejunum, ileum, cecum, and proximal and distal colon were collected, rinsed, and transported in buffer solution (pH 7.4 at 38.5°C). Tissues were incubated in Ussing chambers under short-circuit conditions with buffer solutions designed to mimic the mucosal and serosal energy source that would be available in vivo (glucose for tissues from the small intestine and short-chain fatty acids for tissues that would be exposed to fermentation; rumen, omasum, and large intestinal tissues). The serosal to mucosal flux of 14C-mannitol and 3H-inulin was measured for each region. Although we detected treatment × period interactions for BW and starter intake, dietary treatments did not differ within a week. Overall, the time that ruminal pH was <5.5 was less before weaning than after weaning. We observed a differential response for the appearance of Cr in urine for WN and CON calves, where the appearance of Cr (mg/48h) in urine decreased for both treatments from d 14 to 28, but increased from d 28 to 42 for WN, whereas Cr appearance continued to decrease for CON. The flux of mannitol and inulin did not differ between treatments but did differ among region of the GIT, with rumen, duodenum, and jejunum having the greatest permeability. These data suggest that permeability of the GIT decreases with age but weaning may disrupt this process. The rumen, duodenum, and jejunum appear to be the regions with greatest permeability.

Some Anthocyanins Could Be Efficiently Absorbed across the Gastrointestinal Mucosa: Extensive Presystemic Metabolism Reduces Apparent Bioavailability

Despite the accumulating evidence supporting the health effects of anthocyanins, their plasma concentrations were found to be very low. However, 30 and 56% of cyanidin 3-glucoside (Cy-3-glc) and pelargonidin 3-glucoside (Pg-3-glc) were found as protocatechuic acid (PCA) and 4-hydroxybenzoic acid, respectively, in plasma following oral administration in humans. Second, 12.4% of (13)C was recovered from urine and breath following oral ingestion of [(13)C]-Cy-3-glc in humans. The actual percentage of [(13)C]-Cy-3-glc absorbed across the gastrointestinal wall could be higher because of the involvement of enterohepatic recycling in the disposition of anthocyanins. In animal studies, high total urinary recoveries were found following oral ingestion of (14)C-labeled anthocyanins. Third, anthocyanins seem to be efficiently absorbed following in situ gastric and intestinal perfusions in rats. Therefore, some anthocyanins could be efficiently absorbed from the gastrointestinal lumen, undergo extensive first-pass metabolism, and enter the systemic circulation as metabolites.

Clinical evaluation for T-1982 (cefbuperazone) in the field of pediatric infection

T-1982 (cefbuperazone), a new injectable cephamycin antibiotic, was employed for bacterial infections in children, and the following results were obtained. 1. When administered intravenously at a dose of 20 mg/kg to a 6-year-old female child, serum levels were 62 micrograms/ml at 30 minutes, 39 micrograms/ml at 1 hour, 17.6 micrograms/ml at 2 hours, 6.8 micrograms/ml at 4 hours and 2.9 micrograms/ml at 6 hours with serum half-life (T 1/2) of 76 minutes. Urinary excretion rates were 41.0, 5.3% and 2.4% respectively at 0-2, 2-4 hours and 4-6 hours, and urinary levels were 820 micrograms/ml, 182 micrograms/ml and 310 micrograms/ml, respectively. The total urinary recovery within 6 hours was 48.7%. 2. A total of 11 cases of pediatric infections was treated with T-1982. The clinical efficacy evaluated for 9 cases, excluding 2 cases of non-bacterial infections, was as follows; excellent in 4, good in 1 out of 5 cases of pneumonia, good in 1 case of cervical purulent lymphadenitis, and excellent in 1, good in 1, poor in 1 out of 3 cases of urinary tract infection. 3. As side effect, mild diarrhea in 1 case and slight elevation of GOT, GPT in 2 cases were observed. 4. These results suggest that T-1982 is of good use for bacterial infections in children and the expected efficacy is obtained at a dose of 20 mg/kg 3 times a day.

Urinary Excretion Kinetics of 3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) and Its Phase I and Phase II Metabolites in Humans following Controlled MDMA Administration

3,4-Methylendioxymethamphetamine (MDMA) is excreted inhuman urine as unchanged drug and phase I and II metabolites. Previous urinary excretion studies after controlled oral MDMA administration have been performed only after conjugate cleavage. Therefore, we investigated intact MDMA glucuronide and sulfate metabolite excretion. We used LC-high-resolution MS and GC-MS to reanalyze blind urine samples from 10 participants receiving 1.0 or 1.6 mg/kg MDMA orally. We determined median C(max),t(max), first and last detection times, and total urinary recovery; calculated ratios of sulfates and glucuronides; and performed in vitro-in vivo correlations. Phase II metabolites of 3,4-dihydroxymethamphetamine (DHMA),4-hydroxy-3-methoxymethamphetamine (HMMA),3,4-dihydroxyamphetamine (DHA), and 4-hydroxy-3-methoxyamphetamine were identified, although only DHMA sulfates, HMMA sulfate, and HMMA glucuronide had substantial abundance. Good correlation was observed for HMMA measured after acid hydrolysis and the sum of unconjugated HMMA, HMMA glucuronide, and HMMA sulfate (R(2) = 0.87). More than 90% of total DHMA and HMMA were excreted as conjugates. The analyte with the longest detection time was HMMA sulfate. Median HMMA sulfate/glucuronide and DHMA 3-sulfate/4-sulfate ratios for the first 24 h were 2.0 and 5.3, respectively, in accordance with previous in vitro calculations from human liver microsomes and cytosol experiments. Human MDMA urinary metabolites are primarily sulfates and glucuronides,with sulfates present in higher concentrations than glucuronides. This new knowledge may lead to improvements in urine MDMA and metabolite analysis in clinical and forensic toxicology, particularly for the performance of direct urine analysis.

Correlation of bioavailability in man with simulated absorption data for three doxantrazole preparations.

The in vitro and in vivo availability of doxantrazole, a potential antiallergic compound has been evaluated. A solution was significantly less bioavailable than either tablet or suspension formulations and it is suggested that this is associated with the large volume of the solution vehicle altering the hydrophilicity of the gastrointestinal fluids. In vitro availability was determined from absorption rate constants and absorption profiles obtained using the Sartorius absorption and solubility simulators. A statistically significant correlation was found between the percentage absorbed in vitro at 1 h and both total urinary recovery and area under plasma curve values in vivo. It is considered that in vitro determination of diffusion through artificial lipid membranes may be a useful predictive method of in vivo availability.

Pharmacokinetics of alkylresorcinol metabolites in human urine

Wholegrain cereals are reported to promote beneficial health effects. Wholegrain wheat and rye are almost exclusive sources of alkylresorcinols, and intact alkylresorcinols together with their plasma and urinary metabolites, 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA) and 3,5-dihydroxybenzoic acid (DHBA), have been proposed as biomarkers of the intake of these foods in humans. The pharmacokinetics of alkylresorcinols and their metabolites in plasma have been determined but not that of the urinary metabolites. We aimed to characterise the urinary pharmacokinetics of alkylresorcinol metabolites in humans to evaluate their potential as biomarkers of wholegrain wheat and rye. A group of fifteen volunteers followed a low-alkylresorcinol diet for 2d before ingesting a single dose of rye bread, containing 100mg alkylresorcinols. Urine was collected between baseline (0h) and 25h after administration. Thereafter alkylresorcinol metabolites were quantified by HPLC with coulometric electrode array detection. Maximum excretion rates were observed at 5-6h for both metabolites, DHPPA being predominant over DHBA and also possessing a greater area under the curve0-25h. Total urinary recovery between 0 and 25h yielded 43% of ingested alkylresorcinols, and at 25h significant amounts of metabolites were still retained in the body, suggesting that even a spot urine sample may be sufficient to indicate whether or not wholegrain wheat or rye is a daily dietary component. These results support the use of urinary DHPPA and DHBA as biomarkers of wholegrain wheat and rye and enable new potential for studying the association between wholegrain intake and diseases, even in the absence of dietary data.

Human urinary excretion of NC100692, an RGD-peptide for imaging angiogenesis

The 99mTc complex of NC100692 is being evaluated as a diagnostic agent for the detection of angiogenesis. In the present study, human urine samples from a clinical Phase I study were analysed using reversed-phase liquid chromatography coupled with an ion-trap mass spectrometer (LC–MS) in order to estimate the amount of intact NC100692 and any metabolites excreted in urine following intravenous injection of 150 μg 99mTc-NC100692. Only intact NC100692 was observed in these urine samples, no metabolites were detected, in contrast to data earlier reported for rat urine where two metabolites and no NC100692 were observed. Within 4–8 h after injection, 30 ± 6% of the injected NC100692 was excreted in the urine, with the majority (23 ± 5%) recovered within the first hour post-injection. There was good agreement between the calculated urinary recoveries of NC100692 and total radioactivity in urine samples voided approximately 1 h post-injection. NC100692 constituting 98 ± 24% of the total urinary 99mTc recovery indicating that the 99mTc excreted during this period was most likely excreted as 99mTc-NC100692. The ratio of NC100692 to 99mTc decreased in the urine samples as a function of time following injection for all subjects; this change is most likely due to reduced accuracy in the results at low levels of NC100692.

The comparative bioavailability of an extended‐release niacin and lovastatin fixed dose combination tablet versus extended‐release niacin tablet, lovastatin tablet and a combination of extended‐release niacin tablet and lovastatin tablet

Lovastatin and extended-release (ER) niacin in a fixed dose combination (Advicor) is approved for the treatment of dyslipidemia. Since both drugs are extensively metabolized, this study investigated the bioavailability and pharmacokinetics of their co-administration following single-dose administration. In a 4-way crossover study 40 subjects received: two 1000/20 Advicor tablets (ADV), two 1000 mg niacin ER tablets (NSP), two 20mg lovastatin tablets (Mevacor; MEV), and two niacin ER 1000 mg tablets with two lovastatin 20mg tablets (NSP+MEV). Plasma was assayed for niacin, nicotinuric acid (NUA), lovastatin, lovastatin acid and HMGCoA reductase inhibition. Urine was assayed for niacin and its metabolites, NUA, N-methylnicotinamide and N-methyl-2pyridone-5-carboxamide. Least square mean ratios and 90% confidence intervals for C(max) and AUC((0-t)) were determined for NSP+MEV versus MEV or NSP, ADV versus MEV or NSP, and ADV versus NSP+MEV. Co-administration of niacin and lovastatin did not significantly influence C(max) and AUC((0-t)) of lovastatin, niacin, NUA and total urinary recovery of niacin and metabolites. A 22 to 25% decrease in lovastatin acid C(max) was observed while lovastatin acid AUC((0-t)) was not affected. The HMGCoA reductase inhibition C(max) and AUC((0-t)) were not affected indicating that the difference in lovastatin acid C(max) was not clinically relevant.

Absorption of isoflavones in humans: effects of food matrix and processing

If soy isoflavones are to be effective in preventing or treating a range of diseases, they must be bioavailable, and thus understanding factors which may alter their bioavailability needs to be elucidated. However, to date there is little information on whether the pharmacokinetic profile following ingestion of a defined dose is influenced by the food matrix in which the isoflavone is given or by the processing method used. Three different foods (cookies, chocolate bars and juice) were prepared, and their isoflavone contents were determined. We compared the urinary and serum concentrations of daidzein, genistein and equol following the consumption of three different foods, each of which contained 50 mg of isoflavones. After the technological processing of the different test foods, differences in aglycone levels were observed. The plasma levels of the isoflavone precursor daidzein were not altered by food matrix. Urinary daidzein recovery was similar for all three foods ingested with total urinary output of 33–34% of ingested dose. Peak genistein concentrations were attained in serum earlier following consumption of a liquid matrix rather than a solid matrix, although there was a lower total urinary recovery of genistein following ingestion of juice than that of the two other foods.

Determinants for Urinary and Plasma Isoflavonesin Humans After Soy Intake

: Consumption of soy foods leads to a biphasic appearance pattern of isoflavones (IFLs) in blood and urine, with peaks appearing at 1-2 h and 4-8 h after intake, but its causes are not understood. IFLs were measured repeatedly from plasma and/or urine after intake of soy foods, IFL glucosides, or aglycons without or with a mildly or radically reduced gut flora as a result of oral antibiotic (AB) treatment, or this combined with mechanical bowel preparation (AB+MBP). The typical biphasic IFL pattern in blood and/or urine was observed when a soy protein drink without (control) or with AB treatment or when IFL glucosides or aglycons were consumed. Soy intake combined with AB+MBP or consumption of puerarin led to a shift of the second peak to much later times. The first peak was absent after puerarin intake. Total urinary IFL recovery was more than 50% lower in the first 24 h, but overall 61% higher after AB+MBP vs. the control. When the area under the curves for corresponding time intervals were compared, individual or total urinary IFL excretion rates were highly correlated with individual or total plasma IFL levels (r = 0.85-0.91; P < 0.001). At the same urinary excretion rate three times more genistein than daidzein remained in the circulation. We conclude that urinary IFL excretion rates reflect circulating IFL levels, with daidzein appearing less in blood and more in urine than genistein. The first and second IFL peaks are due to uptake in the small and large intestine, respectively. The latter is the major locus of uptake (90%) at usual dietary IFL doses (0.15-1.5 mmol/kg body weight). A reduced gut flora delayed IFL uptake but led overall to increased urinary recovery because of less bacterial degradation in the intestine.

Pharmacokinetics and tissue distribution of a new mesalamine rectal gel in ulcerative colitis patients

Objective Mesalamine (5-ASA) has a topical action in the treatment of ulcerative colitis (UC) but many adverse events are likely related to its systemic availability. It is therefore important to assess systemic exposure to 5-ASA and tissue concentrations produced by a new dosage form. This project assessed the pharmacokinetics (PK) of 5-ASA from a new rectal gel in UC patients. Methods 6 UC patients received a single 4 g dose of 5-ASA (Canasa-gel®) and, starting 48 hours later, 4 g HS x 4 days. Blood was collected at intervals for 30 hours and urine for 48 hours after first and last doses. Rectal biopsies were taken 8 hours after the last dose. 5-ASA was assayed by a fully validated LC-MS-MS method. Results After single dose, means (sd) were 2821 (1293) ng/mL for Cmax, 37638 (24370) ng h/mL for AUC0-∞, and 6.3 (8.2) h for half-life. After multiple doses means (sd) were 102 (152) ng/mL for Cmin, 2104 (743) ng/mL for Cmax, 21975 (10808) ng h/mL for AUC0-∞, and 4.0 (2.6) h for half-life. Mean (sd) total (unchanged and metabolized) urinary recovery of 5-ASA after single dose was 718 (388) mg. Mean (sd) rectal 5-ASA concentration was 25.2 (28.5) ng/mg tissue. Conclusion The pharmacokinetics from this new gel compare favorably with those from enema and suppository. It delivers adequate amount of 5-ASA to disease tissues. Based on PK, this new dosage form should be at least as safe and effective as enema and suppository. Clinical trials are required to confirm this conclusion. Clinical Pharmacology & Therapeutics (2004) 75, P38–P38; doi: 10.1016/j.clpt.2003.11.146

Simvastatin Does Not Affect CYP3A Activity, Quantified by the Erythromycin Breath Test and Oral Midazolam Pharmacokinetics, in Healthy Male Subjects

Potential for inhibition of CYP3A activity by simvastatin, an HMG-CoA reductase inhibitor, was evaluated in 12 healthy male subjects who received placebo or 80 mg of simvastatin, the maximal recommended dose, once daily for 7 consecutive days. On day 7, an intravenous injection of 3 microCi [14C N-methyl]erythromycin for the erythromycin breath test (EBT) was coadministered with a 2 mg oral solution of midazolam. The values for percent 14C exhaled during the first hour (for EBT) and the pharmacokinetic parameters of midazolam (AUC, Cmax, t1/2) were not affected following multiple once-daily oral doses of simvastatin 80 mg. The 95% confidence interval was 0.97 to 1.18 for EBT and 0.99 to 1.23 for midazolam AUC. In addition, the total urinary recoveries of midazolam and its 1'-hydroxy metabolites (free plus conjugate) obtained from both treatments were not statistically different (p > 0.200). These data demonstrate that multiple dosing of simvastatin, at the highest recommended clinical dose, does not significantly alter the in vivo hepatic or intestinal CYP3A4/5 activity as measured by the commonly used EBT and oral midazolam probes.

Metabolism of n-Butyl Benzyl Phthalate in the Female Wistar Rat. Identification of New Metabolites

n-Butyl benzyl phthalate (BBP), a plasticizer used in polyvinylchloride (PVC) and other polymers, has been orally administered to female Wistar rats with four doses (150, 475, 780 and 1500 mg/kg body weight/day) for 3 consecutive days. Metabolites recovered in urines were analysed by gas chromatography–mass spectrometry (GC–MS) after 24, 48 and 72 hours. Six metabolites were identified. Mono- n-butyl phthalate (MBuP) and mono- n-benzyl phthalate (MBeP) represented respectively 29–34% and 7–12 % of the total recovered metabolites. Hippuric acid, the main metabolite of benzoic acid, represented the second major metabolite (51–56%). Phthalic acid, benzoic acid and an ω-oxidized metabolite of MBuP were also recovered in urine but in small quantities. BBP was never identified in urines. Total urinary metabolites recovery represented 56% of the dose administered in the first 24 hours. However, total recovery decreased when the dose increases (43% at 780 mg/kg body weight/ day, only 30% at 1500 mg/kg body weight/day). Whatever the time was, BBP metabolites recovered in urines were all present and in the same proportions for the two lowest doses. Discrepancy in metabolites quantities expressed as percentages of the dose observed in urine of rat treated with the highest BBP dose disappeared with time as MBuP, MBeP and hippuric acid recovery has significantly increased at day 3. Metabolic profile of BBP in female rats has been established. The aim of the present study is to identify further the active(s) agent(s) involved in the BBP malformations and teratogenic effects.

Urinary excretion of hydroxycinnamates and flavonoids after oral and intravenous administration

The urinary recoveries of the hydroxycinnamates, ferulic acid (3-methoxy, 4-hydroxy cinnamic acid), and chlorogenic acid (the quinic acid ester of 3,4-dihydroxycinnamic acid), and three structurally related flavonoids were studied in the rat. For the latter, the aglycone quercetin was compared with its 3-glucoside (isoquercitrin) and 3-rhamnoglucoside (rutin). Doses of 50 mg/kg were administered via the oral and intravenous routes and urine collected over the subsequent 24-h period. Reverse phase HPLC with photo-diode array detection was used to analyze the unchanged compound and their metabolites excreted in the urine. Ferulic acid and isoquercitrin were orally absorbed (5.4 and 0.48% of administered dose, respectively) and are therefore bioavailable. In contrast, neither unchanged chlorogenic acid, rutin, quercetin, nor the conjugated metabolites in the form of glucuronide or sulphate were detected in the urine after oral dosing. All the flavonoids studied produced low total urinary recoveries after intravenous administration, 9.2% for quercetin-3-rhamnoglucoside, 6.7% for the 3-glucoside, and 2.4% for the aglycone, indicating that extensive metabolism to low molecular weight compounds or excretion via other routes may be occurring. Overall it can be stated that renal excretion is not a major pathway of elimination for intact flavonoids and hydroxycinnamates in the rat.

Quantitative studies on the urinary metabolic fate of 2-chloro-4-trifluoromethylaniline in the rat using 19F-NMR spectroscopy and directly coupled HPLCNMR-MS

1. The metabolism and urinary excretion of 2-chloro-4-trifluoromethylaniline has been studied in the rat using F-NMR spectroscopy and directly coupled HPLC-NMRMS methods. The compound was dosed to three male Sprague-Dawley rats (50 mg kg i.p.) and urine collected over 0-8, 8-24 and 24-48 h post-dosing. 2. A total urinary recovery of 56.3 2.2% of the dose was achieved up to 48 h after dosing. The major metabolite in the urine was identified as 2-amino-3-chloro-5- trifluoromethylphenylsulphate accounting for a total of 33.5 2.2% of the dose. 3. Further metabolites detected and characterized included 2-chloro-4-trifluoromethylphenylhydroxylamine glucuronide (13.2 0.5% of the dose), 2-amino-3- chloro-5-trifluoromethylphenylglucuronide (3.8 0.4% of the dose) and 2-chloro-4- trifluoromethylaniline- N -glucuronide (3.6 0.1% of the dose). Several minor metabolites were also found and identified, including 2-chloro-4-trifluoromethylphenylsulphamate, which together accounted for 2.1 0.4% of the dose. 4. Directly coupledHPLC-NMR-MS and F-NMR spectroscopy is shown to provide an efficient approach for the unequivocal and rapid determination of the quantitative urinary metabolic fate and excretion balance of a fluorinated xenobiotic without the necessity for specific radiolabelling.

19F-NMR and directly coupled HPLC-NMR-MS investigations into the metabolism of 2-bromo-4- trifluoromethylaniline in rat: a urinary excretion balance study without the use of radiolabelling

1. The metabolic fate and urinary excretion of 2-bromo-4-trifluoromethylaniline has been studied in rat using 19F-NMR spectroscopic and directly coupled HPLC-NMRMS methods. The compound was dosed to Sprague-Dawley rats (50 mg kg−, i.p.) and urine collected over 0-8, 8-24 and 24-48 h post-dosing. 2. A total urinary recovery of 53.5 ± 7.0% of the dose was achieved up to 48 h after dosing. The major metabolite in the urine was identified as 2-amino-3-bromo-5-trifluoromethylphenylsulphate accounting for a total of 35.7 ± 6.2% of the dose. 3. Further metabolites detected were 2-bromo-4-trifluoromethylphenylhydroxylamine-N-glucuronide (9.7 ± 0.2% of the dose), 2-bromo-4-trifluoromethylaniline-N-glucuronide (3.0 ± 0.3%) and 2-amino-3-bromo-5-trifluoromethylphenylglucuronide (2.8 ± 0.4). Minor metabolites, including 2-bromo-4-trifluoromethylphenylhydroxylamine-O-glucuronide, 2-amino-3-bromo-5-trifluoromethylphenol and 2-bromo-4- trifluoromethylphenylsulphamate, in total accounted for 2.3 ± 0.9% of the dose. 4. Directly coupled HPLC-NMR-MS and 19F-NMR spectroscopy proved to be efficient techniques for the unequivocal and rapid determination of the urinary metabolic fate and excretion balance of fluorinated xenobiotics without the need for radiolabelling.

NMR spectroscopic studies on the metabolism and futile deacetylation of phenacetin in the rat

1. 1H-NMR spectroscopy of urine was used to determine the % deacetylation and re-acetylation of 2H-labelled (in the acetyl) phenacetin metabolites in the rat. 2. Male Sprague- Dawley rats were each dosed with either phenacetin or phenacetin C2H8 at 50 mg kg−1. The total urinary recoveries for phenacetin and phenacetin-C2H8 were 47.6 ± 16.7 and 50.1 ± 16.2% respectively (not significantly different, p > 0.05). Paracetamol sulphate and glucuronide are the major urinary metabolites of both protio and deuteriophenace tin. 3. The futile deacetylationgiven by the urinary recovery of protio-acetyl metabolitesof phenacetin-C2H8 was 29.6 ± 0.9% for paracetamol sulphate and 36.6 ± 3.1% for paracetamol glucuronide. These observations demonstrate a high level of futile deacetylation in the paracetamol conjugates formed by metabolism of phenacetin-C2H8 and this may indicate a high metabolic flux through the nephrotoxic intermediate 4-aminophenol. 4. The level of futile deacetylation for phenacetin was significantly higher than that found previously in studies of labelled paracetamol in rat or man, and may be important in understanding the higher nephrotoxicity of phenacetin as compared with paracetamol.