Application of a simple methodology to analyze Hydroxypropyl-ß-Cyclodextrin in urine using HPLC-LS in early Niemann-Pick disease type C patient.

A new methodology, based on high resolution liquid chromatography with light scatterin detector, is applied to analyze Hydroxypropyl-beta-Cyclodextrin (HPßCD) in urine samples of a child affected by Niemann-Pick Type C disease. The treatment not only stopped disease progression, but has also increased the life expectancy and quality of our patient. The pharmacokinetic of HPßCD in the patient was studied with a 92.8% of HPßCD recovered. At 88 h, no HPßCD was found in the urine. During the treatment, HPßCD has not shown toxicity. Before application of the new treatment, injections were given every two weeks but, we have demonstrated that this can be increased to every four days.

Quantitative determination of pravastatin and its metabolite 3α-hydroxy pravastatin in plasma and urine of pregnant patients by LC-MS/MS.

This report describes the development and validation of a chromatography/tandem mass spectrometry method for the quantitative determination of pravastatin and its metabolite (3α-hydroxy pravastatin) in plasma and urine of pregnant patients under treatment with pravastatin, as part of a clinical trial. The method includes a one-step sample preparation by liquid-liquid extraction. The extraction recovery of the analytes ranged between 93.8 and 99.5% in plasma. The lower limits of quantitation of the analytes in plasma samples were 0.106 ng/mL for pravastatin and 0.105 ng/mL for 3α-hydroxy pravastatin, while in urine samples they were 19.7 ng/mL for pravastatin and 2.00 ng/mL for 3α-hydroxy pravastatin. The relative deviation of this method was <10% for intra- and interday assays in plasma and urine samples, and the accuracy ranged between 97.2 and 106% in plasma, and between 98.2 and 105% in urine. The method described in this report was successfully utilized for determining the pharmacokinetics of pravastatin in pregnant patients enrolled in a pilot clinical trial for prevention of preeclampsia.

A dispersive liquid-liquid microextraction method based on the solidification of a floating organic drop combined with HPLC for the determination of lovastatin and simvastatin in rat urine.

A dispersive liquid-liquid microextraction method based on solidification of floating organic drop combined with HPLC was developed for the determination of lovastatin and simvastatin in rat urine for the first time. 1-Dodecanol and methanol were used as the extraction and disperser solvents, respectively. Several important parameters influencing the micro-extraction efficiency were studied and systematically optimized, including the type and volume of extraction solvent and disperser solvent, extraction time, pH and salt concentration. The analytes were separated on a Kromasil C18 column at 30°C with a mobile phase of methanol and 0.2% acetic acid in water (83:17, v/v) and detected at 238 nm. Under the optimal conditions, the maximum number of enrichment factors for both analytes was 27. The linear ranges were 20.08-1004 and 20.00-1000 µg/L with the correlation coefficients ranging from 0.9990 to 0.9994 for lovastatin and simvastatin, respectively. The volume of organic solvent consumed in extraction was <0.3 mL, and the extraction time was 10 min. The newly developed environment-friendly sample pretreatment method will be a good alternative to conventional techniques, such as solid-phase extraction, liquid-liquid extraction and protein precipitation, for the HPLC determination of lovastatin and simvastatin in biological samples.

Hollow-fiber-supported liquid membrane microextraction of amlodipine and atorvastatin.

A simple, environmentally friendly, and efficient method, based on hollow-fiber-supported liquid membrane microextraction, followed by high-performance liquid chromatography has been developed for the extraction and determination of amlodipine (AML) and atorvastatin (ATO) in water and urine samples. The AML in two-phase hollow-fiber liquid microextraction is extracted from 24.0 mL of the aqueous sample into an organic phase with microliter volume located inside the pores and lumen of a polypropylene hollow fiber as acceptor phase, but the ATO in three-phase hollow-fiber liquid microextraction is extracted from aqueous donor phase to organic phase and then back-extracted to the aqueous acceptor phase, which can be directly injected into the high-performance liquid chromatograph for analysis. The preconcentration factors in a range of 34-135 were obtained under the optimum conditions. The calibration curves were linear (R(2) ≥ 0.990) in the concentration range of 2.0-200 µg/L for AML and 5.0-200 µg/L for ATO. The limits of detection for AML and ATO were 0.5 and 2.0 µg/L, respectively. Tap water and human urine samples were successfully analyzed for the existence of AML and ATO using the proposed methods.

Effect of hepatic and renal impairment on the pharmacokinetics of dalcetrapib: altered distribution of the active thiol?

BACKGROUND AND OBJECTIVE: Dalcetrapib, a cholesteryl ester transfer protein (CETP) modulator, is a thioester pro-drug that is rapidly hydrolysed to generate a pharmacologically active thiol. The thiol covalently binds to plasma proteins as mixed disulfides, extensively distributes into plasma lipoprotein fractions, and is principally cleared by metabolism, including extensive first-pass metabolism. Here we report two studies assessing the effects of hepatic and renal impairment on the pharmacokinetics of the thiol and its primary metabolites. METHODS: Adults with hepatic or renal impairment and healthy controls were recruited in two separate non-randomized, open-label studies. Eligible subjects were aged 18-70 years (hepatic impairment study) or 18-75 years (renal impairment study) with a body mass index 18-40 kg/m(2). Healthy controls were matched by age, bodyweight and sex. Each participant received a single 600 mg oral dose of dalcetrapib. Plasma and urine sampling was performed up to 3-4 days post-dalcetrapib administration for analysis of the pharmacokinetics of the thiol and its primary S-methyl and S-glucuronide metabolites. In the renal impairment study, CETP activity and mass, and lipid profiles were also assessed. RESULTS: Twenty-eight subjects were enrolled in the hepatic impairment study (mild or moderate hepatic impairment, n = 8 in each group; controls, n = 12). Thirty-five subjects participated in the renal impairment study (mild, moderate or severe renal impairment, n = 8 in each group; controls, n = 11). In patients with moderate hepatic impairment, the area under the plasma concentration-time curve from time zero to infinity (AUC(∞)) for thiol exposure was increased 34 % (geometric mean ratio [GMR] 1.34, 90 % CI 1.02-1.76), compared with matched controls. Regression analysis revealed a weak inverse relationship between thiol exposure and creatinine clearance (p = 0.0137, r(2) = 17.1 %). In patients with moderate or severe renal impairment, thiol exposures were 62 % (AUC(∞) GMR 1.62, 90 % CI 0.81-3.27) and 81 % (AUC(∞) GMR 1.81, 90 % CI 1.21-2.71) higher, respectively, than matched controls. Exposures of the S-glucuronide and S-methyl metabolites were also higher in hepatic and renal impairment groups. In the renal impairment study, CETP activity was decreased following administration of dalcetrapib, with no clear differences between groups. CONCLUSION: Hepatic and renal impairment both altered dalcetrapib pharmacokinetics and increased thiol exposure, with the extent of the effect dependent on the severity of impairment. The effect of renal impairment may be linked to altered distribution of the thiol, which illustrates the importance of assessing distribution to understand the causes and consequences of altered pharmacokinetics of thiol drugs in patient populations.

Discovery of safety biomarkers for atorvastatin in rat urine using mass spectrometry based metabolomics combined with global and targeted approach.

In order to develop a safety biomarker for atorvastatin, this drug was orally administrated to hyperlipidemic rats, and a metabolomic study was performed. Atorvastatin was given in doses of either 70 mg kg(-1) day(-1) or 250 mg kg(-1) day(-1) for a period of 7 days (n=4 for each group). To evaluate any abnormal effects of the drug, physiological and plasma biochemical parameters were measured and histopathological tests were carried out. Safety biomarkers were derived by comparing these parameters and using both global and targeted metabolic profiling. Global metabolic profiling was performed using liquid chromatography/time of flight/mass spectrometry (LC/TOF/MS) with multivariate data analysis. Several safety biomarker candidates that included various steroids and amino acids were discovered as a result of global metabolic profiling, and they were also confirmed by targeted metabolic profiling using gas chromatography/mass spectrometry (GC/MS) and capillary electrophoresis/mass spectrometry (CE/MS). Serum biochemical and histopathological tests were used to detect abnormal drug reactions in the liver after repeating oral administration of atorvastatin. The metabolic differences between control and the drug-treated groups were compared using PLS-DA score plots. These results were compared with the physiological and plasma biochemical parameters and the results of a histopathological test. Estrone, cortisone, proline, cystine, 3-ureidopropionic acid and histidine were proposed as potential safety biomarkers related with the liver toxicity of atorvastatin. These results indicate that the combined application of global and targeted metabolic profiling could be a useful tool for the discovery of drug safety biomarkers.

Synergistic influence of Abcb1 and Abcc2 on disposition and sterol lowering effects of ezetimibe in rats.

Pharmacokinetics of the sterol-lowering drug ezetimibe (EZ) is influenced by intestinal ABCB1 and ABCC2. This study in Lew.1W rats with "chemical" and genetic Abcb1 and Abcc2 deficiency was initiated to evaluate the individual contribution of both efflux carriers to the overall disposition and sterol-lowering effects of EZ. Disposition and sterol-lowering effects of EZ (5 mg/kg, 14 days) were measured in wild-type (WT) and Abcc2-deficient (Abcc2-) rats (N = 8 per group) and in animals treated with PSC833 (20 mg/kg) to generate "chemical" Abcb1-deficiency (Abcb1-, Abcb1-/Abcc2-). EZ serum levels decreased in the order WT (3.11 +/- 1.09 ng/mL), Abcb1- (1.94 +/- 1.10 ng/mL), Abcc2- (1.42 +/- 0.42 ng/mL, p = 0.003 vs. WT), Abcb1-/Abcc2- (1.17 +/- 0.53 ng/mL, p = 0.002 vs. WT) whereas the serum EZ glucuronide levels increased as follows: WT (23.2 +/- 24.6 ng/mL), Abcb1- (119 +/- 74.5 ng/mL, p = 0.002 vs. WT), Abcc2- (195+/-76.5 ng/mL, p < 0.001 vs. WT), Abcb1-/Abcc2- (676 +/- 207 ng/mL, p < 0.001 vs. WT, Abcb1- and Abcc2-). Abcb1 and Abcc2 protein deficiency resulted synergistically in lower fecal but increased renal excretion of total EZ although to a much lower extent. The sterol-lowering effects of EZ were significantly correlated to serum levels of EZ. In conclusion, Abcb1 and Abcc2 deficiency leads to lower levels of the active EZ and in turn to decreased sterol-lowering effects.

Disposition of the cholesterol absorption inhibitor ezetimibe in mdr1a/b (-/-) mice.

The lipid lowering agent ezetimibe (EZ) and its intestinally formed glucuronide (GLUC) were shown to be substrates of the efflux transporters P-glycoprotein (P-gp) and the multidrug resistance associated protein 2 (MRP2) which markedly influences the disposition and efficacy of EZ in man. This study aims to elucidate the unique meaning of P-gp in the pharmacokinetics of EZ in mice. In brief, serum concentrations, organ distribution and elimination of EZ were determined in 10 male wild-type and mdr1a/b (-/-) mice after oral treatment with EZ (10 mg/kg, 10 days). EZ and GLUC were quantified in serum, urine, feces and various tissues using a validated LC-MS/MS method. Compared to wild-type mice, mdr1a/b knockout was associated with significantly increased serum concentrations of GLUC (5.58 +/- 2.07 versus 2.09 +/- 0.83 ng/ml, p < 0.001) but not of EZ (0.92 +/- 0.73 versus 0.55 +/- 0.40 ng/ml, n.s.). Consequently, urinary excretion of GLUC was about three-fold increased (9.96 +/- 0.27 versus 3.10 +/- 1.37 microg/day, p = 0.049) whereas renal clearance and the amount excreted via feces remained unchanged. Both EZ and GLUC were not over-proportionally distributed into investigated organs. P-glycoprotein primary influences the oral absorption of ezetimibe in mice. Distribution, renal and fecal excretion of the drug seems not to be markedly affected by P-glycoprotein.

Stacking and quantitative analysis of lovastatin in urine samples by the transient moving chemical reaction boundary method in capillary electrophoresis.

A simple, sensitive, and useful concentration method for lovastatin (Lvt) in urine has been developed based on the transient moving chemical reaction boundary method (tMCRBM) in capillary electrophoresis. The MCRB is formed with acidic sample buffer (Gly-HCl) and alkaline running buffer (Gly-NaOH). The following optimal conditions were determined for stacking and separation: electrophoretic buffer of 100 mM Gly- NaOH (pH 11.52), sample buffer of 20 mM Gly-HCl (pH 4.93), fused-silica capillary of 76 cm x 75-microm i.d (67 cm from detector), sample injection at 14 mbar for 3 min. A 21- to 26-fold increase in peak height was achieved for detection of Lvt in urine under the optimal conditions compared with normal capillary zone electrophoresis. By combining the sample pretreatment procedure with the stacking method, the sensitivity of Lvt in urine was increased by 105- to 130-fold. The limits of detection (LOD) and quantification (LOQ) for Lvt in urine were decreased to 8.8 ng/mL and 29.2 ng/mL, respectively. The intra-day and inter-day precision values (expressed as RSD) were 2.23-3.61% and 4.03-5.05%, respectively. The recoveries of the analyte at three concentration levels changed from 82.65 to 100.49%.

A LC-MS/MS method to quantify the novel cholesterol lowering drug ezetimibe in human serum, urine and feces in healthy subjects genotyped for SLCO1B1.

Ezetimibe (Ezetrol) is a novel cholesterol lowering drug which disposition is not fully understood in man. We developed a selective and high-sensitive assay to measure serum concentration-time profiles, renal and fecal elimination of ezetimibe in pharmacokinetic studies. Ezetimibe glucuronide, the major metabolite of ezetimibe was determined by enzymatic degradation to the parent compound. Ezetimibe was measured after extraction with methyl tert-butyl ether using 4-hydroxychalcone as internal standard and liquid chromatography coupled via an APCI interface with tandem mass spectrometry (LC-MS/MS) for detection. The chromatography (column XTerra) MS, C(18), 2.1 mm x 100 mm, particle size 3.5 microm) was done isocratically with acetonitrile/water (60/40, v/v; flow rate 200 microl/min). The MS/MS analysis was performed in the negative ion mode (m/z transition: ezetimibe 408-271, internal standard 223-117). The validation ranges for ezetimibe and total ezetimibe were as follows: serum 0.0001-0.015 microg/ml and 0.001-0.2 microg/ml; urine and fecal homogenate 0.025-10 microg/ml and 0.1-20 mg/ml, respectively. The assay was successfully applied to measure ezetimibe disposition in two subjects genotyped for the hepatic uptake transporter SLCO1B1.

Effects of habitual chitosan intake on bone mass, bone-related metabolic markers and duodenum CaBP D9K mRNA in ovariectomized SHRSP rats.

We have demonstrated that the habitual intake of chitosan can decrease bone mass in ovariectomized (OVX) SHRSP rats fed a low-Ca diet (0.1%). In the present study, we examined both the etiology of bone loss induced by dietary chitosan and the preventive effect of vitamin C supplementation. Rats were OVX and maintained on one of the following diets for 6 wk: 10% cellulose (CE). 10% chitosan (CH) or 10% chitosan with sodium ascorbate (CHVC). CH caused a significant reduction in bone mineral density (BMD) and stiffness in femurs and the fourth lumbar vertebrae (L4). There was no significant difference in intestinal Ca absorption between CH and CE, whereas CH intake significantly reduced intestinal P absorption. The bone loss in CH rats was accompanied with an increase in urinary Ca excretion and a decrease in serum Ca as well as a significant increment In serum PTH and 1,25(OH)2D3. The vitamin D receptor and calcium binding protein D9K mRNAs were also significantly increased in the duodenum of CH rats. Vitamin C supplementation to CH caused an increase in the Ca and P contents of femurs as well as BMD of the L4, with a decrease in urinary Ca excretion. These results indicate that dietary chitosan with low Ca intake possibly induces the loss of bone mass by enhancing urinary Ca excretion rather than by inhibiting Ca absorption, and that vitamin C supplementation could prevent bone loss caused by chitosan through the increment of retained Ca followed by suppression of urinary Ca excretion.

Assessing the plasma pharmacokinetics, tissue distribution, excretion and effects on cholesterol pharmacokinetics of a novel hydrophilic compound, FM-VP4, following administration to rats.

PURPOSE: The purpose of this project was to 1) assess the disposition kinetics of [3H]-cholesterol following co-administration with a novel hydrophilic compound, FM-VP4, and 2) determine the pharmacokinetics, tissue distribution and excretion of [3H]FM-VP4 following single oral (150 mg/kg which includes 100 mCi of radiolabel) and intravenous (15 mg/kg which includes 10 mCi of radiolabel) doses. METHODS: Following an overnight fast (12-16 h) and 48 h post-surgery, adult male Sprague Dawley rats were divided into six treatment groups (n=4/group). Groups received single oral doses of 25 mCi/ml [3H]cholesterol alone or with 5, 10, 20, 50 and 100 mg/kg FM-VP4 at 0700 h. Ten percent Intralipid was used to solubilize and co-administer [3H]-cholesterol and FM-VP4. LC-MS analysis confirmed minimal cholesterol and vegetable stanol content within 10% Intralipid. Thin layer chromatography was used to confirm that the majority of radioactivity measured in plasma was associated with either esterified or unesterified cholesterol. In a second study pharmacokinetics of [3H]FM-VP4 were studied following intravenous or orally gavaged doses (n=8). Tissues, urine and feces were also collected in FM-VP4 kinetics study to measure tissue distribution of radioactivity. Plasma [3H]-cholesterol and [3H]FM-VP4 were tested for radioactivity. RESULTS: FM-VP4 co-administration significantly decreased [3H]-cholesterol AUC0-48h and Cmax, and increased CL/F and Vd/F of [3H]-cholesterol as compared to controls in a dose-dependent manner. Following oral administration of [3H]FM-VP4, the majority of radioactivity following was recovered in the feces and gastrointestinal (GI) tract. The compound exhibited an oral bioavailability of 6.5%. Following IV administration, a two-compartment pharmacokinetic model was observed and the majority of the radioactivity was recovered in the GI tract. CONCLUSIONS: FM-VP4 reduces plasma concentration of [3H]-cholesterol in fasting rats. [3H]FM-VP4 has a very low oral bioavailability.

[High-performance liquid chromatographic method for the determination of fenofibric acid and reduced fenofibric acid in human blood, plasma and urine].

In this study, a very reliable HPLC method was developed for the determination of fenofibric acid and reduced fenofibric acid in the biological samples described as follows. After addition of the internal standard solution and 0.5 M HCl to the biological sample, fenofibric acid, reduced fenofibric acid and the internal standard were extracted with a mixed solvent of n-hexane and ethyl acetate (90:10) from the mixture. The acids were back-extracted from the organic phase with 0.1 M Na2HPO4 and then re-extracted from the aqueous phase with a mixed solution of n-hexane and ethyl acetate (95:5) after addition of 0.5 M HCl. The organic phase was evaporated to dryness under the vacuum. The residue was dissolved in MeOH and diluted with distilled water. An aliquot of the resulting solution was injected on the HPLC. High reproducibility was observed in this HPLC method (C.V.% less than 4%). Moreover it was confirmed that the conjugates in the urine could be hydrolyzed by incubation at 37 degrees C for 18 h after addition of 400 IU of beta-glucuronidase.

Disposition of ingested olestra in weanling mini-pigs.

The disposition of ingested olestra in Hanford mini-pigs was examined by following a single oral gavage dose of radiolabelled (U-14C-sucrose) olestra Eight dosed animal (four/sex) and one undosed animal were killed 1, 3 and 7 days after dosing, and tissues were collected and counted. Urine and faeces were collected continuously and counted. Tissue lipids were extracted and analysed for intact radiolabelled olestra by size exclusion chromatography. Sucrose will be excreted in urine if olestra is absorbed and metabolized. Mean recovery of radiolabel was 96.6% of the administered dose. Of the recovered radiolabel, more than 99.4%, on average, was not absorbed and found in faeces, or cage and animal wash solutions. The absorbed radiolabel (0.6%), was distributed across the carcass, all tissues and blood, or excreted in urine. This radiolabel primarily came from the metabolism of glucose and fructose resulting from the hydrolysis of the trace levels of penta- and lower sucrose esters present in the test material. No radiolabel was found in the olestra-containing fraction of liver lipids, the primary measure of absorbed and non-metabolized olestra, at a detection limit of 0.0002% of dose. A conservative estimate of the amount of 14C-sucrose excreted in the urine was 0.0012%. The total absorption of intact olestra was thus less than 0.0014% of the dose, the sum of the two measures. These results indicate that intact olestra is essentially not absorbed by the weanling mini-pig, an animal with a young developing gastrointestinal tract similar to that of young children (2-5 yr).