Early Detection of Growth Hormone Secretagogue Receptor Antagonists Exploiting Their Atypical Behavior in Competitive Assays.

We report the proof-of-concept of a bioaffinity format designed for the early detection of growth hormone secretagogue receptor (GHS-R1a) antagonists in urine samples. We exploit here their atypical behavior in competitive experiments with labeled ghrelin (GHR), namely, the strong promoting effect on the GHR/GHS-R1a interaction at low molar ratios GHR/antagonist. The antagonists potentiate the GHR/GHS-R1a interaction, and they display the same effect on the interaction of GHS-R1a with other agonists listed as doping agents. The developed assay allows the estimation of affinity constants of ligand/receptor and antagonist/receptor binding and is amenable to optical, electrochemical, and mass-sensitive detection. The estimated affinity constants for GHR/GHS-R1a and antagonist/GHS-R1a in the absence of G proteins are in good agreement with recently reported data.

Effect of the angiotensin receptor-neprilysin inhibitor sacubitril/valsartan on the pharmacokinetics and pharmacodynamics of a single dose of furosemide.

AIMS: Sacubitril/valsartan is indicated for the treatment of heart failure and reduced ejection fraction (HFrEF). Furosemide, a loop diuretic commonly used for the treatment of HFrEF, may be coadministered with sacubitril/valsartan in clinical practice. The effect of sacubitril/valsartan on the pharmacokinetics and pharmacodynamics of furosemide was evaluated in this open label, two-period, single-sequence study in healthy subjects. METHODS: All subjects (n = 28) received 40 mg oral single-dose furosemide during period 1, followed by a washout of 2 days. In period 2, sacubitril/valsartan 200 mg (97/103 mg) was administered twice daily for 5 days and a single dose of 40 mg furosemide was coadministered on day 6. Serial plasma and urine samples were collected to determine the pharmacokinetics of furosemide and sacubitril/valsartan and the pharmacodynamics of furosemide. The point estimates and the associated 90% confidence intervals for pharmacokinetic parameters were evaluated. RESULTS: Coadministration of furosemide with sacubitril/valsartan decreased the maximum observed plasma concentration (Cmax ) [estimated geometric mean ratio (90% confidence interval): 0.50 (0.44, 0.56)], area under the plasma concentration-time curve (AUC) from time 0 to infinity [0.72 (0.67, 0.77)] and 24-h urinary excretion of furosemide [0.74 (0.69, 0.79)]. When coadministered with sacubitril/valsartan, 0-4-h, 4-8-h and 0-24-h diuresis in response to furosemide was reduced by ~7%, 21% and 0.2%, respectively, while natriuresis was reduced by ~ 28.5%, 7% and 15%, respectively. Post hoc analysis of the pivotal phase III Prospective comparison of ARNI with ACEI to Determine Impact on Global Mortality and morbidity in Heart Failure trial (PARADIGM-HF) indicated that the median furosemide dose was similar at baseline and at the end of the study in the sacubitril/valsartan group. CONCLUSIONS: Sacubitril/valsartan reduced plasma Cmax and AUC and 24-h urinary excretion of furosemide, while not significantly affecting its pharmacodynamic effects in healthy subjects.

Vitamin D-Binding Protein Is a Potential Urinary Biomarker of Irbesartan Treatment Response in Patients with IgA Nephropathy.

AIMS: The purpose of this study was to identify predictive markers of irbesartan (an angiotensin receptor blocker) treatment response in immunoglobulin A nephropathy (IgAN) patients. METHODS: Urine samples were collected both before and after irbesartan treatment in IgAN patients and compared with urine from healthy volunteers. The total urinary protein produced in 24 h was measured to determine therapeutic response. The urinary proteome was evaluated by two-dimensional gel electrophoresis coupled with MALDI-TOF-MS/MS analysis. Western blotting was used to verify protein expression. A receiver operating characteristic curve was used to evaluate the sensitivity and specificity of candidate biomarkers. RESULTS: Four differentially expressed proteins were identified as vitamin D-binding proteins (VDPs). Western blot showed that urinary VDPs were significantly elevated in nonresponsive versus responsive IgAN patients. The sensitivity, specificity, and accuracy of urinary VDP as a predictive biomarker of irbesartan nonresponsiveness in IgAN were 65%, 85%, and 75%, respectively. CONCLUSION: Our results revealed that urinary VDP might be a useful biomarker for predicting irbesartan treatment response.

Lower CYP2C9 activity in Turkish patients with Behçet's disease compared to healthy subjects: a down-regulation due to inflammation?

BACKGROUND: We previously reported on a Swedish patient with Behçet's disease (BD) who was an ultra-rapid metaboliser of drugs catalysed by CYP2C9. Was this extreme metabolism caused by the disease? AIM: This study aims to compare the genotype/phenotype of CYP2C9 in patients with BD and healthy subjects. As the occurrence of BD is high in Turkey, all subjects were recruited from this country. METHODS: Genotyping of CYP2C9 was performed using standard PCR-RFLP and allele-specific PCR methods. Phenotyping of CYP2C9 was performed by administration of a 50-mg single oral dose of losartan and by calculating the urinary metabolic ratio (MR) of probe drug to its metabolite E-3174. Quantitation was performed by HPLC. RESULTS: The frequency of CYP2C9*2 and *3 was not significantly different between the Behçet's disease patients (12.5 and 8.7%) and the healthy subjects (8.9 and 8.2%). The geometric mean losartan MR was higher in the 52 patients (1.75) than in the 96 healthy subjects (1.02) (p = 0.002; t-test). Within the genotypes *1/*1, there was a significant difference of MR between patients and healthy subjects (P = 0.006). All but three of the Behçet's disease patients were treated with colchicine. In nine subsequent patients, we found no significant effect of 2 weeks of treatment with colchicine on the CYP2C9 MR. CONCLUSION: Contrary to expectation, the CYP2C9 activity was lower in Turkish BD patients compared to healthy subjects. As this seems not to be due to colchicine treatment, our hypothesis is that inflammation related to BD might have caused the down-regulation of the CYP2C9 activity due to immune cytokine reactions. The ultra-rapid metabolism of CYP2C9 substrate drugs in the Swedish patient was not due to her BD.

Matrix Effect Compensation in Small-Molecule Profiling for an LC-TOF Platform Using Multicomponent Postcolumn Infusion.

The possible presence of matrix effect is one of the main concerns in liquid chromatography-mass spectrometry (LC-MS)-driven bioanalysis due to its impact on the reliability of the obtained quantitative results. Here we propose an approach to correct for the matrix effect in LC-MS with electrospray ionization using postcolumn infusion of eight internal standards (PCI-IS). We applied this approach to a generic ultraperformance liquid chromatography-time-of-flight (UHPLC-TOF) platform developed for small-molecule profiling with a main focus on drugs. Different urine samples were spiked with 19 drugs with different physicochemical properties and analyzed in order to study matrix effect (in absolute and relative terms). Furthermore, calibration curves for each analyte were constructed and quality control samples at different concentration levels were analyzed to check the applicability of this approach in quantitative analysis. The matrix effect profiles of the PCI-ISs were different: this confirms that the matrix effect is compound-dependent, and therefore the most suitable PCI-IS has to be chosen for each analyte. Chromatograms were reconstructed using analyte and PCI-IS responses, which were used to develop an optimized method which compensates for variation in ionization efficiency. The approach presented here improved the results in terms of matrix effect dramatically. Furthermore, calibration curves of higher quality are obtained, dynamic range is enhanced, and accuracy and precision of QC samples is increased. The use of PCI-ISs is a very promising step toward an analytical platform free of matrix effect, which can make LC-MS analysis even more successful, adding a higher reliability in quantification to its intrinsic high sensitivity and selectivity.

A novel dilute and shoot HPLC assay method for quantification of irbesartan and hydrochlorothiazide in combination tablets and urine using second generation C18-bonded monolithic silica column with double gradient elution.

Irbesartan (IRB) and hydrochlorothiazide (HCT) are angiotensin-II receptor antagonist and thiazide-class diuretic compounds, respectively, which are in use in the treatment of hypertension. A novel dilute-and-shoot HPLC assay method for simultaneous quantification of IRB and HCT in fixed-dose combination tablets and urine samples was described. The separation of IRB, HCT and agomelatine (internal standard) was carried out using a second generation C18-bonded monolithic silica column (Chromolith(®) High Resolution RP-18e, 100×4.6mm, Merck KGaA), utilizing both mobile phase and flow rate gradient elution programs. The analytes were detected at 230 nm wavelength using photodiode array detector within 24 minutes with high resolution, observing about 50 percent more peak capacity when using second generation C18-bonded monolithic silica column. Urine samples were introduced into the system effortlessly, with only filtration and subsequent dilution. Validation studies were performed according to the official recommendations of USP and ICH, and the developed method was successfully applied to pharmaceutical tablets and urine samples.

Simultaneous determination of olmesartan and amlodipine in human plasma and urine by ultra performance liquid chromatography tandem mass spectrometry.

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine olmesartan and amlodipine levels in human plasma and urine simultaneously. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column and mass spectrometric analysis was performed using a QTrap5500 mass spectrometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 447→207 and 409→238 were used to quantify olmesartan and amlodipine, respectively. This assay method has been fully validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect and recovery. The linearity of this method was found to be within the concentration range of 0.2-500ng/mL and 4-5000ng/mL for olmesartan in human plasma and urine and 0.1-50ng/mL and 2-1000ng/mL for amlodipine in human plasma and urine. Only 2min were needed for an analytical run. This assay was used to support a clinical study where multiple oral doses were administered to healthy Chinese subjects to investigate the pharmacokinetics of olmesartan and amlodipine.

Ionic liquids dispersive liquid-liquid microextraction and high-performance liquid chromatographic determination of irbesartan and valsartan in human urine.

An environmentally friendly ionic liquids dispersive liquid-liquid microextraction (IL-DLLME) method coupled with high-performance liquid chromatography (HPLC) for the determination of antihypertensive drugs irbesartan and valsartan in human urine samples was developed. The HPLC separations were accomplished in less than 10 min using a reversed-phase C(18) column (250 × 4.60 mm i.d., 5 µm) with a mobile phase containing 0.3 % formic acid solution and methanol (v/v, 3:7; flow rate, 1.0 mL/min). UV absorption responses at 236 nm were linear over a wide concentration range from 50 µg/mL to the detection limits of 3.3 µg/L for valsartan and 1.5 µg/L for irbesartan. The effective parameters on IL-DLLME, such as ionic liquid types and their amounts, disperser solvent types and their volume, pH of the sample and extraction time were studied and optimized. The developed IL-DLLME-HPLC was successfully applied for evaluation of the urine irbesartan and valsartan profile following oral capsules administration.

[Relationship between warfarin dosing and activity of CYP2C9 assessed by the content of losartan and its metabolite E-3174 in the urine of patients with mechanical prosthetic heart valves].

We evaluated effect of activity of cytochrome P450 CYP2C9 on maintenance doses of warfarin in 33 patients with implanted artificial heart valves. Losartan test was used for measurement of concentration of active metabolite E-3174 in urine. Concentration of E-3174 below 2500 ng/ml in patients with genotype CYP2C981/*1 with sensitivity 87% and specificity 66% predicted requirement of low doses (< 5 mg/day) of warfarin in the late postoperative period (odds ratio 14, 95% confidence interval 1.135 to 172.75).

Development of a HPLC method for the determination of losartan urinary metabolic ratio to be used for the determination of CYP2C9 hydroxylation phenotypes.

BACKGROUND: Losartan is metabolized to losartan carboxylic acid (E-3174) by the polymorphic cytochrome CYP2C9. The aim of the study was to develop a high-performance liquid chromatographic (HPLC) method with fluorescence detection for simultaneously measuring losartan and its metabolite E-3174 in urine to evaluate the losartan urinary metabolic ratio (MR: losartan/E-3174) for CYP2C9 phenotyping in humans. METHODS: The compounds were separated in a reversed-phase chromatographic column and detected by fluorescence at a wavelength of 250 nm for excitation and of 370 nm for emission. RESULTS: No analytical interferences with endogenous compounds were found, and the extraction recoveries were over 88%. Limits of quantification of 2 ng mL-1 for losartan and 5 ng mL-1 for E-3174 were achieved, as well as good reproducibility with coefficients of variation of <9% in all cases. Analyses with the present HPLC method show significant differences (p<0.05) in losartan MRs between the four CYP2C9 genotype groups in 13 Spanish healthy volunteers. CONCLUSIONS: The method developed is simple and affordable, as well as sensitive and reliable to calculate the MR. Therefore, it appears to be useful for CYP2C9 phenotyping using losartan as a drug test in populations, such as Hispanics with different allele combinations.

Effects of CYP2C9*1/*3 and *1/*13 on the pharmacokinetics of losartan and its active metabolite E-3174.

OBJECTIVE: The effects of CYP2C9*1/*3 and *1/*13 genotypes were evaluated on the pharmacokinetics of losartan and its active metabolite, E-3174, in Korean subjects. METHODS: Losartan (50 mg) was administered in 43 Korean volunteers with different CYP2C9 genotypes (CYP2C9*1/*1, *1/*3 and *1/*13). Losartan and E-3174 levels in the plasma and urine were analyzed by HPLC using fluorescence. RESULTS: The CYP2C9*1/*13 subjects showed lower oral clearance (p < 0.001) and greater AUC0-∞ (p < 0.01) of losartan and higher Cmax (p < 0.01) and longer half-life (p < 0.001) of E-3174 than the CYP2C9*1/*1 subjects, but AUC0-∞ of E-3174 was not different. The CYP2C9*1/*3 subjects showed lower oral clearance (p < 0.001) of losartan and higher Cmax (p < 0.01) and longer half-life (p < 0.01) of E-3174 than the CYP2C9*1/*1 subjects. However, AUC0-∞ of losartan was greater in CYP2C9*1/*3 subjects than in CYP2C9*1/*1, but these results were not significant (p < 0.05, but statistical power < 0.8). In addition, AUC0-∞ of E-3174 was not different. There were no significant differences in pharmacokinetic parameters between the CYP2C9*1/*13 and CYP2C9*1/*3 subjects. CONCLUSION: These results suggest that CYP2C9*1/*3 and CYP2C9*1/*13 are similarly associated with decreased formation of E-3174 from losartan, but the clinical effects of losartan may not be reduced by CYP2C9*1/*3 and CYP2C9*1/*13.

Assessment of the drug-drug interactions between fimasartan and hydrochlorothiazide in healthy volunteers.

AIM: Fimasartan is a selective angiotensin II receptor blocker. Hydrochlorothiazide (HCTZ), which is used to treat hypertension and edematous conditions, is coadministered with many antihypertensive agents. METHODS: An open-label, randomized, multiple-dosing, 2-arm, 1-sequence, 2-period study was conducted to assess the effects of fimasartan (240 mg) on HCTZ (25 mg) or vice versa in 18 and 14 healthy male volunteers, respectively. During each drug administration period, drugs were given once daily for 7 days, with a 7-day washout period between the 2 administration periods. RESULTS: The respective geometric mean ratios of fimasartan for AUC τ,ss and C max,ss with HCTZ were 1.30 [90% confidence interval (CI), 0.84-2.01] and 1.17 (90% CI, 0.93-1.47) compared with fimasartan alone. The respective geometric mean ratios of HCTZ for AUC τ,ss and C max,ss with fimasartan were 0.94 (90% CI, 0.84-1.04) and 0.88 (90% CI, 0.80-0.97) compared with HCTZ alone. Plasma renin activity indicated no significant differences between fimasartan monotherapy and coadministered treatment. CONCLUSIONS: Fimasartan administered alone or in combination with HCTZ was well tolerated at the described dosages. Coadministration of fimasartan increased the urinary excretion of HCTZ and urine volume, but these observations are unlikely to have any clinical relevance.

Safety, tolerability, pharmacokinetics, and pharmacodynamics of fimasartan following single and repeated oral administration in the fasted and fed states in healthy subjects.

BACKGROUND AND OBJECTIVES: Fimasartan (BR-A-657) is a novel, non-peptide angiotensin II receptor antagonist with a selective type I receptor blockade effect. Two first-in-human studies investigated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of fimasartan. METHODS: Fasted single oral tablet doses of fimasartan 20-480 mg or placebo were administered to 40 healthy male subjects (aged 19-54 years) in a double-blind, randomized, sequential-group design. Subjects receiving fimasartan 240 mg also received the same treatment in the fed state after an interval of 7 days. In another study, oral tablet doses of fimasartan 120 and 360 mg or placebo were given once daily for 7 days to groups of eight fasted healthy male subjects (aged 20-55 years) in a double-blind, randomized, sequential-group design. Safety and tolerability were assessed. The PK and PD of fimasartan were also evaluated and compared for the different doses. RESULTS: Fimasartan was safe and well tolerated, but with an increased incidence of low BP and postural dizziness for the 360 mg dose after repeated administration. Fimasartan produced increases in plasma renin activity, angiotensin I and II, which were not dose dependent. Maximal increases occurred between 6 and 8 hours post-dose, lasting up to 48 hours. Fimasartan was absorbed rapidly after all doses and had a multiphasic distribution. Two peaks in the plasma concentration-time profile were observed in most subjects. Steady state was achieved after three doses, and accumulation was minimal after repeated doses for 7 days (24-30%). The effective half-life ranged from 9.84 to 13.2 hours. The systemic exposure of fimasartan was dose proportional, and no marked food effect was noted after administration of 240 mg in the fed state. Urinary excretion of fimasartan was very low (1.74-2.51%), suggesting non-renal elimination. CONCLUSION: Fimasartan had a good safety profile and was well tolerated after fasted single oral doses of 20-480 mg, a fed single oral dose of 240 mg, and fasted repeated oral doses of 120 and 360 mg in healthy subjects. In addition, the PK and PD of fimasartan in this population were well characterized. Further studies are needed to evaluate the safety, efficacy, and dose-response relationship of fimasartan in patients with hypertension.

Simultaneous quantitative determination of olmesartan and hydrochlorothiazide in human plasma and urine by liquid chromatography coupled to tandem mass spectrometry.

A specific, sensitive and rapid method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was developed for the simultaneous determination of olmesartan (OLM) and hydrochlorothiazide (HCTZ) in human plasma and urine. Solid-phase extraction (SPE) was used to isolate the analytes from biological matrices followed by injection of the extracts onto a C(18) column with isocratic elution. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using negative electrospray ionization (ESI). The method was validated over the concentration range of 1.00-1000 ng/mL and 5.00-5000 ng/mL for OLM in human plasma and urine as well as 0.500-200 ng/mL and 25.0-25,000 ng/mL for HCTZ in human plasma and urine, respectively. Inter- and intra-run precision of OLM and HCTZ were less than 15% and the accuracy was within 85-115% for both plasma and urine. The average extraction recoveries were 96.6% and 92.7% for OLM, and 87.2% and 72.1% for HCTZ in human plasma and urine, respectively. The linearity, recovery, matrix effect and stability were validated for OLM/HCTZ in human plasma and urine.

Determination of losartan, telmisartan, and valsartan by direct injection of human urine into a column-switching liquid chromatographic system with fluorescence detection.

Column-switching high-performance liquid chromatographic (HPLC) method has been developed and validated for quantification of losartan, telmisartan, and valsartan in human urine. Urine samples were diluted on the extraction mobile phase (1:4, v/v) and a volume of 20 microL of this mixture were directly injected onto the HPLC system. The analytes were extracted from the matrix using an on-line solid-phase extraction procedure involving a precolumn packed with 25 microm C(18) alkyl-diol support (ADS), and a solution 2% methanol in 5mM phosphate buffer (pH 3.8) at a flow-rate of 0.8 mL/min for isolation and preconcentration of losartan, telmisartan, and valsartan. The enriched analytes were back-flushed after, onto the analytical column with a mixture of 5mM phosphate buffer (pH 3.8)-acetonitrile-methanol (65:20:15, v/v/v) at a flow-rate of 3.0 mL/min and detected by fluorescence at 259 and 399 nm as excitation and emission wavelength respectively. The separation of losartan, telmisartan, and valsartan was achieved on a Chromolith RP-18e monolithic column. The method provides extraction recoveries from spiked urine samples greater than 93%. Intra-day and inter-day precision were generally acceptable; the intra-day-assay C.V. was <3.5 for all compounds and the inter-day-assay C.V. was < 3.7%. The estimated calibration range was 0.001-2.5 microg/mL(-1) with excellent coefficient of determination (>0.9981). The detection limits for losartan, telmisartan, and valsartan at a signal-to-noise ratio of 5:1 were 0.002, 0.0002 and 0.001 microg/mL(-1) when a sample volume of 20 microL was injected. The proposed method permitted the simultaneous determination of losartan, telmisartan, and valsartan in 8 min, with an adequate precision and sensitivity. However, the overlap of the sample cleanup step with the analysis increases the sampling frequency to 12 samples/h. The developed column-switching method was successfully applied for the determination of these analytes in human urine samples of patients submitted at ARA-IIs therapy.

Direct analysis of valsartan or candesartan in human plasma and urines by on-line solid phase extraction coupled to electrospray tandem mass spectrometry.

AT1 receptor blockers are agents for the treatment of hypertension. Rapid and precise assay methods are needed to evaluate possible sub- or overdosage. A direct on-line solid phase extraction coupled to tandem mass spectrometry was developed and validated to determine valsartan (5-2000ng/mL) or candesartan (1-200ng/mL) in human plasma and urines. Both intra- and inter-assay accuracy and precision were in the range 89.2-111% and 0.9-16.9% RSD. Total run time was 4.5min. This on-line clean-up-MS method was found to be robust for the analysis of a high number of samples with unvarying performance.

In vitro and in vivo pharmacokinetic characteristics of clazosentan, an intravenous endothelin receptor antagonist, in humans.

OBJECTIVE: In this study, the distribution, metabolism and excretion of the endothelin receptor antagonist clazosentan were investigated. SUBJECTS AND METHODS: 4 healthy male subjects received an intravenous 3-h infusion at a rate of 0.2 mg/kg/h of 14C-labeled clazosentan and blood, urine and feces samples were collected for a period of 8 days. Experiments were performed to investigate the plasma protein binding, the binding to red blood cells and the inhibition potential of cytochrome P450 isoenzymes of clazosentan. RESULTS: Clazosentan was mainly excreted unchanged into feces whereas about 15% of the radioactive dose was recovered in urine. No metabolites representing more than 5% of total radioactivity were identified. No relevant inhibition of the human cytochrome P450 isoenzymes, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4, was observed in vitro at clazosentan concentrations largely exceeding those observed in clinical trials. In human blood, clazosentan was highly bound to plasma proteins and did hardly penetrate into red blood cells. CONCLUSION: The primary route of excretion of clazosentan was via the feces, mainly as unchanged drug.

Separation and quantitation of several angiotensin II receptor antagonist drugs in human urine by a SPE-HPLC-DAD method.

In this work, an SPE-HPLC method coupled to photodiode array detection was validated in human urine matrix, in order to monitor four antihypertensive angiotensin II receptor antagonist drugs in patients under cardiovascular treatment. For that purpose, experimental design was used. Quantitation was accomplished by the internal standard method. The obtained LOQs were 95, 113, 125, and 85 ng/mL for eprosartan, telmisartan, irbesartan, and valsartan, respectively. The intraday and interday precision and accuracy at four concentration levels in the working range (LOQ-15 microg/mL) were always lower than 11% RSD and 8% relative error. The urine samples proved to be stable during 4 h at room temperature, after three thaw-freeze cycles, and for 2 months at -20 degrees C. No interferences from other endogenous compounds or co-administered drugs were found. The method has been successfully applied to monitor the renal elimination of eprosartan and valsartan during 24 h.

Quantitative determination of olmesartan in human plasma and urine by liquid chromatography coupled to tandem mass spectrometry.

A specific, sensitive and fast method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was developed for the determination of olmesartan in human plasma and urine. Solid-phase extraction (SPE) was used to isolate the compounds from biological matrix followed by injection of the extracts onto a C(18) column with isocratic elution. The method was validated over the concentration range of 0.2-1000 and 5-10,000 ng/mL for olmesartan in human plasma and urine, respectively. The method was applied to the pharmacokinetic study of olmesartan medoxomil in healthy Chinese male and female subjects.

Inhibitory effect of valproic acid on cytochrome P450 2C9 activity in epilepsy patients.

Drug interactions constitute a major problem in the treatment of epilepsy because drug combinations are so common. Valproic acid is a widely used anticonvulsant drug with a broad therapeutic spectrum. Case reports suggest interaction between valproic acid and other drugs metabolized mainly by cytochrome P450 isoforms. The aim of this study was to evaluate the inhibitory effect of valproic acid on cytochrome P450 2C9 (CYP2C9) activity by using losartan oxidation as a probe in epilepsy patients. Patients were prescribed sodium valproate (mean 200 mg/day for the first week and 400 mg/day in the following period) according to their clinical need. A single oral dose of 25 mg losartan was given to patients before and after the first dose, first week and 4 weeks of valproic acid treatment. Losartan and E3174, the CYP2C9-derived carboxylic acid metabolite of losartan in 8 hr urine were assayed by using high pressure liquid chromatography. Urinary losartan/E3174 ratio did not change significantly on the first day (0.9, 0.3-3.5; median, range), and first week (0.6, 0.2-3.8; median, range), while a significant increase was observed after 4 weeks of valproic acid treatment (1.1, 0.3-5.7; median, range) as compared to that of measured before valproic acid administration (0.6, 0.1-2.1; median, range) (P = 0.039). The degree of inhibition was correlated with the steady-state plasma concentrations of valproic acid (r(2) = 0.70, P = 0.04). The results suggest an inhibitory effect of valproic acid on CYP2C9 enzyme activity in epilepsy patients at steady state. The risk of pharmacokinetic drug-drug interactions should be taken into account during concomitant use of valproic acid and CYP2C9 substrates.