Yubing Zhu

Measurement of fexofenadine concentration in micro-sample human plasma by a rapid and sensitive LC-MS/MS employing protein precipitation: application to a clinical pharmacokinetic study.

A simple, rapid and sensitive liquid chromatography/positive ion electro-spray tandem mass spectrometry method (LC-MS/MS) was developed and validated for the quantification of fexofenadine with 100 microL human plasma employing glipizide as internal standard (IS). Protein precipitation was used in the sample preparation procedure. Chromatographic separation was achieved on a reversed-phase C(18 )column (5 microm, 100 x 2.1 mm) with methanol : buffer (containing 10 mmol/L ammonium acetate and 0.1% formic acid; 70 : 30, v/v) as mobile phase. The total chromatographic runtime was approximately 3.0 min with retention time for fexofenadine and IS at approximately 1.9 and 2.1 min, respectively. Detection of fexofenadine and IS was achieved by LC-MS/MS in positive ion mode using 502.1 --> 466.2 and 446.0 --> 321.1 transitions, respectively. The method was proved to be accurate and precise at linearity range of 1-600 ng/mL with a correlation coefficient (r) of > or =0.9976. The validated method was applied to a pharmacokinetic study in human volunteers following oral administration of 60 or 120 mg fexofenadine formulations, successfully.

A simple and sensitive HPLC–ESI-MS/MS method for the determination of andrographolide in human plasma

A liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for the determination of andrographolide in human plasma was established. Dehydroandrographolide was used as the internal standard (I.S.). The plasma samples were deproteinized with methanol and separated on a Hanbon C(18) column with a mobile phase of methanol-water (70:30, v/v). HPLC-ESI-MS/MS was performed in the selected ion monitoring (SIM) mode using target ions at [M-H(2)O-H](-), m/z 331.1 for andrographolide and [M-H](-), m/z 331.1 for the I.S. Calibration curve was linear over the range of 1.0-150.0ng/mL. The chromatographic separation was achieved in less than 6.5min. The lower limits of quantification (LLOQ) was 1.0ng/mL. The intra and inter-run precisions were less than 6.95 and 7.22%, respectively. The method was successfully applied to determine the plasma concentrations of andrographolide in Chinese volunteers.

Bioequivalence and pharmacokinetic comparison of two mycophenolate mofetil formulations in healthy Chinese male volunteers: An open-label, randomized-sequence, single-dose, two-way crossover study

BACKGROUND Mycophenolate mofetil (MMF) is an ester prodrug of mycophenolic acid (MPA), so clinical studies measure the circulating plasma MPA concentration instead of MMF. MPA is extensively glucuronidated by several uridine diphosphate glycosyltransferases into an inactive 7-O-glucuronide and a pharmacologically active acylglucuronide. Considering the effect of racial differences and genetic factors on the pharmacokinetic (PK) properties of drugs, it is necessary to study them in Chinese populations. OBJECTIVES The aim of this study was to compare the clinical bioequivalence and PK properties of a test (dispersible tablets) and reference (capsules) formulation of MMF 1.0 g in healthy Chinese volunteers. We also established a validated HPLC method for the determination and quantification of MPA in human plasma. The study was required to obtain Chinese regulatory approval for the test formulation. METHODS This open-label, randomized-sequence, single-dose, 2-way crossover study was conducted at the First Hospital of Nanjing Medical University, Nanjing, China. Eligible subjects were healthy male volunteers who were randomly assigned at a 1:1 ratio to receive a single 1.0-g dose of the test or reference formulation, followed by a 1-week washout period and administration of the alternate formulation. The plasma concentration of MPA, which is the active metabolite of MMF, was determined using a validated HPLC method. For analysis of PK properties, blood samples were collected at 0, 10, 20, 30, and 45 minutes, and 1, 1.5, 3, 5, 8, 11, 18, 36, and 48 hour(s). The PK parameters, including C(max), T(max), t((1/2)), AUC(0-48), and AUC(0-infinity), were determined from the plasma concentrations of the 2 formulations by noncompartmental analysis. Tolerability was assessed at baseline (be- fore administration) and at 30 minutes and 1, 5, 18, and 48 hours after administration by monitoring vital signs. Laboratory tests (hematology, blood biochemistry, hepatic function, and urinalysis) were performed for the identification of adverse events (AEs) (eg, leukopenia, thrombocytopenia, anemia). Patient interviews were conducted to assess the occurrence of AEs such as diarrhea, abdominal pain, nausea, vomiting, and secondary infections. The formulations were considered to meet the regulatory requirements of bioequivalence if the log-transformed ratios of C(max) and AUC were within the predetermined equivalence range (80%-125%) as established by the US Food and Drug Administration (FDA). RESULTS Eighteen healthy Chinese male volunteers (mean [range] age, 23.5 [22-30] years; weight, 63.3 [56-68] kg; height, 171 [165-184] cm) were enrolled and completed the study. The main PK parameters of the MMF test and reference formulations were as follows: mean (SD) T(max), 0.68 (0.21) and 0.81 (0.18) hour, respectively; C(max), 25.58 (4.79) and 26.47 (3.67) mg/L; AUC(0-48), 59.19 (9.23) and 58.32 (9.28) mg/L/h; t((1/2)), 15.12 (3.17) and 16.04 (4.22) hours; AUC(0-infinity)), 63.28 (10.23) and 62.41 (10.28) mg/L/h. The mean (SD) relative bioavailability was 101.5% (10.3%). No statistically significant differences were found based on ANOVA. The ratios of C(max) (0.97) and AUC (1.01) for the test and reference formulations were within the FDA bioequivalence definition intervals of 80% to 125%. No AEs were reported by subjects or found on analysis of vital signs or laboratory tests. CONCLUSIONS In this study in healthy Chinese male volunteers, results from the PK analysis suggested that a single dose of the test and reference formulations of MMF 1.0 g met the regulatory requirements of bioequivalence, based on the FDA regulatory definition (rate and extent of absorption). Both formulations were well tolerated.

Pharmacokinetics of betamethasone and betamethasone 17-monopropionate in Chinese healthy volunteers after intramuscular injection of betamethasone phosphate/betamethasone dipropionate.

The aim of this study was to evaluate the pharmacokinetic profiles of betamethasone (BOH, CAS 378-44-9) and betamethasone 17-monopropionate (B17P), the active metabolites of betamethasone phosphate (BSP) and betamethasone dipropionate (BDP), respectively, after administration of betamethasone i.m. (BSP 2 mg and BDP 5 mg). After ten healthy volunteers had received a single-dose intramuscular adminitration of betamethasone i.m., blood samples were collected pre-dose and for 336 h postdose. The plasma levels of B17P and BOH were measured by liquid chromatography-tandem mass spectrometry (LC-MS/ MS). When compared to BOH, B17P exhibited a longer time to maximum concentration (15.0 +/- 9.0 h vs. 2.8 +/- 1.7 h), a lower Cmax (0.6 +/- 0.2 ng/mL vs. 14.5 +/- 3.7 ng/mL), and a much longer half-life (80.8 +/- 22.7 h vs. 9.6 +/- 3.6 h). Betamethasone i.m. produced rapid onset and sustained action through an initial rapid-increased plasma concentration of BOH and a sustained plasma concentration of B17P, respectively.

Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Trimetazidine in Human Plasma

A sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of trimetazidine (CAS 13171-25-0) in human plasma, using pseudoephedrine as internal standard (IS). Plasma samples were simply pretreated with methanol for deproteinization. The chromatographic separation was performed on a C18 column with a mobile phase of 3 mmol/L ammonium acetate solution-methanol (15:85, v/v) at a flow rate of 0.3 mL/min. The chromatographic separation was achieved in less than 3.2 min. The linearity was established over the concentration range of 1-100 ng/mL. Both intra- and inter-batch standard deviations were less than 9.5%. The method was successfully applied to study the relative bioavailability of trimetazidine hydrochloride tablets in healthy Chinese volunteers and the pharmacokinetic parameters of the reference and test tablets were compared.

Bioequivalence of two formulations of glucosamine sulfate 500-mg capsules in healthy male chinese volunteers: An open-label, randomized-sequence, single-dose, fasting, two-way crossover study

BACKGROUND Glucosamine sulfate is used for the treatment of arthrosis, especially osteoarthritis of the knee joint. The available evidence suggests differences in its pharmacokinetics in Chinese subjects compared with non-Chinese subjects. OBJECTIVE The aim of this study was to compare the pharmacokinetics and relative bioavailability of a test and reference formulation of glucosamine sulfate 500 mg after single oral administration in healthy Chinese volunteers. METHODS This open-label, randomized-sequence, single-dose, 2-way crossover study was performed at the First Hospital of Nanjing Medical University, Nanjing, China. Eligible subjects were healthy male volunteers who were randomly assigned at a 1:1 ratio to receive a single 500-mg dose of the test or reference capsule formulation, followed by a 1-week washout period and administration of the alternate formulation. The study drugs were administered after a 12-hour overnight fast. Glucosamine sulfate was assayed using a liquid-chromatography tandem mass spectrometry method. For analysis of pharmacokinetic properties, including C(max), AUC(0-t), and AUC(0-infinity)), blood samples were obtained at intervals over a 14-hour period after study drug administration. The formulations were considered bioequivalent if the log-transformed ratios of C(max) and AUC were within the predetermined equivalence range (70%-143% for C(max) and 80%-125% for AUC) as established by the State Food and Drug Administration (SFDA) of China. Tolerability was assessed by monitoring vital signs and laboratory tests (hematology, blood biochemistry, hepatic function, and urinalysis), and by questioning subjects about adverse events (AEs). RESULTS Twenty-two healthy male Chinese subjects were enrolled (mean [range] age, 24 [22-26] years; weight, 63.9 [58.5-69.3] kg; height, 172 [167-177] cm); all completed the study. No period or sequence effect was observed. The 90% CIs for the log-transformed ratios of C(max), AUC(0-t), and AUC(0-infinity)) were 93.4 to 127.3, 92.4 to 114.5, and 92.7 to 114.6, respectively (all, P = NS). The AUC(0-infinity) of the test and reference formulations was 1.83 (0.66) and 1.77 ( 0.72) microg/h/mL, respectively. No AEs were observed or reported during the study. CONCLUSIONS In this small study in healthy male Chinese volunteers, a single 500-mg dose of the test formulation met the SFDAs regulatory definition for bioequivalence to the reference formulation. Both formulations were well tolerated.

Pharmacokinetics and bioavailability study of two ondansetron oral soluble film formulations in fasting healthy male Chinese volunteers.

Background Ondansetron oral soluble film is designed to be applied on top of the tongue without requiring water to aid dissolution or swallowing, which is especially fitting for nausea and vomiting patients. Purpose This study was conducted to compare the bioavailability of two 8 mg ondansetron oral soluble film formulations. Patients and methods This randomized, open-label, two-period crossover study was performed under fasting conditions. A total of ten eligible subjects were randomly assigned at a 1:1 ratio to receive a single 8 mg dose of the test and reference ondansetron oral soluble film formulations, followed by a 1-week washout period and administration of the alternate formulation. The concentrations of ondansetron were assayed using an liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS) method. For analysis of pharmacokinetic properties, including the peak concentration of Tmax (Cmax), AUC from time 0 (baseline) to t hours (AUC0–t), and AUC from baseline to infinity (AUC0–∞), blood samples were obtained at intervals over the 24-hour period after studying drug administration. Tolerability was assessed by monitoring vital signs and laboratory tests (hematology, blood biochemistry, hepatic function, and urinalysis) and by questioning subjects about adverse events. Results The mean (standard derivation [SD]) relative bioavailability was 96.5 (23.7%). The 90% confidence intervals (CIs) for the log-transformed ratios of Cmax and AUC0–t were 84.71%–103.28% and 91.38%–108.60%, respectively (P>0.05). Similar results were found for the data without log-transformation. No statistically significant differences were found based on analysis of variance. No significant adverse events occurred or were reported during the study. Conclusion As the 90% CIs based on the differences between the test and reference formulation were within the 80%–125% range for both the Cmax and AUC0–t, we concluded that the two formulations were bioequivalent with respect to the rate or the extent of absorption. Both formulations are well tolerated.

Relative Bioavailability of Two Formulations of Nevirapine 200-mg Tablets in Healthy Chinese Male Volunteers: A Single-Dose, Randomized-Sequence, Open-Label, Two-Way Crossover Study

BACKGROUND Nevirapine was the first member of the nonnucleoside reverse transcriptase inhibitor class to be approved for the treatment of HIV infection. It binds directly to the allosteric site on the reverse transcriptase and inhibits the activity of both RNA- and DNA-dependent DNA polymerases. OBJECTIVE This study compared the pharmacokinetics and relative bioavailability of a test and reference formulation of nevirapine 200-mg tablets after single oral doses in healthy Chinese men to meet regulatory criteria for marketing of the new generic formulation. METHODS This single-dose, randomized-sequence, open-label, 2-way crossover study was conducted at the Nanjing First Hospital of Nanjing Medical University, Nanjing, China. Healthy male Chinese volunteers were randomized in a 1∶1 ratio to receive a single 200-mg (3.2-mg/kg) tablet of the test or reference formulation, followed by a 2-week washout period and administration of the alternate formulation. The study drugs were administered after a 10-hour overnight fast. Concentrations of nevirapine were assayed using an HPLC-UV method. For analysis of nevirapine pharmacokinetic parameters, blood samples were obtained before dosing and at regularly scheduled intervals over 168 hours after administration. The 2 formulations would be assumed to be bioequivalent for regulatory purposes if the 90% CIs for the log-transformed ratios of nevirapine AUC and C(max) were within the range established by the US Food and Drug Administration (0.80-1.25). Tolerability was evaluated throughout the study based on vital signs, physical examinations, 12-lead ECGs, and subject interviews concerning adverse events (AEs). RESULTS Twenty Chinese male subjects were enrolled in and completed the study. Their mean age was 23 years (range, 21-25 years), mean weight was 63 kg (range, 56-70 kg), and mean height was 171 cm (range, 166-176 cm). No period or sequence effect was observed. The mean (SD) t(½) was 38.12 (2.23) hours for the test tablet and 36.79 (5.06) hours for the reference tablet; T(max) was 3.1 (0.7) and 3.0 (0.7) hours, respectively; C(max) was 2.52 (0.31) and 2.60 (0.48) mg · L(-1); AUC(0-168) was 155.66 (22.41) and 150.66 (22.11) mg · h · L(-1); and AUC(0-∞) was 163.30 (22.88) and 157.75 (22.87) mg · h · L(-1). Mean relative bioavailability was 103.6% (8.6%). The 90% CIs for the log-transformed ratios of C(max) (93.51-102.13) and AUC(0-168) ( 99.84-106.74) were within the predetermined range for the assumption of bioequivalence. One subject reported mild headache after receiving the test formulation; the relationship of this AE to study drug was considered uncertain. No serious or clinically significant AEs were observed or reported during the study. CONCLUSIONS In this single-dose study in healthy fasted Chinese males, the test tablet met the regulatory criterion for assumption of bioequivalence to the reference tablet. Both formulations were well tolerated in the population studied. SFDA registration no: 2009L04358.

Effects of CYP2C19 Genetic Polymorphisms on the Pharmacokinetic and Pharmacodynamic Properties of Clopidogrel and Its Active Metabolite in Healthy Chinese Subjects

PURPOSE Some studies in the white population have shown that carriers of at least 1 loss-of-function allele in the gene that encodes the cytochrome P-450 2C19 isozyme (CYP2C19) have lower levels of the clopidogrel active metabolite (CAM) and a reduced antiplatelet effect of clopidogrel. However, data are limited regarding the association between CYP2C19 genetic variants and exposure to CAM and on the pharmacodynamic properties of CAM in the Chinese population. Data from the white population cannot be extrapolated to the Chinese population because of the marked interethnic differences in CYP2C19 variants. This study was aimed to investigate the influence of CYP2C19 genetic polymorphisms on the pharmacokinetic properties of CAM and the antiplatelet effect of clopidogrel in healthy Chinese volunteers, and to provide evidence for the role of a CYP2C19 genotyping test in predicting the antiplatelet effect of clopidogrel in the Chinese population. METHODS Twenty healthy subjects received a single 300-mg dose of clopidogrel and were assigned to 1 of 3 groups according to CYP2C19 genotype: CYP2C19 *1/*1 (normal metabolizers [NM]; n = 8), CYP2C19 *1/*2 or *3 (intermediate metabolizers [IM]; n = 10) and CYP2C19 *2/*2 or *3 and *3/*3 (poor metabolizers [PM]; n = 2). Blood samples were collected at baseline and at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours after administration. The plasma concentrations of clopidogrel and CAM were analyzed by LC-MS/MS, and adenosine diphosphate-induced platelet aggregation was measured by light-transmittance aggregometry. FINDINGS There were no significant differences in Cmax and AUC0-t of clopidogrel prodrug in the NM group compared with the IM and PM groups. The mean CAM Cmax value was significantly higher in the NM group than in IM and PM groups (45.39 [12.57] vs 29.15 [7.92] ng/mL [P = 0.003] and 19.55 [2.19] ng/mL [P = 0.004], respectively). The mean CAM AUC0-t value was significantly higher in the NM group than in the IM and PM groups (61.05 [21.63] vs 37.67 [11.01] ng · h/mL [P = 0.007] and 27.08 [2.72] ng · h/mL [P = 0.016]). The NM group exhibited a significantly higher percentage of inhibition of platelet aggregation than did the IM or PM group (P = 0.001). The correlations between the pharmacokinetic properties (Cmax, AUC0-t) of CAM and the pharmacodynamic data (maximal and inhibition of platelet aggregation) were significant (both, Pearson r > 0.5 and P < 0.01). IMPLICATION In these healthy Chinese subjects, carriers of CYP2C19 loss-of-function allele(s) had significantly reduced exposure of CAM and decreased levels of inhibition of platelet aggregation with clopidogrel; these genotypes therefore might be a determinant for the formation of CAM and its antiplatelet effects. Study identifier: ChiCTR-OCH-14004382.

Relative Fasting Bioavailability of Two Formulations of Nateglinide 60 mg in Healthy Male Chinese Volunteers: An Open-Label, Randomized-Sequence, Single-Dose, Two-Way Crossover Study

BACKGROUND Nateglinide, N-(trans-4-isopropylcyclohexyl-carbonyl)-d-phenylalanine, is a potent insulin secretagogue designed to restore early-phase insulin secretion. It increases pancreatic insulin secretion by competitively binding to sulfonylurea receptors inhibiting adenosine triphosphate-sensitive potassium channels and thus reducing blood glucose levels. The drug has a rapid onset (causing immediate insulin release) and a short duration (allowing insulin to return to baseline levels between meals) of insulinotropic action. OBJECTIVE To meet the requirements for marketing a new generic product, this study was designed to compare the pharmacokinetic parameters and relative fasting bioavailability of new generic (test) formulation of nateglinide with the reference formulation of nateglinide in healthy Chinese male volunteers. METHODS This open-label, single-dose, randomized-sequence, 2-way crossover study was performed at Nanjing First Hospital of Nanjing Medical University. Eligible subjects were healthy male volunteers who were randomly assigned in a 1:1 ratio to receive a single 60-mg (0.88 mg/kg) dose of the 2 formulations, followed by a 1-week washout period and then administration of the alternate formulation. Study drugs were administered after a 10-hour overnight fast. Concentrations of nateglinide were determined by using a validated LC-MS method. For analysis of pharmacokinetic properties, including C(max), AUC(0-10), and AUC(0-∞), blood samples were obtained at intervals over the 10-hour period after study drug administration. As established by the State Food and Drug Administration, the formulations were assumed bioequivalent if 90% CIs for the test/reference ratios of ln-transformed values of C(max) and AUC (obtained by using ANOVA) were within the predetermined equivalence range (80%-125%). Tolerability was assessed by monitoring vital signs and laboratory tests (hematology, blood biochemistry, hepatic function, and urinalysis) and by questioning subjects about adverse events. RESULTS The 90% CIs for nateglinide were as follows: C(max), 98.4% to 118.6%; AUC(0-10), 99.5% to 110.3%. Both C(max) and AUC(0-10) met the predetermined criteria for assuming bioequivalence. The relative bioavailability of the test formulation was estimated to be 102.1% (13.5%). One volunteer (5%) experienced a headache after administration of the test formulation. This resolved spontaneously within 1 hour and was considered by the investigators to be mild. No serious adverse events were reported. No period or sequence effects were observed. CONCLUSIONS In this study of healthy Chinese male volunteers, a single 60-mg dose of nateglinide (test formulation) met the regulatory criteria for assuming bioequivalence to the established reference formulation. Both formulations were well tolerated. Chinese Clinical Trials registration number: ChiCTR-TRC-11001754.