Bioequivalence study of two clopidogrel film-coated tablet formulations in healthy volunteers

The present study was performed to compare the bioavailability of two clopidogrel 75 mg film-coated tablet formulations (test formulation and reference formulation). This study was a randomized, single-blind, two-period, two-sequence cross-over study which included 24 healthy adult male and female subjects under fasting condition. The pharmacokinetic parameters were assessed based on the concentrations of clopidogrel (CAS 120202-66-6) parent compound. In each of the two study periods (separated by a washout of one week) a single dose of test or reference drug was administered. Plasma concentrations of the drug were determined by LC-MS/MS method. The pharmacokinetic parameters assessed in this study were area under the plasma concentration-time curve from time zero to 24 h (AUCt), area under the plasma concentration-time curve from time zero to infinity (AUCinf), the peak plasma concentration of the drug (Cmax), time needed to achieve the peak plasma concentration (t(max)), and the elimination half life (t1/2). The geometric mean ratios (90% CI) of the test drug/reference drug for clopidogrel parent compound were 95.19% (81.63-110.90%) for AUCt, 95.55% (80.50-113.42%) for AUCinf, and 100.18% (80.87-124.09%) for Cmax. The 90% confidence intervals calculated for AUCt and Cmax of clopidogrel parent compound were within the standard bioequivalence range (80-125% for AUC and Cmax). It was concluded that the two clopidogrel film-coated tablets (test and reference drug) were bioequivalent in terms of the rate and extent of absorption.     

A novel enzyme-linked immunosorbent assay method for the detection of human neutrophil antigen-2a antibodies

BACKGROUND: Antibodies to human neutrophil antigen (HNA)-2a are responsible for a number of immune-mediated neutropenia disorders. Although several methods exist for the identification of anti-HNA-2a, all these methods have several limitations. In this study, a solid-phase enzyme-linked immunosorbent assay (ELISA) using recombinant HNA-2a antigen (rHNA-2a) allowing rapid detection of HNA-2a antibodies was developed.
STUDY DESIGN AND METHODS: Soluble rHNA-2 was generated by transfection of insect cells with CD177 vector. Purified rHNA-2a was immobilized on microtiter wells coated with anti-CD177 and was applied to analyze 10 sera containing HNA-2a antibodies. For the evaluation of the ELISA method, results were compared with the standard assay, MAIGA (monoclonal antibody antigen capture assay) for detection of neutrophil antibodies.
RESULTS: The specificity of HNA-2a antibodies in all sera was confirmed by immunoblotting. Sera were then tested simultaneously in ELISA and MAIGA assays. Nine of 10 sera showed positive reactions in ELISA, whereas only 9 of 10 sera reacted in the standard MAIGA assay. All HNA-2a antibodies were detectable in MAIGA when diluted sera were applied. No reaction was observed with different sera containing neutrophil-reactive antibodies (6 anti-HNA-1a, 4 anti-HNA-1b, and 20 anti-HLA Class I and II) in ELISA. All HNA-2a antibodies were detectable in MAIGA when diluted sera were applied. Notably, sera containing anti-proteinase 3 (PR3) from patients with Wegener's granulomatosis reacted in MAIGA. In contrast, this antibody showed no reaction in ELISA with purified rHNA-2a.
CONCLUSIONS: These results demonstrated that ELISA with rHNA-2a provides a good method for detecting HNA-2a antibodies in human serum. This assay enables to exclude the presence of autoantibody against PR3 in patient's sera, which cannot be differentiated from anti HNA-2a with current serologic methods.     

Blockade of maternal anti-HPA-1a–mediated platelet clearance by an HPA-1a epitope–specific F(ab')2 in an in vivo mouse model of alloimmune thrombocytopenia

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is most commonly caused by transplacental passage of maternal human platelet-specific alloantigen (HPA)-1a antibodies that bind to fetal platelets (PLTs) and mediate their clearance. SZ21, a monoclonal antibody (MoAb) directed against PLT glycoprotein IIIa, competitively inhibits the binding of anti-HPA-1a alloantibodies to PLTs in vitro. The purpose of this investigation was to determine whether SZ21 F(ab')₂ fragments might be therapeutically effective in inhibiting or displacing maternal HPA-1a antibodies from the fetal PLT surface and preventing their clearance from circulation.
STUDY DESIGN AND METHODS: Resting human PLTs from HPA-1ab heterozygous donors were injected into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Purified F(ab')₂ fragments of SZ21 or control immunoglobulin G (IgG) were injected intraperitoneally 30 minutes before introduction of HPA-1a antibodies. Blood samples were taken periodically and analyzed by flow cytometry to determine the percentage of circulating human PLTs.
RESULTS: Anti-HPA-1a IgG from NAIT cases were able to efficiently clear HPA-1a–positive PLTs from murine circulation. Administration of SZ21 F(ab')₂ fragments not only inhibited binding of HPA-1a antibodies to circulating human PLTs, preventing their clearance, but also displaced bound HPA-1a antibodies from the PLT surface.
CONCLUSION: F(ab')₂ fragments of HPA-1a–selective MoAb SZ21 effectively inhibit anti-HPA-1a–mediated clearance of human PLT circulating in an in vivo NOD/SCID mouse model. These results suggest that agents that inhibit binding of anti-HPA-1a to PLTs may have therapeutic potential in the treatment of NAIT.     

Postnatal development of γ-GT activity in rat brain microvessels corresponds to capillary growth and differentiation

It has been demonstrated histochemically that endothelial cells of the cerebral capillaries show a high activity in γ-glutamyl transpeptidase (γ-GT), an enzyme which takes part in the transfer of large neutral amino acids across the blood-brain barrier. Reports of the disappearance of enzyme activity in endothelial cells (EC) grown in culture¹² suggest that the presence of astroglial cells (AG) is required for the expression of γ-GT in these cells. The present study deals with the developmental changes in γ-GT activity in capillaries of rat cerebral cortex during ontogenesis (i.e. on days 2, 7, 11, 14, 21 and 60 after birth).

γ-GT activity is determined by measuring the enzyme kinetics of the histochemical reaction on isolated brain capillaries using a flying spot microscope densitometer. Enzyme activity is expressed as an increase in relative optical density (at 500 nm) in arbitrary units/min/μm² during the 2 min immediately after initiating incubation and corresponds to the γ-GT active area (in μm²) of the capillary segment. During early postnatal development, a biphasic change of γ-GT activity in capillaries of rat cerebral cortex is observed. The first phase (i.e. the postnatal period between the 2nd and 12th day) is characterized by a significant decrease in γ-GT activity, which coincides with the onset of the rapid mitotic proliferation of cortical endothelial cells. During the second phase (i.e. the postnatal period between the 12th and 21st day), a fast increase in the γ-GT activity can be measured. Then enzyme activity reaches the adult level between the 21st and 60th postnatal day.

With regard to changes in capillary length density and in astroglial proliferation the results correspond to the differentiation and maturation of the blood-brain barrier and confirm evidence stressing the important role of astroglial cells for the expression of γ-GT activity.     

Efficacy and Tolerability of Amlodipine in the General Practice Treatment of Essential Hypertension in an Asian Multinational Population

OBJECTIVE: To evaluate the efficacy and tolerability of once-daily amlodipine (Pfizer Pharmaceuticals Inc.) alone or in combination with other antihypertensive drugs in an Asian population with essential hypertension. PATIENTS: An open study was undertaken in 165 male and 158 female patients with uncomplicated hypertension (diastolic blood pressure 95 to 115mm Hg). Patients were recruited from 41 general practices in seven Asian countries and received amlodipine 5mg daily for 4 weeks and then 10mg once daily for a further 4 weeks if the target diastolic blood pressure of /=10mm Hg had not been achieved. This one-step dose-adjustment period was followed by a 4-week maintenance period on a constant dose. Amlodipine was the sole medication in 284 patients and was added to other antihypertensive drugs in 39 patients uncontrolled on previous medication. RESULTS: 263 patients, including 131 males, were evaluated for efficacy at the final treatment visit. 166 (63%) patients achieved the target reduction in diastolic blood pressure with amlodipine 5mg once daily, while 84 patients achieved the target reduction with 10mg once daily. Systolic and diastolic blood pressure reductions were similar irrespective of gender or age, and there were no significant changes in resting heart rate in any subgroup. In 68 patients who underwent ambulatory monitoring, the systolic and diastolic blood pressures were reduced by once-daily amlodipine throughout the 24-hour period without change in the intrinsic circadian pattern. Amlodipine was well tolerated in all patient subgroups; adverse events accounted for less than 1% of treatment discontinuations, and there were no hospitalisations or deaths during the study. Investigators rated both the antihypertensive efficacy and tolerability of amlodipine as excellent or good in 93% of patients. CONCLUSION: In 263 Asian patients with uncomplicated essential hypertension treated in general practice, once-daily amlodipine in a dose of 5 or 10mg provided significant antihypertensive efficacy either as monotherapy or in combination with other antihypertensive drugs while maintaining a favourable tolerability profile regardless of gender or age.     

Alloimmunization against ly, a Low-Frequency Antigen on Platelet Glycoprotein Ib/IX as a Cause of Severe Neonatal Alloimmune Thrombocytopenic Purpura

Neonatal alloimmune thrombocytopenia (NAIT) is usually induced by platelet-specific antibodies against HPA-la (Zw^a) or HPA-5b (Br^a). Recently, low-frequency alloantigens on the platelet glycoprotein (GP) IIb/IIIa complex have been discovered as a cause for NAIT. In this report, a new low-frequency platelet-specific alloantigen, ly, is described which induced severe NAIT. The corresponding antigen was detected in 1/249 unrelated German blood donors. Antibody binding assays with trypsin-digested platelets (ELISA, immunoprecipitation with biotin-labelled platelets) indicate that the antigen is not localized on the glycocalicin moiety of GP Ibα, but may be situated on the remnant moiety of GP Ibα, GP IX or GPIbβ. Apparently, ly is not related to the HPA-2 (Ko) antigen system.     

NA gene frequencies in the German population, determined by polymerase chain reaction with sequence-specific primers

BACKGROUND: The granulocyte antigens NA1 and NA2 are often targets of granulocyte antibodies causing immune neutropenia. Currently, NA typing relies on the properties of the typing sera or antibodies and the techniques used. Therefore, the technique of polymerase chain reaction with sequence-specific primers (PCR-SSP) was adapted for DNA-based NA typing and was used for determining the NA gene frequencies in the German population. STUDY DESIGN AND METHODS: The genomic DNA of 160 unrelated healthy individuals was typed for NA1 and NA2 by PCR-SSP. In 60 granulocyte samples, the NA phenotype was additionally determined by the antigen capture assay and the granulocyte immunofluorescence test (GIFT) and correlated with the genotyping results. RESULTS: Results of the antigen capture assay and PCR-SSP correlated precisely, whereas nine individuals were typed heterozygous only by GIFT. The gene frequencies were 0.35 for NA1 and 0.65 for NA2. CONCLUSION: The NA2 gene is more frequent in the German population than the NA1 gene, as determined by genotyping using PCR-SSP. In contrast to GIFT, which showed an error rate for NA typing of 15 percent, PCR-SSP and the antigen-capture assay are more reliable methods of NA typing of granulocytes.     

Lowering of theophylline clearance by isoniazid in slow and rapid acetylators.

The effect of isoniazid (INH) pretreatment (400 mg daily for 2 weeks) on the elimination kinetics of theophylline (given intravenously as aminophylline equivalent to 151.2 mg theophylline) was investigated in 13 healthy male non-smokers. Amongst the 13 subjects studied, seven were rapid and six were slow acetylators. The mean clearance of theophylline was significantly lowered after INH (2.20 +/- 0.24 l h-1) (mean +/- s.e. mean) compared with the baseline value (2.80 +/- 0.24 l h-1). The volume of distribution at steady state was also lowered significantly after INH (0.42 +/- 0.01 l kg-1 vs 0.47 +/- 0.02 l kg-1). Consequently, there was no significant prolongation of theophylline half-life after INH (7.0 +/- 0.3 h vs 6.7 +/- 0.4 h control). The lowering of theophylline clearance by INH may be related to acetylator status since slow acetylators showed a greater interaction than rapid acetylators. However, this difference was not statistically significant.     

Peak flow rate in normal Indonesian children aged 8 to 15 years.

Although normal values for peak flow rates have been available for Caucasian children and Indonesian adults for several years, similar reliable standard values are not available for Indonesian children. It is the objective of this study to assess the measurements of peak flow rate obtained from a sample of healthy Indonesian school children in Denpasar aged 8 to 15 years. The relationship between peak flow rate (PFR), age, weight and height was studied in 304 male and 254 female children ranging from 8-15 years of age at 2 primary and junior high schools in Denpasar. Strict criteria of "normality" were employed in the selection of the subjects. Measurements were taken with the subject standing and the highest value of three peak expiratory rate manouvers was recorded. Regression analysis was used to calculate the predicted normal value of PFR and also to assess the relationship of PFR to age and weight. The results are recorded in a normogram. Possible cause of the differences between our findings and those of other authors are discussed. In conclusion the author is of the opinion that the normogram (Godfrey) supplied with the Mini Wright apparatus can be used in predicting normal values of PFR for Indonesian children.