Rosuvastatin, a synthetic lipid-lowering agent acts selectively by competitive inhibition of 3-hydroxy- 3-methylglutaryl-coenzyme A. It is indicated as an adjunct to diet in patients with hypercholesterolemia and mixed dyslipidemia.The purpose of this study was to demonstrate bioequivalence between a generic rosuvastatin 40 mg tablet (Zentiva, Prague, Czech Republic) and a reference product (Crestor, AstraZeneca, Luton, UK), under fasting conditions as required by the European Medicinal Agency.A single-oral 40 mg-dose, randomized, open-label, 2-way crossover design study was conducted in 42 healthy volunteers under fasting conditions. Rosuvastatin was administered following an overnight-fast in two occasions with a 14-day washout period in-between. Blood samples were collected in EDTA-K2 tubes prior to dosing and over a 96-hour period. Rosuvastatin was measured in plasma using an automated LC-MS/MS assay (range 81.02 - 40,512.00 pg/ml). Pharmacokinetics were performed using non-compartmental analyses approach to evaluate AUC(last), AUC∞ and C(max). ANOVA was performed on the ln-transformed data and the 90% Confidence Interval (CI) was determined. Bioequivalence will be concluded if the 90% CI falls within 80.00 - 125.00% for AUC(last) and C(max). Safety and tolerability were also evaluated.39 volunteers completed the study and were considered for the pharmacokinetic and statistical analyses. Descriptive safety data analyses were performed on all subjects. All pharmacokinetic parameters met the acceptance criteria as the 90% CI were within 80.00 - 125.00%. Both formulations were well tolerated and no serious adverse events were reported.This study showed that the test and reference products met the regulatory criteria for bioequivalence following a 40 mg oral dose under fasting conditions.
ABSTRACT: After ejaculation, mammalian sperm must undergo a preparation period known as “capacitation” to become capable of fertilizing the oocyte. Although physiological capacitation occurs in the female genital tract, the process can be reproduced in vitro by incubation in appropriate media. However, the signaling events regulating capacitation are poorly understood, especially in boar sperm. Calcium is thought to be of fundamental importance in capacitation. Our laboratory recently identified a tyrosine‐phosphorylated protein of M r 32 000 (“p32”) from boar sperm, and its appearance is closely related to capacitation. The objective of this study was to understand the mechanism regulating the appearance of our p32 tyrosine phosphoprotein. Since calcium has been linked to sperm capacitation and protein tyrosine phosphorylation in other species, we hypothesized that extracellular calcium is involved in the appearance of the p32. Sperm were incubated in either noncapacitating medium (NCM) or capacitating medium (CM) for various times. Proteins were extracted with sodium dodecyl sulfate (SDS), separated by SDS‐polyacrylamide gel electrophoresis (PAGE), and then immunoblotted with an antiphosphotyrosine antibody. To assess intracellular calcium levels, fresh sperm were loaded with the fluorescent calcium indicator indo‐1, and relative fluorescence was measured by flow cytometry. Analysis demonstrated that relative intracellular calcium levels increased during incubation in capacitating conditions but not in NCM, which coincides with the appearance of the p32. The p32 tyrosinephosphorylated protein appeared only in the presence of calcium, and the calcium ionophore Br‐A23187 accelerated its appearance. Consistent with our hypothesis, the appearance of the p32 was inhibited by extracellular calcium chelators (ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′,‐tetraacetic acid [EGTA], EDTA, and 1,2‐bis‐(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid potassium salt [BAPTA‐K + ]), showing the importance of calcium in protein tyrosine phosphorylation related to capacitation in boar sperm.
Mammalian sperm must undergo capacitation, a preparation period in the female reproductive tract or in vitro, in order to fertilize. We have previously described a Mr 32 000 tyrosine phosphorylated protein, "p32," that appears in pig sperm during capacitation. The identity of p32 remains unknown; if and how it is involved during capacitation is not understood. The objective of the present study was to identify p32 by proteomic techniques. Western blotting of proteins separated successively under nonreducing and then reducing conditions showed the appearance of the tyrosine phosphorylated p32 only when sperm were incubated in capacitating conditions. The spot was sequenced by mass spectrometry/mass spectrometry and identified as "sp32," a protein implicated in proacrosin maturation. The same membranes probed with anti-sp32 antibody demonstrated that sp32 is present in both noncapacitating and capacitating conditions and revealed exactly the same spot as p32. Immunoprecipitation with either anti-phosphotyrosine or anti-sp32 antibody corroborated these results. Indirect immunofluorescence with anti-phosphotyrosine antibody or anti-sp32 antibody show similar labeling of capacitated sperm, supporting the hypothesis that p32 is a tyrosine phosphorylated form of sp32. After ionophore treatment to induce the acrosome reaction, anti-sp32 and anti-phosphotyrosine labeling on the acrosome disappeared. These results demonstrate that sp32, a (pro)acrosin binding protein, is the p32, a tyrosine phosphorylated protein related to capacitation. We will now focus on the significance of tyrosine phosphorylation on sp32 function during fertilization-related events.
Mammalian sperm undergo capacitation in the female reproductive tract or under defined conditions in vitro. Although capacitation is now considered to be mediated by intracellular signaling events, including protein phosphorylation, the regulation of the transduction mechanisms is poorly understood. The objective of the present study was to evaluate the importance of medium components on capacitation of porcine sperm, the appearance of an Mr 32 000 sperm protein (p32), and activity of a tyrosine kinase (TK-32). As determined by the ability of the sperm to undergo the A23187-induced acrosome reaction, pig sperm require bicarbonate and calcium but not BSA for capacitation in vitro. The appearance of p32 was assessed by immunoblotting SDS-extracted and separated sperm proteins using an anti-phosphotyrosine antibody. The appearance of p32 requires calcium, although p32 appears even in the absence of bicarbonate in the incubation medium, demonstrating that the appearance of this tyrosine phosphoprotein is not a final end point of pig sperm capacitation. An in-gel tyrosine kinase renaturation assay showed that TK-32 activity depends on calcium and bicarbonate in the incubation medium. Immunoprecipitation experiments using an anti-phosphotyrosine antibody and inhibitor demonstrated that p32 and TK-32 are different proteins. These data indicate that the signal transduction mechanisms of capacitation in pig sperm are different from those in other mammals, suggesting that certain species specificity may exist with respect to this phenomenon.
Leydig cells present in the testicular interstitium are the main site of testosterone production in males. During fetal and neonatal life, a specific population of Leydig cells (fetal Leydig cells, FLC) is responsible for androgen production and masculinization of the male foetus. During postnatal life, the FLC population regresses and is substituted by a distinct population (adult Leydig cells, ALC) derived from undifferentiated stem cells. ALC provide the testosterone essential for completion of male sex differentiation and male reproductive function. Platelet-derived growth factors (PDGF) and their tyrosine kinase receptors (PDGF-R) are known to be implicated in various functions in numerous tissues including the testis. In the testis, expression of PDGF and PDGF-R is developmentally regulated and is cell-specific. The PDGF-A ligand is produced by Sertoli cells while Leydig cells express PDGF-Rα. The PDGF-A/PDGF-Rα system is essential for normal testis development and for differentiation of both populations of Leydig cells since in the absence of PDGF-A or PDGFRα, Leydig cells fail to differentiate. No information, however, is available regarding the molecular mechanisms regulating PDGF-Rα expression in Leydig cells. The aim of this study was to characterize the mouse PDGFRα promoter. We have isolated 4 kb of the PDGF-Rα promoter from mouse genomic DNA, a fragment previously shown to be sufficient for proper expression in Leydig cells in vivo. Since we found that PDGF-Rα is expressed in the TM3 Leydig cell line both at the mRNA and protein levels, we used this cell line to map the regulatory elements important for PDGF-Rα promoter activity. A series of 5′ progressive deletion constructs (−2540 bp, −859 bp, −478 bp, −203 bp, −134 bp, −113 bp, −70 bp, −52 bp) were generated and transfected in TM3 cells. Using this approach, we found that a 60 bp region located between −134 bp to −70 bp is responsible for conferring about 80 % of PDGF-Rα promoter activity in TM3 Leydig cells. Sequence analysis of this region revealed the presence of potential binding sites for several transcription factors previously reported to be present in the testis. These include Pax5, FoxJ2, Smad3, Mok2, Ik2, and members of the RUSH family. To better define the regulatory elements within the 60 bp region, we have used a linker-scanning approach to mutate by sitedirected mutagenesis the 60 bp region 3 bp at a time in the context of the - 4 kb promoter. A total of 20 site-directed mutants were thus generated and transfected in TM3 Leydig cells. We found that 2 mutants had a dramatic effect on PDGF-Rα promoter activity, decreasing it by 75–80 %. The first mutant is located at −103 bp and eliminates a putative binding site for the transcription factors Pax5 and Mok2 while the second mutant, at −87 bp, abolishes a site for members of the Fox family (FoxJ2). The implication of these transcription factors, however, remains to be established. In conclusion, we have mapped the regulatory regions essential for transcription of the PDGF-Rα gene which encodes a critical regulator of Leydig cell differentiation and function. C. Dubé is recipient of CIHR/Wyeth Pharmaceuticals postdoctoral fellowship. Supported by CHIR. (poster)
