Simultaneous pharmacokinetic assessment of cefadroxil and clavulanic acid in human plasma by LC-MS and its application to bioequivalence studies

A simple, rapid and selective liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry （LC-APCI-MS） assay method has been developed and fully validated for the simultaneous quantification of cefadroxil （CF） and clavulanic acid （CA） in human plasma. Analytes and internal standard （IS） were extracted from human plasma by solid- phase extraction （SPE） technique using Sam prep （3 mL, 100 mg） extraction cartridge. The extracted samples were chromatographed on a reverse phase Cls column using a mixture of methanol： acetonitrile： 2 mM ammonium acetate （pH 3.5） （25：25：50, v/v/v） as the mobile phase at a flow rate of 0.8 mL/min. Quantification of the analytes and IS were carried out using single quadrupole LC-APCI-MS through selected-ion monitoring （SIM） at m/z 362 and m/z 198, for CF and CA, respectively. Method validation was performed as per the FDA guidelines and the results met the acceptance criteria. Plasma concentration of CF and CA followed by the oral administration of CF/CA （500/125 mg） pill to healthy male volunteers （n= 12） was measured. Area under plasma concentration-time curve from 0 to 12 h （AUC0-12 h） and 0 h extrapolated to infinity （AUC0-∞） were calculated. The ratio of AUC0-12 h/AUC0-∞ was found to be 〉 85% for all the subjects, as recommended by the FDA guidelines.

Amoxicillin–clavulanic acid induced sperm abnormalities and histopathological changes in mice

Objective: To explore the genotoxic potential and histopathological changes induced in liver, kidney, testis, brain and heart after using the antibiotic drug amoxicillin/clavulanic acid(4:1).Methods: The study included chromosomal aberration analysis in bone-marrow and mouse spermatocytes, induction of sperm morphological abnormalities and histopathological changes in different body organs. The drug was administrated orally at a dose of81 mg/kg body weight twice daily(Total = 162 mg/kg/day) for various periods of time equivalent to 625 mg/men(twice daily).Results: The results revealed non-significant chromosomal aberrations induced after treatment with amoxicillin/clavulanic acid(AC) in both bone marrow and mouse spermatocytes after 7 and 10 days treatment. On the other hand, statistically significant percentages of sperm morphological abnormalities were recorded. Such percentage reached 8.10 ± 0.55, 9.86 ± 0.63 and 12.12 ± 0.58 at the three time intervals tested(7, 14 and 35 days after the 1 st treatment respectively)(treatment performed for 5 successive days) compared with 2.78 ± 0.48 for the control. The results also revealed histopathological changes in different body organs after AC treatment which increased with the prolongation of the period of therapy. Congestion of central vain, liver hemorrhage and hydropic changes in hepatocytes were noticed in the liver. Degenerative changes were found in kidney glomerulus and tubules while testis showed atrophy of seminiferous tubules, and reduction of spermatogenesis. AC also induced neurotoxicity and altered brain neurotransmitter levels. Hemorrhage in the myocardium, disruption of cardiac muscle fibers and pyknotic nuclei in cardiomyocytes were recorded as side effects of AC in heart tissue.Conclusions: The results concluded that AC treatment induced sperm morphological abnormalities and histopathological changes in different body organs. Clinicians must be aware of such results while describing the drug.

Proteome-wide alterations in an industrial clavulanic acid producing strain of Streptomyces clavuligerus

The usefulness of genetic/metabolic engineering for further improvement of industrial strains is subject of discussion because of the general lack of knowledge on genetic alterations introduced by iterative cycles of random mutagenesis in such strains.An industrial clavulanic acid(CA)-overproducer Streptomyces clavuligerus DEPA was assessed to understand proteome-wide changes that have occurred in a local industrial CA overproducer developed through succesive mutagenesis programs.The proteins that could be identified corresponded to 33 distinct ORFs for underrepresented ones and 60 ORFs for overrepresented ones.Three CA biosynthetic enzymes were overrepresented in S.clavuligerus DEPA;carboxyethylarginine synthase(Ceas2),clavaldehyde dehydrogenase(Car)and carboxyethyl-arginine betalactam-synthase(Bls2)whereas the enzymes of two other secondary metabolites were underrepresented along with two important global regulators[two-component system(TCS)response regulator(SCLAV_2102)and TetR-family transcriptional regulator(SCLAV_3146)]that might be related with CA production and/or differentiation.g-butyrolactone biosynthetic protein AvaA2 was 2.6 fold underrepresented in S.clavuligerus DEPA.The levels of two glycolytic enzymes,2,3-bisphosphoglycerate-dependent phosphoglycerate mutase and phosophoglycerate kinase were found decreased while those of dihydrolipoyl dehydrogenase(E3)and isocitrate dehydrogenase,with two isoforms were found as significantly increased.A decrease of amino acid metabolism,methionine biosynthesis in particular,as well as S-adenosylmethionine synthetase appeared as one of the prominent mechanisms of success of S.clavuligerus DEPA strain as a prolific producer of CA.The levels of two enzymes of shikimate pathway that leads to the production of aromatic amino acids and aromatic secondary metabolites were also underrepresented.Some of the overrepresented stress proteins in S.clavuligerus DEPA included polynucleotide phosphorylase/polyadenylase(PNPase),ATP-dependent DNA helicase,two isoforms of an anti-sigma factor and thioredoxin reductase.Downregulation of important proteins of cell wall synthesis and division was recorded and a protein with b-lactamase domain(SCLAV_p1007)appeared in 12 isoforms,5 of which were drastically overrepresented in DEPA strain.These results described herein provide useful information for rational engineering to improve CA production in Streptomyces clavuligerus.