Quantitative determination of piritramide in human plasma and urine by off- and on-line solid-phase extraction liquid chromatography coupled to tandem mass spectrometry.

A method for the liquid chromatography/tandem mass spectrometric (LC/MS/MS) quantification of piritramide, a synthetic opioid, in plasma after conventional off-line solid-phase extraction (SPE) and in urine by on-line SPE-LC/MS/MS in positive electrospray mode was developed and validated. Applicability of the on-line approach for plasma samples was also tested. Deuterated piritramide served as internal standard.

HPLC-MS/MS analysis of willow bark extracts contained in pharmaceutical preparations.

Preparations containing willow bark extract are popular herbal remedies, but they are mostly standardised with respect to only one compound (usually salicin). RP-HPLC using a C18-column eluted with water:methanol:tetrahydrofuran and coupled to electrospray triple-quadrupole MS and MS/MS was used for the characterisation of dried extracts of Salix spp. and for the identification of their constituents.

Stereospecific pharmacokinetic characterisation of phenprocoumon metabolites, and mass-spectrometric identification of two novel metabolites in human plasma and liver microsomes.

Phenprocoumon belongs to the group of vitamin K antagonists (VKAs), for example warfarin and acenocoumarol. It is widely used for therapeutic anticoagulation and clinically administered as a racemate. Both enantiomers are partially metabolized by the polymorphic CYP2C9 enzyme.

Achiral-chiral LC/LC-MS/MS coupling for determination of chiral discrimination effects in phenprocoumon metabolism.

Many physiological processes show a high degree of stereoselectivity, including the metabolism of xenobiotics as catalyzed by cytochrome P450 enzymes. An analysis of these chiral discrimination effects in drug metabolism is essential for an in-depth understanding of metabolic pathways that differ between enantiomers of a given chiral drug or metabolite thereof. Achiral chromatographic separation and structural identification followed by chiral analysis of metabolites from blood specimens usually requires a time-consuming multistage analytical technique.

Genetic polymorphisms of cytochrome P450 2C9 causing reduced phenprocoumon (S)-7-hydroxylation in vitro and in vivo.

The effect of cytochrome P450 (CYP) 2C9 polymorphisms on the stereoselective biotransformation of the oral anticoagulant phenprocoumon (PPC) to inactive, monohydroxylated metabolites was studied in vitro and in vivo. In human liver microsomes, the (S)-7-hydroxylation--being the major metabolic pathway--was significantly compromised in a gene-dose-dependent manner in samples expressing the CYP2C9*2 or CYP2C9*3 allele. The CYP2C9*3/*3 genotype corresponded to an almost fourfold lower (S)-7-hydroxylation rate than CYP2C9*1/*1 (wild-type).

Mass spectrometric pathway monitoring of secondary metabolites: systematic analysis of culture extracts of Streptomyces species.

Streptomyces spheroides, Streptomyces rishiriensis, and Streptomyces roseochromogenes are producers of the aminocoumarin-type antibiotics novobiocin, coumermycin A(1), and clorobiocin, respectively, all of which are bacterial gyrase inhibitors. In an attempt to develop a general analytical method for pathway monitoring of secondary metabolites from culture extracts of these strains, we used superior mass spectrometric methods. The aim was to develop and apply a technique for the rapid analysis of Streptomyces culture extracts with respect to those substances, thereby providing a method for screening extracts of genetically modified strains for new pharmaceutically active antibiotics with improved pharmacological effects.

Effects of CYP2C9 polymorphisms on the pharmacokinetics of R- and S-phenprocoumon in healthy volunteers.

CYP2C9 catalyses the biotransformation of the oral anticoagulants S-warfarin and R- and S-acenocoumarol. According to data obtained in vitro, phenprocoumon is also metabolized by CYP2C9 but the impact of the CYP2C9 polymorphism on phenprocoumon pharmacokinetics has not been studied. Twenty-six healthy heterozygous and homozygous carriers of the CYP2C9 alleles *1 (wild-type), *2 (Arg144Cys), and *3 (Ile359Leu) received a single oral dose of 12 mg of racemic phenprocoumon.

Determination of (R)- and (S)-phenprocoumon in human plasma by enantioselective liquid chromatography/electrospray ionisation tandem mass spectrometry.

Phenprocoumon is a commonly used oral anticoagulant of the coumarin type, and has found extensive clinical use in the treatment of thrombophlebitis, pulmonary embolism and atrial fibrillation. In the course of a clinical study to investigate the influence of genetic polymorphisms of the CYP2C9 enzyme on phenprocoumon metabolism, we developed a new enantioselective liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/MS/MS) method to quantify (R)- and (S)-phenprocoumon in human plasma. HPLC separation of the enantiomers was achieved on a Chira-Grom-2 column under isocratic conditions using a water/acetonitrile/formic acid eluent.

CloN2, a novel acyltransferase involved in the attachment of the pyrrole-2-carboxyl moiety to the deoxysugar of clorobiocin.

The aminocoumarin antibiotic clorobiocin contains a 5-methylpyrrole-2-carboxylic acid unit, attached via an ester bond to the 3-OH group of the deoxysugar moiety. To investigate candidate genes responsible for the formation of this ester bond, a gene inactivation experiment was carried out in the clorobiocin producer Streptomyces roseochromogenes var. oscitans DS 12.

CloR, a bifunctional non-heme iron oxygenase involved in clorobiocin biosynthesis.

The aminocoumarin antibiotics novobiocin and clorobiocin contain a 3-dimethylallyl-4-hydroxybenzoate (3DMA-4HB) moiety. The biosynthesis of this moiety has now been identified by biochemical and molecular biological studies. CloQ from the clorobiocin biosynthetic gene cluster in Streptomyces roseochromogenes DS 12976 has recently been identified as a 4-hydroxyphenylpyruvate-3-dimethylallyltransferase.

Organic nitrates. II. Synthesis and biological activities of 4-nitrooxymethylphenyl-1,4-dihydropyridines.

Both 2-nitrooxymethyl-4-phenyl- (2) and 4-nitrooxymethylphenyl-1,4-dihydropyridines (3) represent new combinations of two different vasodilating structures. 2 could not be isolated due to its spontaneous lactonization. Derivatives of 3 were obtained via Hantzsch synthesis using nitrooxymethylated benzaldehydes.

[A program for prevention of pseudomonas infections and immunoprophylaxis with PSAEVA vaccine].

The authors present three main trends of prevention of Gram-negative opportunistic infections investigated at the Cardiosurgical Clinic of the Institute of Clinical and Experimental Medicine. An important part is played by active and systematic surveillance with early signalling and analysis of new infections with subsequent aimed interventions against concrete sources of infection and mechanisms of transmission. It is necessary to think more of activation of endogenous infection the specific features of which make repression difficult and motivate investigations of preventive approaches.

[A tonsillitis epidemic caused by adenoviruses].

An epidemic of lacunar angina which affected, in relatively close common life, mostly only one of nine military units living in the same area. The failure of penicillin and negative finding of streptococci resulted in a retrospective establishment of the agent, which with all probability was adenovirus type 4. It has become obvious that the typical picture of lacunar or even pseudomembranous angina is not a reliable sign indicating streptococcal etiological even in a mass infection.

Complex surveillance of streptococcus pyogenes. VI. Relationship between hypersensitivity to zymosan and indicators of immunity against group A streptococci.

A prospective study involving 20 volunteers was designed to test relationships between the sensitivity to zymosan and the indicators of nonspecific immunity against streptococci (inhibitory activity of blood and production of mediators). The immunization with Mannozym produced a conversion of zymosan-induced MIF with a subsequent improvement of immunity indicators in 4 out of 7 primovaccinated and 2 revaccinated subjects. This effect was detectable in some vaccines within 2-4 days after immunization and persisted for up to 5 months.

Complex surveillance of Streptococcus pyogenes V. Protective features complex surveillance of streptococcus of cell-mediated immunity against group a streptococci.

Exoproducts of incubated white blood elements from persons hypersensitive to zymosan were found to promote the phagocytic activity of human microphages (and guinea pig macrophages) under conditions of a combined model of "MIF-opsonophagocytic test". The stimulation resulted in a bacteriostatic effect, was non-type-specific and improved microphage potential to inactivate streptococci opsonized with anti-M antibodies in low titre. The microphages pre-exposed to anti-M sera remained unaffected, the effect was non-species-specific and nontype-specific and the stimulation resulted from the action of albumin fraction exoproducts.

Studies on prophylactic efficacy of N-2-hydroxyethyl palmitamide (Impulsin) in acute respiratory infections. Serologically controlled field trials.

The results of three serologically controlled double blind field trials in army units are presented. The evaluation of results according to morbidity, regardless of aetiology, showed a significant reduction in acute respiratory diseases (ARD) after administration of Impulsin. In the 1973 trial (901 volunteers), 22.

Complex surveillance of Streptococcus pyogenes. IV. The phenomenon of interference of human serum under the conditions of the bactericidal test.

The phenomenon of interference of human sera under the conditions of the bactericidal test was described. In the presence of these sera, secondarily non-resistant strains of Streptococcus pyogenes were capable of vigorous growth and anti M 12 bactericidal activity was impaired. The factor was found in the albumin fraction and has probably the character of a blocking cytophilic lymphokin.

Complex surveillance of Streptococcus pyogenes. III. Immunological characteristics of streptococcal resistance in blood and new aspects of type nonspecific protection.

The influence of nonspecific immunity factors in indifferent human blood on the resistance of M non-typable field streptococci and collection M strains in the absence of anti-M antibodies was studied. The experimental results indicate that primary resistance is conditioned on balanced equipment of the strain with M protein and the nonspecific cofactor receptor [R]. Disbalanced strains were strongly phagocytized, with participation by nonspecific opsonins (complement C3 and nonspecific cofactor), irrespective of their M protein content.

Complex surveillance of Streptococcus pyogenes. II. Resistance in human blood and bactericidal activity receptors of field and collection streptococci.

A comparative study of a series of 31 epidemic streptococci and 36 collection M protein producers showed no statistically significant difference in their resistance in human blood. Even highly resistant field strains did not produce M protein (as the main bactericidal activity receptor) under laboratory conditions, however, although under natural conditions they left an anti-M response in the exposed population. Streptococcal resistance is not generally associated with M protein, but is influenced by another factor.

Complex surveillance of Streptococcus pyogenes. I. Immunological surveys of anti-M antibodies and possibilities of long-term epidemiological prognosis.

The results of complex surveillance of Streptococcus pyogenes, based on mass infections in the Czechoslovak army over a period of 11 years, are submitted. The epidemiological method revealed an incidence of cycles, lasting several years, of predominant pairs of T types, most of which could not be typed by their M protein. Auxiliary immunological surveys allowed the biological M activity of widespread strains to be estimated and probably form a basis for considerations on a negative long-term epidemiological type prognosis.