Monitoring sub-nanogram amount of acetylspiramycin in human urine using flow injection analysis with chemiluminescence detection.

AIM: To establish a new and simple flow injection method for the rapid determination of acetylspiramycin (ASPM). METHODS: ASPM was determined by chemiluminescence (CL) method combined with flow injection (FI) technology, which was based on the inhibitive effect of ASPM on the chemiluminescence reaction of the luminol-K3Fe (CN)6 system. RESULTS: The decrease of chemiluminescence intensity was proportional to the logarithm of ASPM concentration (0.1-100) microgram.L-1, the detection limit was 40 ng.L-1 (3 sigma). The whole process, including sampling and washing, could be completed in 0.5 min with a RSD less than 3.0% (n = 5). CONCLUSION: The FI-CL method is of both high sensitivity and good selectivity giving a throughput of 120 h-1. The proposed method was applied successfully to the determination of ASPM in pharmaceutical preparations and human urine without any pre-treatment. It was found that the ASPM concentration reached its maximum after being orally administrated for two hours.

Determination of three major components of bitespiramycin and their major active metabolites in rat plasma by liquid chromatography-ion trap mass spectrometry.

The rapid, selective and sensitive liquid chromatographic-ion trap mass spectrometric (LC-MS(n)) method was developed and validated for determination of three major components (isovaleryspiramycins, ISV-SPMs) of a novel macrolide antibiotic bitespiramycin and their major active metabolites (spiramycins, SPMs) in rat plasma. The analytes ISV-SPMs, SPMs, internal standard roxithromycin and azithromycin were extracted from plasma samples by liquid-liquid extraction, and chromatographed on a C(18) column using two mobile phase systems. Detection was carried out on an ion trap mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI). Three components (ISV-SPM I, II, III or SPM I, II, III) could be simultaneously determined within 6.5 min. Linear calibration curves were obtained in the concentration ranges of 4-200 ng/ml for ISV-SPM I and SPM I, 12-600 ng/ml for ISV-SPM II and SPM II, and 18-900 ng/ml for ISV-SPM III and SPM III. The intra- and inter-run precision (RSD), calculated from quality control (QC) samples were less than 8.8 and 10.4% for ISV-SPMs, and 9.3 and 11.2% for SPMs, respectively. The method was applied for the evaluation of the pharmacokinetics of bitespiramycin in rats following peroral/intravenous administration.

The use of an experimental metritis model to study antibiotic distribution in genital tract secretions in the ewe.

The influence of experimentally-induced metritis on spiramycin disposition in genital secretions was investigated in six ovariectomized ewes. A crossover study design was selected to compare control with metritis pharmacokinetics. A clinically-relevant metritis was obtained under progestagen priming by inoculation in the uterine lumen of a bacterial suspension of Actinomyces pyogenes and Fusobacterium necrophorum Ewes were given a single iv administration of spiramycin at a dose of 20 mg.kg-1. Plasma and genital secretions were regularly sampled up to 96 h post-injection and spiramycin activity was measured using a microbiological method. Experimental metritis did not affect plasma spiramycin disposition and the antibiotic was more concentrated and lasted longer in genital secretions than in plasma regardless of the animal's state of health. The area under the concentration-time curve of spiramycin in genital secretions was twofold higher (p < 0.05) in infected than in healthy ewes (3361 +/- 112 micrograms.h.g-1 and 175 +/- 41 micrograms.h.g-1 respectively). The mean residence time of spiramycin in genital secretions was significantly longer in diseased ewes (32 +/- 4 h) than in control ewes (23 +/- 4 h). The maximum concentration of spiramycin in genital secretions was equal for both studies but occurred later in infected ewes (2.7 +/- 1.0 h versus 8.6 +/- 4.5 h). It was concluded that a uterine infection had a marked influence on the disposition of spiramycin in genital tract secretions and that this uterine infection model in the ewe merits consideration for the study of drug treatments of genital tract infection.

Kinetics of spiramycin/metronidazole (Rodogyl) in human gingival crevicular fluid, saliva and blood.

The peripheral distribution of spiramycin and metronidazole, which are combined in the proprietary drug "Rodogyl", has been studied in gingival fluid, saliva and blood after a single administration to 12 healthy volunteers and after repeated administration to 4 patients with recurring severe periodontitis. Analysis of the 2 antibiotics have been performed at regular intervals during the 24-h period immediately following the administration to the volunteers and after the 1st and the 15th days of repeated administration to the patients. The results show that gingival fluid contains concentrations of spiramycin and metronidazole higher than those needed to inhibit the growth of periodontopathic bacteria. Spiramycin was found at higher concentrations in GCF than in blood, although this feature was not found for metronidazole, which was administered simultaneously and showed similar concentrations in both fluid and serum. Such high concentrations persist for a long time, and suggest the potential of this compound in the treatment of severe cases of periodontitis.

Pharmacokinetics of spiramycin in the rhesus monkey: transplacental passage and distribution in tissue in the fetus.

Transplacental transfer of spiramycin was investigated in a rhesus monkey model to study whether the antibiotic reaches therapeutic levels in the fetus. Spiramycin concentrations were measured by bioassay and high-performance liquid chromatography. Pharmacokinetic parameters were determined for bioactive spiramycin as measured by the bioassay. Pharmacokinetic pilot studies showed that spiramycin distribution follows a two-compartment model in rhesus monkeys. Following a single intravenous dose of 50 or 250 mg, dose-dependent kinetics were observed. At a dose of 50 mg, 10% of the dose was excreted unchanged in the urine. At the higher dose of 250 mg, an oliguric effect was observed. Spiramycin concentrations in fetal serum were measured over time while the maternal concentration was maintained at a constant level. During a 5-h experiment, a maximum fetal-maternal serum ratio of 0.27 was found. In three fetuses, concentrations in serum and tissue were measured following intravenous administration of 50 mg of spiramycin twice daily to the mother for at least 7 days. The fetal-maternal serum ratios were found to be 0.4 to 0.58 after intravenous administration of the final dose of 50 mg to the mother. It appeared that spiramycin accumulated in the soft tissues, especially in the liver and spleen, of both the mother and the fetus. The concentration in placental tissue appeared to be 10 to 20 times that of the concentration in fetal serum. The concentration of spiramycin in amniotic fluid was about five times higher than the concentration in fetal serum. Another important observation was that absolutely no spiramycin was found in the brain.

Determination of spiramycin and neospiramycin in plasma and milk of lactating cows by reversed-phase high-performance liquid chromatography.

After chloroform extraction, the rapid and sensitive determination of spiramycin and neospiramycin can be performed with AASP-diol clean-up cartridges prior to reversed-phase C18 high-performance liquid chromatography. The limits of quantification of spiramycin in plasma and milk are 0.023 and 0.013 microgram/ml, respectively, and those of neospiramycin, are 0.058 and 0.006 microgram/ml, respectively. Application of the method to the analysis of plasma and milk samples obtained from pharmacokinetic studies is described. Spiramycin has a terminal half-life of 14.27 h in plasma and 34.59 h in milk, while neospiramycin has a half-life of 25.62 h in plasma and 105.85 h in milk.

Pharmacokinetics and tissue residues of spiramycin in cattle after intramuscular administration of multiple doses.

Pharmacokinetic variables of spiramycin and its distribution in muscle, liver, kidney, and injection sites were studied in 18 mixed-sex 1-year-old calves to assess drug withdrawal time after 2 IM administrations of 100,000 IU of spiramycin/kg of body weight at 48-hour intervals. Presence of a compound, other than spiramycin I (ie, neospiramycin), was observed in tissues used for withdrawal time determination. High concentrations observed at the injection sites decreased slowly to maximal residue limit with half-life of 109.5 hours for neospiramycin and 77.5 hours for spiramycin. At 14 days, neospiramycin concentrations were higher in kidney than in liver and half-life was different between these 2 tissues. Two methods of withdrawal time determination were used and the part of the samples without residue detected, in the calculation, was discussed. Withdrawal time of 35 days can be proposed on the basis of average daily intake determined for spiramycin, with concentration at injection sites representing 10% of the whole muscle concentration.

Effect of sexual steroid hormones on spiramycin disposition in genital tract secretions of the ewe.

The present work investigated the influence of sexual steroid compounds (estradiol 17 beta and fluorogestone) on antibiotic passage across the uterine barrier. Five healthy and mature ewes, with controlled hormonal impregnation, were given a single iv injection of spiramycin, a macrolide antibiotic, at a dose of 64,000 IU/kg. Plasma and uterine secretions were regularly sampled before the injection and for 30 h post-injection. Blood was collected from the jugular vein and uterine secretions were obtained by uterine flushing with a sterile saline solution containing 0.2% inulin. Spiramycin was concentrated in the uterine secretions, whatever the hormonal status; the secretions-to-plasma ratio was 4.68 +/- 1.88 under estrogen priming and 2.68 +/- 0.91 under progestagen priming. The area under the concentration-time curve (AUC) and the mean residence time (MRT) were significantly higher (p less than 0.001) in uterine secretions than in plasma. The AUC in uterine secretions was significantly higher (p less than 0.05) under estrogen priming (439.07 +/- 241.25 IU.h/mL) than under progestagen priming (141.41 +/- 89.37 IU.h/mL). The spiramycin MRTs in uterine secretions were 11.92 +/- 4.08 and 12.06 +/- 3.35 h for both estrogens and progestagen treatment, respectively. These experiments demonstrate that estrogens increase uterine bioavailability, but not the residence time, of spiramycin when administered by a systemic route.

Comparison of automated liquid chromatographic and bioassay methods for determining spiramycin concentration in bovine plasma.

The performance of a liquid chromatographic (LC) method for spiramycin measurement in bovine plasma has been compared with that of a microbiological method. Plasma samples were obtained from cattle administered spiramycin intravenously. Comparison tests used were intraclass correlation (r1), correlation (r), and Student's paired t-test. For concentrations lower than 2.5 IU/mL, microbiological values were higher than LC values. This difference in results modified pharmacokinetic interpretation and might be explained by the presence of microbiologically active metabolites.

Respiratory tract distribution and bioavailability of spiramycin in calves.

Pharmacokinetic determinants of spiramycin and its distribution into the respiratory tract were studied in 2 groups of calves, 4 to 10 weeks old. Group-A calves (n = 4) were used to determine pharmacokinetic variables of spiramycin after IV (15 and 30 mg/kg of body weight) and oral administrations of the drug (30 mg/kg) and to measure distribution of spiramycin into nasal and bronchial secretions. Group-B calves (n = 4) were used to determine distribution of spiramycin into lung tissue and bronchial mucosa. Spiramycin disposition was best described by use of an open 3-compartment model. Mean (+/- SD) elimination half-life was 28.7 +/- 12.3 hours, and steady-state volume of distribution was 23.5 +/- 6.0 L/kg. Bio-availability after oral administration was 4 +/- 3%. High and persistent concentrations of spiramycin were achieved in the respiratory tract tissues and fluids. Tissue-to-plasma concentration ratio was 58 for lung tissue and 18 for bronchial mucosa at 3 hours after spiramycin administration and 137 and 49, respectively at 24 hours. Secretion-to-plasma concentration ratio was 4 for nasal secretions and 7 for bronchial secretions, and remained almost constant with time. Thus, spiramycin penetrates well into the respiratory tract, although the value in bronchial secretions is lower than that in lung tissues and bronchial mucosa. Calculations indicate that a loading dose of 45 mg/kg, administered IV, followed by a maintenance dose of 20 mg/kg, IV, once daily is required to maintain active concentrations of spiramycin against bovine pathogens in bronchial secretions.

Spiramycin concentrations in plasma and genital-tract secretions after intravenous administration in the ewe.

Uterine infections are associated with reduced fertility in ruminant species. Spiramycin is a macrolide antibiotic potentially active against most of the microorganisms isolated from secretions of infected genital tracts. The present work investigated the ability of systemically administered spiramycin to enter genital secretions, by determining the disposition kinetics of the antibiotic in both plasma and uterine genital secretions. Five healthy ovariectomized ewes were given a single intravenous (i.v.) injection of spiramycin, at a dose of 20 mg/kg. Plasma and genital secretion samples were collected at predetermined intervals for 5 days post-injection. Blood was collected from the jugular vein while mucus was obtained by inserting polyurethane sponges into the vagina. The spiramycin concentration peak in genital-tract secretions was obtained 2.53 +/- 0.63 h after the i.v. administration. The mean residence time was significantly longer (P less than 0.01) in the mucus (18.31 +/- 3.24 h) than in plasma (6.99 +/- 2.53 h). An average mucus to plasma ratio of 7.87 +/- 3.00 was calculated from the area under concentration-time curves covering the period under study. These data indicate that after systemic administration to ewes, spiramycin is rapidly found in genital-tract secretions, at concentrations which are sufficiently high and persistent to suggest its use in the treatment of post-partum uterine infections.