Pharmacokinetic/pharmacodynamic evaluation of systemic effects of flunisolide after inhalation.

The pharmacokinetics and pharmacodynamics of flunisolide were studied in healthy volunteers after inhalation. In the morning on the day the study began, volunteers inhaled 0.5 mg of flunisolide with and without oral administration of charcoal, or 1 mg, 2 mg, and 3 mg of flunisolide with concomitant administration of charcoal. A placebo group was used to assess the endogenous cortisol, granulocyte, and lymphocyte baseline levels. Flunisolide plasma levels were determined by high-performance liquid chromatography using a tandem mass spectrometer as detector (HPLC/MS/MS). Cortisol plasma levels and differential white blood cell counts were obtained over 12 hours. An integrated pharmacokinetic/pharmacodynamic (PK/PD) model was applied to link the flunisolide plasma concentrations with the effects on lymphocytes, granulocytes, and cortisol. Maximum concentration levels of 3 to 9 ng/mL of flunisolide were observed after 0.2 to 0.3 hours for all of the investigated doses. The terminal half-life ranged from 1.3 to 1.7 hours. There was no statistical difference between treatments in the presence or absence of orally administered charcoal. The pharmacokinetic/pharmacodynamic (PK/PD) models satisfactorily described the time-courses of the effects on granulocytes, lymphocytes, and cortisol suppression. The resulting E50-values (concentrations to induce 50% of the maximum effect) concurred with the reported values of in vitro receptor binding affinities. The duration of the systemic effects were short because of the short half-life of the drug. Cumulative cortisol suppression increased with dose administration and ranged from 20% to 36%. The PK/PD simulations resulted in a smaller degree of cortisol suppression for the drug administered at 10 PM. The cumulative change from baseline was slightly smaller for the effects on granulocytes and lymphocytes than those on cortisol. This information promotes the comparison with other inhaled glucocorticoids.

Leukotriene generation in patients with multiple injuries.

We determined the generation and metabolism of lipoxygenase products in isolated granulocyte fractions of patients with multiple trauma (n = 9) and compared the results with those of healthy volunteers (n = 8). The supernatants of stimulated cells were analyzed by high-performance liquid chromatography. During the first week after injury a significantly reduced capacity to generate LTB4 and an increased metabolism of LTB4 into omega-oxidated products (20-OH-LTB4 and 20-COOH-LTB4) were observed after stimulation of the granulocytes with Ca ionophore. The depressed leukotriene production could be partly abrogated by the addition of arachidonic acid. These findings are comparable with alterations previously described in severely burned patients with postburn sepsis. Additionally, an elevated production of LTC4 by peripheral granulocyte fractions was observed in two patients suffering from adult respiratory distress syndrome (ARDS) as well as an increased number of eosinophils during the time of lung dysfunction. Analysis of bronchoalveolar lavage in patients with multiple trauma (11 patients with ARDS and 11 patients without ARDS) by a specific radio-immunoassay confirmed an elevated production of cysteinyl-leukotrienes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cefaclor, cefetamet and Ro 40-6890 on inflammatory responses of human granulocytes.

The effect of three cephalosporins (cefetamet, cefaclor and Ro 40-6890) upon human granulocytes and their ability to modulate the chemiluminescence response, phagocytose, kill bacteria and generate leukotrienes was studied. In the presence of the cephalosporins there was a significant increase in phagocytosis of Escherichia coli. The bactericidal activity of human granulocytes for several other bacteria was also enhanced. Cefetamet and cefaclor increased the chemiluminescence response of human neutrophils to Pseudomonas aeruginosa and Proteus mirabilis in contrast to Ro 40-68790, which decreased the chemiluminescence response. The cephalosporins decreased the synthesis of leukotrienes from human neutrophils after stimulation with Escherichia coli and Staphylococcus aureus. These data emphasize the immunomodulatory functions of various cephalosporins on cells involved in host defence.

Metabolism of leukotriene B4 via reduction into dihydro-leukotriene B4 in human monocytes, alveolar macrophages, and U-937 cells.

The biological effects of leukotriene B4 (LTB4) within the microenvironment are controlled by rapid inactivation. In this regard human granulocytes convert LTB4 into omega-oxidated products (20-OH-LTB4 and 20-COOH-LTB4); moreover, we recently described the formation of unpolar metabolites of LTB4 in human tonsillar and lung macrophages. By means of high performance liquid chromatography (HPLC) we identified the main metabolite of LTB4 as dihydro-LTB4 (5,12-dihydroxyeicosatrienoic acid). Studies on a lymphocyte (74-78%), monocyte (19-22%), and basophil (less than 4%) containing cell fraction isolated from peripheral blood as well as peripheral monocytes purified by elutriation centrifugation revealed evidence that these cells metabolize LTB4 to a very low degree if incubated immediately after isolation. However, after culture for 24-72 h these cells showed a strongly increased capacity to metabolize LTB4. The pattern of metabolites in this cell fraction was identical to bronchoalveolar macrophages (purity greater than 95%). Similarly, the LTB4-reductase was expressed in differentiated human monocytic U-937 cells almost 5-7 h after the addition of dimethylsulfoxide (1.3%) or phorbol-myristate-acetate (16 nM). The expression of this pathway was blocked in the presence of cycloheximide (10 micrograms/ml) whereas actinomycin (3.8 micrograms/ml) had no effects. Dihydro-LTB4 was further metabolized by granulocytes probably via omega-oxidation; therefore, several metabolites could be detected by radioactive high performance liquid chromatography (HPLC) after incubation of bronchoalveolar cells consisting of macrophages and granulocytes with 3H-LTB4. Our data provide evidence for a unique role of macrophages to control the level of LTB4 by generation as well as metabolism into dihydro-LTB4.

Serum CD14 levels in polytraumatized and severely burned patients.

Recently it has been demonstrated that the CD14 molecule which is expressed on monocytes and macrophages serves as a receptor for lipopolysaccharide (LPS) bound to LPS-binding protein (LBP) and thus mediates LPS-induced tumour necrosis factor (TNF) production. Here we report that CD14 is found as a soluble (s) molecule in serum. In healthy volunteers sCD14 levels (mean +/- s.e.m.) were 3.7 +/- 0.05 micrograms/ml (n = 30, 25-50 years of age) as determined by ELISA (detection limit 20 ng/ml serum) using two monoclonal antibodies in a sandwich technique. In polytraumatized patients (n = 16) significantly decreased levels (1.7 +/- 0.3) were detected immediately after the trauma, which increased to 4.9 +/- 0.3 micrograms/ml within the first 6 days post trauma. sCD14 remained elevated during the first 14 days post trauma in patients with the most severe injuries (injury severity score greater than 45 points), whereas a return to normal levels was observed in patients with an injury score of less than 45 points. In addition, the levels of the high-density lipoproteins that partially inactivate free endotoxin are significantly decreased post trauma. No correlation between parameters of inflammation (C3a and neopterin levels, leucocyte counts, amount of band cells), liver function and sCD14 levels was established. Comparable to polytraumatized patients, increased sCD14 serum levels were observed in five patients with burn trauma (burned area greater than 35%) within the second week post trauma when clinical signs of septicaemia were evident.

Modulation of leukotriene formation by cellular composition and exogenous leukotriene A4.

We investigated the interactions of exogenous leukotriene A4 (LTA4) with isolated cells in the presence or absence of cellular stimuli. The majority of isolated cells are able to transform exogenous LTA4 into LTB4 as well as LTC4. In eosinophils, LTA4 induced 15-hydroxy-eicosatetranoic acid formation and was converted into LTB4. The Ca-ionophore-induced generation of LTB4 from polymorphonuclear leukocytes or from the cell fraction containing lymphocytes, monocytes and basophils was significantly suppressed with LTA4 while the formation of LTC4 was increased. Conversely, the Na-fluoride- and fMLP-induced generation of LTB4 was significantly increased. Our results suggest that the stimulus and the cellular composition determine the pattern of the generated inflammatory mediators.

Influence of ciprofloxacin on leukotriene generation from various cells in vitro.

The effect of ciprofloxacin on leukotriene generation from human polymorphonuclear leucocytes (PMN) and the respective lymphocyte, monocyte and basophil (LMB) containing cell fraction has been studied. Furthermore, the influence on the LTB4-receptor expression of PMNL, as well as on the synthesis of 12-hydroxyeicosatetraenoic acid (12-HETE) from human platelets, was analysed. The antibiotic concentrations ranged from 0.5 to 4 micrograms/10(7) cells. Analysis of the generated leukotrienes was performed by high performance liquid chromatography (HPLC). The calcium-ionophore A23187 induced leukotriene generation from PMNs and LMBs was significantly suppressed by preincubation with ciprofloxacin in a dose-dependent manner. Incubation of PMNs with the same concentrations of ciprofloxacin induced an augmentation of the LTB4-receptor expression. Preincubation of human platelets led to a suppression of the calcium-ionophore induced 12-HETE generation at high concentrations (8 and 4 micrograms/10(8) cells) and to an increased synthesis of 12-HETE at lower concentrations (0.5, 1, and 2 micrograms ciprofloxacin/1 x 10(8) platelets).

In-vitro enhancement of leukotriene B4 receptor expression on human neutrophils by cefadroxil.

The influence of cefadroxil on LTB4-receptor expression of polymorphonuclear leucocytes (PMNs) was studied. Furthermore, the effect of cefadroxil on the leukotriene generation from PMNs and the lymphocyte, monocyte and basophil (LMB) containing cell fraction as well as on the synthesis of 12-hydroxyeicosatetraenoic acid (12-HETE) from human platelets was analysed. Antibiotic concentrations ranged from 50 to 5 micrograms/10(7) cells. Analysis of the generated leukotrienes was performed by high performance liquid chromatography (HPLC). Significant augmentation of the LTB4-receptor expression in human PMNs (range 190%-220%) was observed at concentrations of 50 and 25 micrograms/10(7) cells. The calcium-ionophore A23187 induced LTB4 generation from PMNs as well as 12-HETE synthesis from platelets was not significantly modulated in the presence of cefadroxil. Preincubation of the human LMB fraction led to slight suppression of the ionophore induced LTB4 generation up to 20%.

Generation and metabolism of leukotrienes in granulocytes of patients with cystic fibrosis.

We studied the generation and metabolism of lipoxygenase products in peripheral granulocytes from children suffering from cystic fibrosis (CF). Peripheral granulocytes were stimulated at different times (days) before and during anti-infectious treatment with the Ca ionophore (7.5 microM, 5 and 20 min), opsonized zymosan (2 mg) and arachidonic acid (50 microM); the amount of lipoxygenase products in the cell supernatants was determined by high performance liquid chromatography. Granulocytes from patients with CF, compared to an age-matched control group, showed an increased omega-oxidation of the synthesized leukotriene (LTB4) into 20-OH- and 20-COOH-LTB4 after stimulation with the Ca ionophore (ratio of LTB4 versus omega-oxidated products in patients with CF: 0.77 +/- 0.07, mean +/- SEM, n = 11; control group: 1.07 +/- 0.1, n = 11, p less than 0.01) whereas the combined amounts of LTB4 and its omega-oxidated products did not differ significantly. A comparable profile was observed with opsonized zymosan. Stimulation of the cells with the Ca ionophore combined with arachidonic acid led to a significantly increased formation of lipoxygenase products in the patient group, whereas only a slight enhancement was observed in the control group. During the 14-day anti-infectious treatment a normalization of the altered pattern was observed. 12-Hydroxyeicosatetraenoic acid (12-HETE) production from platelets within the granulocyte fraction was significantly depressed in the CF group compared to the controls (38.5 +/- 12.5 versus 339 +/- 93 ng/5 +/- 10(6) cells, p less than 0.005). Within the CF group a strong correlation between the release of LTB4 and its metabolites, the production of 12-HETE and clinical (e.g. pO2, FEV1) and laboratory findings (e.g. IgE and IgG levels, C-reactive protein) was established. Our data suggest that the inflammatory process in patients with CF is associated with an alteration of the lipoxygenase pathway of granulocytes which correlates with the clinical signs of inflammation.

The neutrophil and leukotrienes--role in health and disease.

The granulocyte plays a major role in inflammatory processes by its capability to produce and release proinflammatory mediators such as the leukotrienes. This class of mediators exerts multiple functions in various physiological and pathophysiological processes. The interdependent interaction among the various mediator cascades, the inflammatory cells as well as the neuroendocrine system are areas of current interest. Appropriate stimuli (immunological, non immunological) by their defined pattern of signal transduction are responsible for priming, cellular activation and deactivation of granulocytes as it is shown for microbial infection. Thus the mediators may provide the prerequisites for a balanced homeostasis during host defense. It is clear that among the complexity of mediators not a single factor but the interaction of multiple mediator cascades reflect the final outcome of the disease process. In this regard the actual concentrations at local sites are obviously more relevant as those in whole body fluids.

Decreased expression of leukotriene B4 receptor sites on polymorphonuclear granulocytes of severely burned patients.

Polymorphonuclear granulocytes were isolated from patients with burn injury and the specific binding of (3H)leukotriene B4 was assessed. We observed a decreased receptor expression as compared to healthy donor cells, which may be the result of receptor downregulation as a consequence of cellular preactivation. In addition, leukotriene B4-synthesis was also reduced and differential cell counts demonstrated a shift from segmented neutrophils to immature cells. In survivors the values returned to normal parameters whereas nonsurvivors who succumbed in the course of generalized sepsis showed depressed cellular functions up to their death.

Tumour necrosis factors modulate the affinity state of the leukotriene B4 receptor on human neutrophils.

Pre-incubation of human polymorphonuclear granulocytes with recombinant human tumour necrosis factors (TNF) revealed a time- and dose-dependent reduction of the expression of leukotriene B4-receptor sites. Analysis of the binding data by Scatchard plots showed a shift from a heterologous receptor population (indicating high- and low-affinity subsets) to a homologous population. From the results it is considered that TNF can influence host defence through the modulation of leukotriene B4 receptor affinity.

The role of lipid mediators in asthma (current views and perspectives).

Among the lipid mediators, leukotrienes and the platelet activating factor (PAF) have been attributed various roles in the immunopathology of allergic diseases. PAF has been shown to be a chemotactic factor for eosinophils, which conversely release leukotriene C4 on activation. PAF also may induce airway hyperreactivity. Leukotrienes (LTB4) are also powerful chemotactic factors and predominantly attract neutrophils, which stimulates dependent release lipid mediators (hydroxyeicosatetraenoic acids--HETES, leukotrienes, PAF) and thus amplify the inflammatory process. Leukotrienes (LTC4, D4, E4) induce bronchoconstriction, and mucus production. Lipid mediators are released by immunological as well as nonimmunological processes, e.g. by inflammatory stimuli. While analytical methods for leukotriene determinations have been established, the analysis of PAF is mainly carried out by biological studies. Further investigations with antagonists may be helpful to clarify the role of the lipid mediators during allergic and inflammatory processes.

Application of high-performance liquid chromatography of some antibiotics in clinical microbiology.

During recent years high-performance liquid chromatography has become an excellent tool for the determination of antibiotics in biological fluids. Compared with biological assays, the major benefits of this method are specificity and rapidity. In particular, the determination of biologically inactive metabolites emphasizes that this technique plays an outstanding role for the analysis of antibiotics. This paper describes how the method can be used in the analysis of several antibiotics and demonstrates the efficacy of this method for clinical microbiology. Methods for the determination in biological fluids of acylaminopenicillins (azlocillin, mezlocillin, piperacillin and aspoxicillin), quinolones (ciprofloxacine, norfloxacine and ofloxacine), a penem (imipenem) and a cephalosporin (cefixime) are summarized. Furthermore, their application to in vitro studies and their trial in clinical studies are described.

Arachidonic acid metabolites from polymorphonuclear leukocytes of healthy donors, severely burned patients and children with cystic fibrosis--routine monitoring by high-performance liquid chromatography.

A rapid and sensitive high-performance liquid chromatographic method for the determination of arachidonic acid metabolites was developed. This method provides a clear and simple separation of the omega-oxidation products as well as of leukotriene B4 and 5S,12S-dihydroxy-6-cis-8-trans-10-trans-14-cis-eicosatetraenoic acid. Furthermore, a solvent switch enables the detection of the monohydroxyeicosatetraenoic acids together with the cysteinyl-leukotrienes as well as leukotriene B4 and the dihydroxyeicosatetraenoic acids within 70 min (including column equilibration for 10 min). The efficiency of the method for the routine monitoring of leukotrienes was performed with human polymorphonuclear leukocytes of healthy donors, severely burned patients and of children with cystic fibrosis. The advantages of this method include high sensitivity and easy handling, which are important for routine analyses of the arachidonic acid metabolites.

Mezlocillin in pleural effusions--analysis by HPLC.

Mezlocillin concentrations in the pleural fluid of six patients (42-76 years of age, suffering from cytologically confirmed malignant pleural effusions) were determined after intravenous infusion of 10 g mezlocillin. Serum and pleural fluid samples were withdrawn 15, 30, 45, 60 min, 2, 4, and 8 h post infusion. Detection of mezlocillin and its metabolites penicilloic acid and penilloic acid was carried out by means of high performance liquid chromatography (HPLC). Mezlocillin concentrations in serum increased up to 778 +/- 270 micrograms/ml after 15 min, steadily decreasing to 55 +/- 50 micrograms/ml (8 hours post infusion) comparable to the known pharmacokinetic behaviour of mezlocillin; in the pleural effusions mezlocillin levels increased up to 100 +/- 38 micrograms/ml after 1 h. This concentration was maintained throughout the following 7 h. Penicilloic levels ranged about 2-4% within serum, whereas levels below 1% were measured in the pleural fluid.

Degradation of acylaminopenicillins with regard to their pH dependency.

Determination of antibiotic concentration is performed in many biological fluids and tissues which all have different pH values. Therefore, we investigated the in vitro stability of three acylaminopenicillins (azlocillin, mezlocillin and piperacillin) in borate buffer by the HLPC technique with regard to pH dependency. HPLC allows the detection of all three substances together with their metabolites, penicilloate and penilloate, within 15 min. Decomposition was monitored at 37 degrees C during a 24 h incubation period (pH values ranged between pH 3.0 and 10.0). The highest degradation rates were observed with a buffer solution of pH = 10.0: 50% of the azlocillin and 83% of the mezlocillin were decomposed after 8 h while under the same conditions, piperacillin was completely decomposed already after 1 h. The highest stability was detected in borate buffer at pH values of 4.0, 5.0, and 6.0. At pH = 3.0, degradation was determined as follows: 31% of the piperacillin, 39% of the mezlocillin, and 45% of the azlocillin were decomposed after 24 h. Penilloic acid was identified as the main metabolite in contrast to buffer solutions with higher pH values which only revealed negligible amounts of this compound.

Concentration of azlocillin in human chondral tissue.

The concentration of azlocillin was determined using high performance liquid chromatography in serum and chondral tissue after intravenous infusion of azlocillin (75 mg/kg body weight). In serum the levels of ten patients (2 to 27 years of age, body weight 12 to 69 kg) decreased from 478 (30 min post infusion) to 120 micrograms/ml (120 min). In contrast, the concentrations in chondral tissue ranged between 24 and 35 mg/g tissue at the corresponding times. Although the mean levels suggest a remarkable penetration of azlocillin into chondral tissue, the high individual differences observed in the tissue levels (2.1 to 138 micrograms/g tissue) require a higher dosage to ensure sufficient antimicrobial therapy in all patients.