Stability of beta-lactamase inhibitors and beta-lactam antibiotics in parenteral dosage forms and in body fluids and tissue homogenates: a comparative study of sulbactam, clavulanic acid, ampicillin and amoxycillin.

The beta-lactamase inhibitors and the beta-lactam antibiotics differ appreciably in chemical stability. A comparative study of four different infusion solutions at 4, 25 and 37 degrees C yielded the following descending sequence of stability: sulbactam (CAS 68373-14-8), ampicillin (CAS 69-53-4), amoxycillin (CAS 61336-70-7) and clavulanic acid (CAS 58001-44-8). Especially noteworthy was that the two beta-lactamase inhibitors, sulbactam and clavulanic acid, behaved very differently. Moreover, sulbactam is markedly more stable than clavilanic acid even when incubated at 37 degrees C in body fluids or tissue homogenates. The differences in the chemical stability of these pharmacological agents should be taken into consideration in the therapeutic use of combination preparations such as sulbactam/ampicillin (Unacid(R)) and clavulanic acid/amoxycillin.

Pharmacokinetics of ampicillin/sulbactam in patients undergoing colorectal surgery: measurements in serum, the colonic wall and in tissue at the incision site.

The concentrations of ampicillin and sulbactam were determined in serum, colonic wall and incision site tissues from 23 patients undergoing elective colorectal surgery after infusion of a high-dose regimen (2 g ampicillin/1 g sulbactam) or a low-dose regimen (1 g ampicillin/0.5 g sulbactam). The results confirmed that ampicilin and sulbactam penetrated well into the tissues studied and reached therapeutically effective concentrations at the various sites. The high dose regimen showed higher concentrations of both compounds in serum and tissues, indicating a longer period of perioperative protection against bacterial pathogens. Thus, about 39 min after the end of the infusion of the high-dose regimen, the mean concentration of ampicillin was 68.8 +/- 31.2 mug/g and of sulbactam 23.4 +/- 6.3 mug/g in the tissue of the colonic wall. Low-dose prophylaxis, showing mean tissue concentrations of ampicillin of 35.6 +/- 7.0 mug/g and of sulbactam of 14.2 +/- 2.4 mug/g about 48 min after the infusion, is appropriate if the duration surgery does not significantly exceed 2 h.

Reduced intracellular activity of antibiotics against Listeria monocytogenes in multidrug resistant cells.

Multidrug resistance is expressed not only by bacteria, but also by tumor cells and by some normal cells of the body. It enables eukaryotic cells to exclude not only cytostatic drugs but also non-cytostatic antibiotics. This was demonstrated in genetically engineered multidrug resistant (MDR) cells infected with the facultative intracellular bacterium Listeria monocytogenes for all macrolide antibiotics tested (azithromycin, clarithromycin, erythromycin, josamycin, roxithromycin and spiramycin). In these cells and in conventionally selected MDR cells higher concentrations of the macrolides were necessary to inhibit the growth of L. monocytogenes than in the respective parental cells. This effect was due to a reduced intracellular accumulation, which was shown with a biological assay for all macrolides tested. For azithromycin, the results of this test were confirmed by measurement of the intracellular concentrations with high-performance liquid chromatography (HPLC). Besides the macrolides, MDR cells excluded also antibiotics of other chemical groups which was shown for ciprofloxacin, clindamycin, rifampicin and the streptogramin derivative RP 59500. In addition, in conventionally selected cells higher concentrations of chloramphenicol, doxycyclin, ofloxacin and trimethoprim than in the respective parental cells were necessary to inhibit the growth of L. monocytogenes. In contrast, when using genetically engineered cells, no significant differences were found for these antibiotics. These differences might be due to a higher expression of multidrug resistance in the conventionally selected cells because these cells were also more effective in excluding rhodamine 123 in a flow cytometric assay. In conclusion, expression of multidrug resistance by eukaryotic cells leads to a reduced concentration of macrolides and other antibiotics in these cells and to an impairment of activity against intracellular bacteria.

Effect of piroxicam on structure and function of joint cartilage.

In vitro experiments were performed on isolated articular chondrocytes under the influence of piroxicam. It was demonstrated that this non-steroidal anti-inflammatory agent affected neither cell proliferation nor the incorporation of 35SO4 into matrix macromolecules. Dogs were treated with piroxicam for 8 weeks. Morphological studies were performed on the tissues of the knee joints. Macroscopic and light microscopic investigations revealed no structural differences between the tissues (synovial membrane and articular cartilage) of control and treated dogs. Even at the ultrastructural level no alterations in the cartilage were observed. The capacity of chondrocytes to incorporate 35SO4 under in vitro conditions was identical in control and experimental animals. It is concluded that piroxicam has no adverse effect on chondrocytes under in vitro conditions or on articular cartilage structure in the in vivo model.

Histopathological study of experimental poststreptococcal pneumonia in mice. Group A, type 50, streptococcal infection of murine nares controls with Staphylococcus aureus and E. coli.

Microscopic methods (light and electron microscopy, histochemistry, immunohistochemistry) have been used to assess previously unknown pulmonary inflammatory responses of specific pathogen-free (SPF) mice secondary to infection via the nares by group A, type 50, streptococci suspended in saline ("strep group mice"). As controls for the strep group mice, the animals were either injected with saline alone via nares (no lesions were seen), or with Staphylococcus aureus in saline ("staph group mice") or with E. coli ("E. coli group mice"). The three different bacterial species caused clearly different histological changes in the lung. In the strep group mice, the microscopic findings were consistent with the diagnosis of lymphocytic interstitial pneumonia of bronchiolovascular bundles, secondary to exaggerated pulmonary recirculation of lymphocytes, concomitant with vasoconstrictive angiopathy of encased pulmonary artery branches and nodular inflammatory cell aggregates in lung parenchyma. These aggregates either consisted predominantly of lymphocytes, or of mixed cells (neutrophils, lymphocytes, macrophages) or of activated macrophages only. In 18 of 22 inflamed lungs of strep group mice, no bacteria could be cultured from lung tissue. In staph group mice the microscopic findings are consistent with the diagnosis of lymphocytic interstitial pneumonia of bronchiolovascular bundles, secondary to exaggerated pulmonary recirculation of lymphocytes only. In 12 of 17 inflamed lungs of staph group mice, no bacteria could be cultured from lung tissue. In E. coli group mice the microscopic findings were consistent with the diagnosis of distal terminal bronchiolitis and early pleural-based pneumonitis, in which lymphocytes and neutrophils mingled with macrophages. In 10 of 11 inflamed lungs of E. coli group mice, no bacteria could be cultured from lung tissue. The morphologic approaches described here may have potential for unravelling the complex inflammatory processes underlying different forms of interstitial and parenchymal pneumonia.

Distribution of orally administered azithromycin in various blood compartments.

The concentrations of azithromycin in whole blood, plasma, erythrocytes and polymorphonuclear leucocytes (PMNLs) were measured in 6 healthy volunteers following the last administration of a three-day regimen of 500 mg once daily. Marked enrichment of azithromycin was observed in PMNLs; the drug concentration amounted to 119 +/- 31 (SD) mg/l 6 hours after the administration. Twelve days thereafter 42 +/- 10 mg/l azithromycin was still measured in the PMNLs, although the drug was no longer demonstrable in plasma (< 0.02 mg/l). The elimination of azithromycin from the PMNLs (half-life 210 +/- 69 hours) was clearly slower than the elimination from plasma (half-life 93 +/- 70 hours). The maximal concentrations of azithromycin in plasma (0.64 +/- 0.27 mg/l) and erythrocytes (0.17 +/- 0.06 mg/l) were much lower and occurred earlier (tmax = 3 hours) than those observed in the PMNLs. The enrichment factor for azithromycin in PMNLs relative to plasma came to 177 +/- 92 at 3 hours or 1814 +/- 706 at 120 hours after the last administration. In a parallel in vitro study, the effect of accumulation of azithromycin in PMNLs on the intracellular survival of ingested staphylococci was investigated. At subinhibitory extracellular concentrations of azithromycin as low as 0.03 mg/l (MIC = 1 mg/l), a significant reduction in bacterial viability was observed, thus demonstrating antibacterial activity of the intracellularly enriched antibiotic.

Comparative pharmacokinetics of fluconazole and of itraconazole in Japanese and in German subjects.

In separate but identically designed studies the oral pharmacokinetics of 100 mg doses of the 2 antimycotics fluconazole and itraconazole were examined in Japanese and German subjects (both n = 12), both fasting and concomitant to a heavy breakfast. The results with the 2 races were compared. With fasting subjects no significant difference was found with either antimycotic for any parameter. With fluconazole the pharmacokinetic parameters after food were essentially the same for the 2 races. The only significant difference, a delay of 1.5 h in the median tmax in the Japanese relative to the Germans, should not be of practical importance. In contrast, the mean AUC for itraconazole in Japanese subjects after the meal was only 54.9% of the value with Germans (p < 0.05); the corresponding Cmax was only 54.7% (p < 0.01). As itraconazole is normally administered together with food, this suggests that there is a possibility of underdosage in Japanese subjects. There appears to be no such problem with fluconazole.

Pharmacokinetics of ampicillin/sulbactam in patients undergoing spinal microneurosurgical procedures.

The fixed combination of ampicillin (2 g)/sulbactam (1 g) was administered as perioperative prophylaxis at induction of anesthesia in 20 patients undergoing spinal microneurosurgery. It was noteworthy that after the short infusion ampicillin and sulbactam penetrated rapidly from blood into the different tissues affected by the surgical procedures. The following mean concentrations were measured in tissues: muscle 32.3+/-6.5 mg/kg ampicillin and 18.6+/-2.9 mg/kg sulbactam (11.1 min), ligament 39.5+/-11.1 mg/kg ampicillin and 25+/-6.5 mg/kg sulbactam (13.8 min), bone 12+/-3.6 mg/kg ampicillin and 7+/-0.8 mg/kg sulbactam (20.6 min), disk 10.2+/-3.3 mg/kg ampicillin and 7.3+/-1.8 mg/kg sulbactam (44.2 min). The mean time of sampling is given in brackets. For a period of at least 2 h the levels of both drugs measured in serum and in the different tissues were above the MICs for bacteria involved in postoperative wound infections. The administration of ampicillin/sulbactam apparently achieved sufficiently, high antibiotic concentrations, even in bradytrophic tissues such as ligament, bone, and disk, and seemed to meet the pharmacological criteria for perioperative prophylaxis in spinal microneurosurgery.

The uptake of fluconazole in finger and toe nails.

OBJECTIVE: The uptake of the antimycotic agent fluconazole in finger and toe nail following various treatment schedules was investigated in order to characterize the pharmacokinetic basis for the systemic treatment of onychomycosis with fluconazole. SUBJECTS: Between 8 and 12 healthy, male and female Caucasian subjects were included in four separate studies. Mean age of the subjects in the single studies ranged between 34 years (study 4, group 2; n = 4 male and 4 female) and 38 years (study 4, group 1; n = 4 male and 4 female). METHODS: Fluconazole was administered orally over 4 weeks in all studies. The treatment schedules were 150 mg once weekly (study 1), 300 mg once weekly (study 2), 50 mg once daily (study 3) and 150 or 300 mg once weekly in a parallel group study (study 4). At fixed times samples of blood, nail cuttings and nail dust were taken, up to two months after end of treatment. Fluconazole was analyzed in blood plasma and in the nail samples using a highly specific and sensitive gas chromatographic procedure. RESULTS: High concentrations of fluconazole were found in distal nail clippings with all three treatments. Mean maximum concentrations which occurred in the third or fourth week of treatment amounted to 2.1 microg/g (150 mg/w), 5.4 microg/g (300 mg/w) and 6.5 microg/g (50 mg/d) in finger nails and to 9.6 microg/g (150 mg/w), 12.3 microg/g (300 mg/w) and 12.2 microg/g (50 mg/d) in toe nails. The nail concentrations were 1-2 times (finger) and 2-3 times (toe) higher than the corresponding fluconazole plasma levels and were within the MIC range for dermatophytes and yeasts occurring commonly in onychomycosis. The residence times of fluconazole in the nail plate after the end of treatment was long, with approximate half-lives of 33 days in finger nail and 30 days in toe nail. In pharmacokinetic terms there was no evidence of advantages of the daily dosage (50 mg) over the once-weekly (300 mg) dosage. Fluconazole was found to penetrate into both finger and toe nails at a very fast rate. On the first two days of the 150 mg/w and 300 mg/w treatments, i.e. after the first dosage, fluconazole concentrations in the distal nail plates amounted to 50-80% of the later observed peak levels. The initial concentrations in the upper dorsal plate were particularly high, with mean peak concentrations of 11.9 microg/g (150 mg) and 33.7 microg/g (300 mg) in finger nails and 5.7 microg/g (150 mg) and 24.4 microg/g (300 mg) in toe nails. CONCLUSIONS: Fluconazole is rapidly and highly distributed into finger and foot nail, reaching there higher concentrations than in the plasma. The rapid initial uptake of fluconazole in nail, which is unlike the uptake of other antifungal agents, suggests the existence of special routes of access to the nail for fluconazole, possibly based on high diffusion rates.

Influence of concomitant food intake on the gastrointestinal absorption of fluconazole and itraconazole in Japanese subjects.

The effect of food intake on the pharmacokinetics of orally administered fluconazole or itraconazole was investigated in a single-dose randomized two-way crossover study in Japanese subjects. 100-mg capsules of fluconazole or itraconazole were given to two parallel groups, each of 12 male and female volunteer subjects, and plasma concentrations of the antimycotics were determined by specific assays. Gastric pH and gastric emptying times were measured by coadministration of a radiotelemetric pH capsule. Intersubject variations in drug plasma concentrations were found to be much higher with itraconazole than with fluconazole. The coefficient of variation of the AUC (0-72) after food amounted to +/- 62% for itraconazole and +/- 16% for fluconazole. The consumption of a heavy breakfast significantly delayed the tmax of both drugs by approximately two hours (p < 0.05). This effect was accompanied by a significant prolongation of gastric emptying times (p < 0.0001). Food intake had essentially no effect on the absorbed amounts of fluconazole. The median relative bioavailability (postprandial vs fasting) based on AUC (0-72) was f = 0.99, with an individual range from 0.72 to 1.15. The corresponding Cmax ratio was 1.04 (range 0.91-1.17). The effect of food on the bioavailability of itraconazole was highly variable: it ranged from marked reductions (f = 0.35) to large increases (f = 3.74) of the AUC (0-72), at a median ratio of f = 1.23. The Cmax ratios postprandial vs fasting ranged between 0.27 and 5.71 (median = 1.04). It is concluded that food had an unpredictable effect on the extent of itraconazole absorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Relative bio-availability of sultamicillin in healthy volunteers following administration of two tablet formulations.

The pharmacokinetics of both ampicillin and sulbactam obtained from a balanced, open two-way crossover study in 20 normal adult volunteers receiving a single 375 mg oral tablet of sultamicillin administered as two different formulations--the original product (Duocid) and a generic formulation that is commercially available in Turkey--were compared. Pharmacokinetic parameters for the two formulations and for both ampicillin and sulbactam were tested for bio-equivalence by the two one-sided Student's t-test (80-120% range; P < 0.1). Area under concentration--time curves and maximum concentrations for both components were found to be non-equivalent for the two formulations, the generic formulation having consistently lower mean values. The results were consistent with studies of the in vitro release of sultamicillin from the two tablets. It is concluded that the generic formulation is pharmacokinetically inferior to the original product.

[Fluconazole level in aqueous humor after oral drug administration in humans]. / Fluconazolspiegel im Kammerwasser nach oraler Wirkstoffgabe beim Menschen.

For 2 years fluconazole, a triazole antimycotic, has been available for treatment of systemic mycosis. Compared to amphotericin B fewer severe side effects have been reported. So far, no data have been published as to its penetration into the human eye. In the present study, 20 cataract patients were given 200 mg fluconazole (0.5 to 8 h preoperatively. During the cataract operation 0.1 ml of the aqueous was removed as well as 10 ml serum. With the help of high-pressure liquid chromatography (HPLC), the concentration of fluconazole in each of the samples was determine. If the aqueous humor was removed at least 2h after fluconazole application, concentrations between 2.7 and 5.4 micrograms/ml were reached (mean 3.7 +/- 2.17) In these cases the concentration in the aqueous humor was 80% of the concentration found in the serum at the same time. If the sample of the aqueous humor was collected only 1 h after application, 40% of the concentration in the serum was found in the aqueous humor. These data prove that fluconazole shows an extremely good penetration through the blood-aqueous barrier. After a single dose of 200 mg, a concentration is reached in the eye that surmounts the minimal inhibiting concentration found for Candida species sensitive to fluconazole. Therefore, fluconazole seems to be a good alternative to amphotericin B for the treatment of infections caused by such fungi.

Effects of non-steroidal anti-inflammatory drugs on human leucocytes.

The action of 4 non steroidal anti-inflammatory drugs (NSAID) on the function of human polymorphonuclear neutrophils (PMN), monocytes and lymphocytes has been investigated and compared to that of prednisolone. Benoxaprofen and diclofenac inhibited the chemotaxis of leucocytes in a dose-dependent fashion in vitro whereas acetylsalicylic acid and indomethacin were inactive or effective on the migration of these cells at high concentrations only. Benoxaprofen did not reduce significantly (variance analysis, p = 0.05) the chemotactic locomotion of PMN or monocytes isolated from volunteers after repeated oral administration of the drug. The other function of the leucocytes investigated, which are essential for the defense of the host, were not suppressed by NSAID at therapeutic concentrations. From these experimental results it was concluded that Nsaid have a differing spectrum of biological activities and can act selectively on functions of leucocytes. However, the relevance of these findings to the effect of drugs on the inflammatory process is still unclear.

Effect of anti-rheumatic drugs on the release of lysosomal enzymes from human leukocytes.

The release of lysosomal enzymes from human polymorphonuclear neutrophils (PMN) into the extracellular medium was selectively induced by the phagocytosis of zymosan particles. The "in vitro" effect on the release of lysosomal enzymes was determined for eight different substances used in the therapy of rheumatoid arthritis. Prednisolone and the three non-steroidal anti-rheumatic agents indomethacin, diclofenac and benoxaprofen only inhibited the release of various lysosomal enzymes at the elevated concentrations of 20-200 micrograms/ml. Of the four drugs examined which are used in basis therapy, Chloroquine and Levamisole exhibited the most clear-cut inhibition of the enzymes (at 20-200 micrograms/ml). The effect of gold salts was uncertain and D-penicillamine only inhibited the release of myeloperoxidase. The release of lysosomal enzymes from neutrophils is then largely resistant to anti-rheumatic agents--at least in the range of therapeutic concentrations. On the basis of these experimental results it seems questionable whether the relatively weak inhibition of lysosomal enzymes observed "in vitro" can contribute to the anti-rheumatic efficacy in patients of the drugs examined.

Action of antirheumatic drugs on the function of human leucocytes.

The effect of 4 antirheumatic drugs, used in the basic therapy of patients with rheumatoid arthritis, on the cellular function of human leucocytes has been studied in vitro. Chloroquine and gold salt aurothioglucose inhibited in a dose-related manner the chemotaxis of polymorphonuclear neutrophils (PMN) and monocytes at therapeutic concentrations. D-Penicillamine only reduced migration of monocytes, but not that of PMN. Chloroquine has been found to suppress the phagocytosis and the microbicide effect of PMN, the transformation of T-lymphocytes and the chemotactic locomotion of the cells in similar concentrations. On the contrary, the concentrations of the gold salt and D-penicillamine inhibitory in these activities were substantially higher than those required for prevention of chemotaxis in vitro. Levamisole, known for immunostimulatory properties in patients, reduced the responsiveness of the leucocytes only at the highest dosage. The relevance of these experimental findings for the therapeutic approach and for their interaction with the host's defense mechanisms has been discussed.

[The effects of plant preparations on cellular functions in body defense]. / Untersuchung des Einflusses von Phytopräparaten auf zelluläre Funktionen der körpereigenen Abwehr.

Two preparations of Echinacea purpurea and a preparation of Eleutherococcus senticosus increased the in vitro phagocytosis of Candida albicans by granulocytes and monocytes from healthy donors by 30-45%. The chemotactic migration of granulocytes in the Boyden Chamber was increased by 45% with an Echinacea purpurea extract. The two herbal preparations had no effect in either direction on intracellular killing of bacteria or yeasts. Echinacea and Eleutherococcus preparations did not induce in vitro transformation of lymphocytes. The mistel toe preparation examined (Viscum album) did not influence the tested functions of granulocytes, monocytes or lymphocytes of healthy donors.

[Stability of beta-lactamase inhibitors and beta-lactam antibiotics in parenteral formulations as well as in body fluids and tissue homogenates. Comparison of sulbactam, clavulanic acid, ampicillin and amoxicillin]. / Stabilität von beta-Lactamase-Inhibitoren und beta-Lactam-Antibiotika in parenteralen Darreichungsformen sowie in Körperflüssigkeiten und Gewebehomogenaten. Vergleichende Untersuchung von Sulbactam, Clavulansäure, Ampicillin und Amoxicillin.

Stability of beta-Lactamase Inhibitors and beta-Lactam Antibiotics in Parenteral Formulations as Well as in Body Fluids and Tissue Homogenates/Comparative studies with sulbactam, clavulanic acid, ampicillin and amoxicillin. The beta-lactamase inhibitors and the beta-lactam antibiotics are markedly different in chemical stability. The comparative examination of 4 different infusion solutions at 4 degrees C, 25 degrees C and 37 degrees C gives the following sequence of decreasing stability: sulbactam (CAS 68373-14-8), ampicillin (CAS 69-53-4), amoxicillin (CAS 61336-70-7) and clavulanic acid (CAS 58001-44-8). It is particularly striking that the two beta-lactamase inhibitors, sulbactam and clavulanic acid, behave very differently. Sulbactam is also much more stable than clavulanic acid to incubation at 37 degrees C in body fluids or in tissue homogenates. The differences in the stability of the individual drugs should be born in mind during clinical use of combination formulations such as sulbactam/ampicillin (Unacid) and clavulanic acid/amoxicillin.

Intracellular activity of azithromycin against Mycobacterium avium complex in human macrophages.

In the concentration range examined (0.5-16 micrograms/ml) the azalide antibiotic azithromycin (CAS 83905-01-5, Zithromax) inhibited the growth of mycobacteria in macrophages over 7 days. The higher concentrations of azithromycin, 8 and 16 micrograms/ml, reduced the number of phagocytized bacteria in macrophages by at least 1 log unit within 4 days. The system used, macrophages from healthy volunteers, is suitable for testing the intracellular activity of drugs against the Mycobacterium avium complex.

Kinetics of the uptake of antimicrobial agents by human polymorphonuclear leucocytes.

The in-vitro rate constants of the cellular uptake and elimination of the antimicrobial agents josamycin (Wilprafen), erythromycin and tetracycline were measured in normal human polymorphonuclear leucocytes (PMNs) using the velocity gradient centrifugation technique with radiolabelled drugs at extracellular concentrations corresponding to therapeutically effective serum levels. The rate of antibiotic uptake increased stepwise in the order tetracycline less than erythromycin less than josamycin. The half-lives of the uptake came to 0.8 min for josamycin, 4.7 min for erythromycin, and 14.8 min for tetracycline. The extraordinarily rapid uptake of josamycin by PMNs corresponds to the high lipophility of the drug. The accumulation of the three tested antibiotics in PMNs occurred much faster than their elimination from the cells, suggesting directional transport of the molecules through the leucocyte membrane and/or rate limiting dissociation from intracellular binding sites. Significant differences between the drugs tested were observed in the temperature dependence of their rates of uptake. The apparent activation energies of cellular uptake amounted to 114.2 kJ mol-1 (josamycin), 68.6 kJ mol-1 (erythromycin) and 52.2 kJ mol-1 (tetracycline). There is experimental support for a contribution of the nucleoside carrier system to the membrane transport of josamycin.