Angela Burian

Impact of pH on bacterial growth and activity of recent fluoroquinolones in pooled urine

Acidification of urine is widely recommended for prevention and treatment of urinary tract infections. We set out to describe the effect of modification of pH on bacterial growth of relevant bacteria as well as on activity of modern fluoroquinolones in urine in vitro. Bacterial growth of Escherichia coli ATCC 25922 and Klebsiella oxytoca ATCC 700324 was determined in pooled human urine adjusted to pH levels between 5.0 and 8.0. Minimal inhibitory concentrations (MICs) and time-kill curves were performed for ciprofloxacin, levofloxacin and moxifloxacin in pH-adjusted urine and Mueller-Hinton Broth (MHB). Uptake of radioactive labeled [C(14)]-ciprofloxacin into bacterial cells was investigated at different pHs. While no difference in bacterial growth of E. coli and K. oxytoca was observed at pH values between 5.0 and 8.0, acidification of urine led to major impairment of antimicrobial activity of all tested fluoroquinolones, indicated by an up to 40-fold increase in MIC compared to MHB and nearly total neutralization of activity in time-kill experiments. The most probable mechanism behind this observation may have been reduced uptake of fluoroquinolones into bacterial cells, as indicated by bacterial uptake of [C(14)]-ciprofloxacin and a reversibility of the effect. The observed reduction in activity of modern fluoroquinolones confirms previous observations from older compounds.

Absolute oral bioavailability and metabolic turnover of β-sitosterol in healthy subjects

The metabolic turnover, absolute oral bioavailability, clearance, and volume of distribution for β-sitosterol were measured in healthy subjects. [14C]β-Sitosterol was used as an isotopic tracer to distinguish pulse doses from dietary sources and was administered by both oral and intravenous routes. The administered doses of [14C]β-sitosterol were in the region of 3 to 4 μg, sufficiently low as not to perturb the kinetics of β-sitosterol derived from the diet. Because the plasma concentrations of [14C]β-sitosterol arising from such low doses were anticipated to be very low, the ultrasensitive isotope ratio analytical method of accelerator mass spectrometry was used. The limit of quantification for [14C]β-sitosterol was approximately 0.1 pg/ml, the oral absolute bioavailability was just 0.41%, clearance was 85 ml/h, volume of distribution was 46 L, and the turnover was 5.8 mg/day. Given the steady-state concentrations of β-sitosterol (2.83 μg/ml), then the dietary load was calculated to be approximately 1400 mg/day.

Simultaneous assessment of the pharmacokinetics of a pleuromutilin, lefamulin, in plasma, soft tissues and pulmonary epithelial lining fluid.

BACKGROUND Lefamulin is a pleuromutilin antibiotic under evaluation for the treatment of bacterial infections, including respiratory tract infections. Currently, there are no high-quality pharmacokinetic data on drug tissue concentrations of lefamulin available. METHODS A single dose of intravenous lefamulin (150 mg) was given to 12 healthy men. The registered EudraCT number for this study was 2010-021938-54. Lefamulin concentrations were simultaneously measured in plasma, skeletal muscle tissue, subcutaneous adipose tissue and epithelial lining fluid (ELF) over 24 h, and corresponding pharmacokinetic parameters were calculated. Microdialysis was used to measure unbound lefamulin concentrations in skeletal muscle tissue and subcutaneous adipose tissue, which were similar to unbound lefamulin concentrations in plasma. Bronchoalveolar lavage was performed 1, 2, 4 and 8 h post-dose to determine lefamulin concentrations in ELF. RESULTS Unbound lefamulin levels showed a 5.7-fold higher exposure in ELF compared with that in plasma, demonstrating good penetration to the target site. CONCLUSIONS Lefamulin may be an addition to the therapeutic armamentarium for the treatment of infections. Simultaneous measurements of unbound drug concentration can guide target attainment for future therapeutic trials.

Impact of pH on Activity of Trimethoprim, Fosfomycin, Amikacin, Colistin and Ertapenem in Human Urine

Objective: Although major impairment of activity at lower pH values has been reported for fluoroquinolones, acidification is a widely recommended practice for the prophylaxis and treatment of uncomplicated urinary tract infections (UTIs). Until now, there is little evidence for the influence of pH on the activity on other antimicrobial classes in urine. Methods: Bacterial growth curves of Staphylococcus aureus (ATCC 29213), Klebsiella oxytoca (ATCC 700324), Proteus mirabilis (ATCC 14153), Escherichia coli (ATCC 25922) and Enterococcus faecalis (ATCC 29212) were performed in Mueller-Hinton broth and in pooled human urine with a pH of between 5.0 and 8.0. Bacterial killing of trimethoprim, fosfomycin, amikacin, colistin and ertapenem against the five strains (where appropriate) was determined consecutively at concentrations equal to the MIC. Results: While no difference in the bacterial growth of E. coli, S. aureus, P. mirabilis and K. oxytoca was observed at different pH values, bacterial growth of E. faecalis was significantly reduced at low pH. Acidification to pH 5 impaired the antimicrobial activity of all investigated antibiotics, i.e. the net effect of bacterial growth and killing resulted in increased colony-forming units/ml at the end of the experiment. Conclusion: The present in vitro findings indicate that acidification of urine during the treatment of UTIs should be carefully considered. While growth curves of one strain supports the concept of therapeutic or prophylactic acidification during UTIs, the most common pathogen, E. coli, was not affected by low pH. Independent of the investigated strain or antibiotic, pH values below 6 lead to a reduction of antimicrobial activity.

Target site pharmacokinetics of linezolid after single and multiple doses in diabetic patients with soft tissue infection

The underlying pathology of diabetic wounds, i.e. impairment of macro‐ and microcirculation, might also impact target site penetration of antibacterial drugs. To compare tissue concentrations of linezolid in infected and not infected tissue 10 patients suffering from type 2 diabetes with foot infection were included in the study. Tissue penetration of linezolid was assessed using in vivo microdialysis at the site of infection as well as in non‐inflamed subcutaneous adipose tissue. All patients were investigated after receiving a single dose of linezolid and five patients in addition at steady state. After a single dose of linezolid significantly higher area under the concentration vs. time curve over 8 hours (AUC0–8) and maximum concentrations (Cmax)‐values were observed in plasma (65.5 ± 21.2 mg*h/L and 16.4 ± 4.6 mg/L) as compared to inflamed (36.3 ± 22.9  mg*h/L and 6.6 ± 3.6 mg/L) and non‐inflamed tissue (33.0 ± 17.7 mg*h/L and 6.7 ± 3.6 mg/L). Multiple administrations of linezolid led to disappearance of significant differences in Cmax and AUC0–8 between plasma, inflamed, and non‐inflamed tissue. Approximately 2‐fold increase of Cmax and AUC0–8‐values in tissue was observed at steady state as compared to the first administration. Penetration of linezolid is not impaired in diabetic foot infection but equilibrium between plasma and tissue might be delayed.

Antimicrobial activity of antibiotics in urine under different physiological conditions

Methods Urine obtained from healthy volunteers was pooled and sterile filtered. Microdilution tests were performed with Escherichia coli ATCC 25922 in MHB and human urine. The pH of urine was adjusted to values ranging from 5 to 8. For simulating different glucose levels in urine of diabetic patients, urine was adjusted to glucose levels of 100 and 1000 mg/dL. Bacterial growth in different media was investigated by growth curves. Results obtained from MIC testing were confirmed by use of bacterial killing curves. Klebsiella oxytoca ATCC 700324 was used to investigate transferability of finding to other strains. Each experiment was performed 5 times.

A randomised, two-period, cross-over, open-label study to evaluate the pharmacokinetic profiles of single doses of two different flurbiprofen 8.75-mg lozenges in healthy volunteers.

Aims: To compare the bioavailability of a new oromucosal formulation of flurbiprofen 8.75-mg lozenges, developed by Alfa Wassermann S.p.A. (test drug) to that of marketed flurbiprofen 8.75-mg lozenges (Benactiv Gola®, reference drug). Methods: This was an open, randomised, two-period, crossover, pharmacokinetic (PK) study in which flurbiprofen plasma levels were compared in 12 healthy volunteers after the administration of single doses (8.75 mg × 2) of two different oromucosal lozenges to be sucked and slowly dissolved in the mouth. A wash-out period of at least 7 days separated the two study periods. Blood samples were collected prior to dosing and at predefined intervals for 24 h after dose. Flurbiprofen plasma concentrations were determined by liquid chromatography/tandem mass spectrometry. PK parameters maximum plasma concentration (Cmax), time to maximum plasma concentration (Tmax), area under the plasma concentration-time curve from time zero to 24 hours (AUC0–t), area under the plasma concentration-time curve from time zero to infinity (AUC0–∞) and half-life were calculated and compared by analysis of variance using treatment, period and sequence as sources of variation. Bioequivalence between the two formulations was based on 90% confidence intervals of the ratio of the geometric means of Cmax and AUC falling within the 0.80–1.25 range as defined in bioequivalence guidelines by regulators. Tolerability of the two formulations was assessed by adverse event monitoring, routine laboratory tests, physical examination, electrocardiographic tracing and vital sign measurements. Results: All enrolled subjects completed the study. Bioequivalence without significant treatment effect was demonstrated between the test drug/reference drug ratios of mean Cmax and AUCs. Moreover, mean Tmax was superimposable. No safety parameter presented a clinically relevant variation after administration of either formulation that were therefore well tolerated. Conclusion: The new formulation of flurbiprofen 8.75-mg compressed lozenges developed by Alfa Wassermann S.p.A. is bioequivalent to the reference product flurbiprofen 8.75-mg lozenges (Benactiv Gola) in healthy volunteers.

Understanding the Activity of Antibiotics in Cerebrospinal Fluid in vitro.

Background: In vitro studies suggest that antimicrobial activity of antibiotics meant to treat central nervous system infections such as meningitis or ventriculitis may be altered by cerebrospinal fluid (CSF). This could explain the reason behind the often observed discrepancies between the activity of antibiotics determined in artificial growth media in vitro, and their sometimes reduced clinical efficacy in CSF in vivo. If conducted in CSF, in vitro microbiological investigations might predict the ability of antibiotic drugs to treat CSF infections better than experiments in artificial growth media. In addition, they are less expensive, critical and time consuming than animal studies, and might potentially be appreciated in drug development as a rapid and cost-effective means to gain valuable information on drugs meant to treat infections residing in CSF. Summary: Data from microbiological in vitro experiments performed in CSF were compiled for fosfomycin, rifampicin, cefepime, cefotaxime, ceftriaxone, ciprofloxacin, gentamicin and vancomycin. Where possible, correlations between in vitro data and evidence from in vivo studies were established. Key Messages: As discussed in the text, no clear correlations between in vitro studies in CSF and clinical outcomes could be identified. Methodological recommendations derived from the collected studies are summarized in order to optimize future research on the topic.

An exploratory microdialysis study investigating the effect of repeated application of a diclofenac epolamine medicated plaster on prostaglandin concentrations in skeletal muscle after standardized physical exercise

AIM Muscle injuries and extensive exercise are associated with cyclo-oxygenase dependent formation of inflammatory prostaglandins. Since the effect of topical administration of non-steroidal anti-inflammatory drugs (NSAIDs) on local cyclo-oxygenase is unknown, the present exploratory, open label, non-randomized study set out to measure exercise induced release of prostaglandins before and after epicutaneous administration of diclofenac. METHODS Microdialysis was used to determine the local interstitial concentration of PGE2 and 8-iso-PGF2α as well as diclofenac concentrations in the vastus lateralis under rest, dynamic exercise and during recovery in 12 healthy subjects at baseline and after a treatment phase applying a total of seven plasters medicated with 180 mg of diclofenac epolamine over 4 days. RESULTS At baseline PGE2 concentrations were 1169 ± 780 pg ml(-1) at rest and 1287 ± 459 pg ml(-1) during dynamic exercise and increased to 2005 ± 1126 pg ml(-1) during recovery. After treatment average PGE2 concentrations were 997 ± 588 pg ml(-1) at rest and 1339 ± 892 pg ml(-1) during exercise. In contrast with the baseline phase no increase in PGE2 concentrations was recorded during the recovery period after treatment (PGE2 1134 ± 874 pg ml(-1)). 8-iso-PGF2α was neither affected by exercise nor by treatment with diclofenac. Local and systemic concentrations of diclofenac were highly variable but comparable with previous clinical pharmacokinetic studies. CONCLUSIONS We can hypothesize an effect of topical diclofenac epolamine plaster on limiting the increase of local concentrations of the pro-inflammatory prostaglandin PGE2 induced in the muscle of healthy human subjects following standardized physical exercise. No effect of diclofenac treatment on 8-iso-PGF2α concentrations was observed, mainly since isoprostane is produced by a free radical-catalyzed lipid peroxidation mechanism independent of cyclo-oxygenases.

A mysterious case of complex regional pain syndrome in a 9-year-old girl

Abstract Purpose: To describe the clinical manifestation and the treatment of complex regional pain syndrome type II in childhood. Methods: Using information on the symptoms, diagnosis, rehabilitation and outcome of a young patient with complex regional pain syndrome type II. Results: A 9-year -old girl had severe pain in the region of the left foot, signs of a common fibular nerve entrapment, hyperalgesia not limited to the distribution of the injured nerve, weakness and temperature asymmetry unknown origin. She consulted few doctor’s before she was given the right diagnosis of complex regional pain syndrome type II. Following the diagnosis the treatment started, it included intensive physiotherapy, electrical therapy and also supportive psychological therapy. Half a year later, the patient was free of the daily pain and returned to all physical activity without any restrictions. Conclusions: The case report illustrates that peripheral nerve compression or injuries specifically, complex regional pain syndrome type II, should be taken into consideration when evaluating children with weakness and pain of the lower or upper limb. Implication of rehabilitation Raising the awareness of complex regional pain syndrome in the childhood is essential for an early diagnosis and appropriate treatment. The treatment options include early and adequate pain management inclusive electrical therapy and physiotherapy. Psychological therapy helps to avoid psychological stress reaction and the disease negative impact on the child’s education and sports and the family social life.