Functional studies on the activity of efflux transporters in an ex vivo model with chicken splenocytes and evaluation of selected fluoroquinolones in this model

The efflux proteins P-glycoprotein (P-gp), BCRP and members of the MRP-family (MRPs) are increasingly recognized as determinants of the absorption, tissue distribution and excretion of numerous drugs. A widely applied in vitro screening method, to assess the effect of these efflux transporters in transmembrane transport of drugs is based on the use of peripheral blood mononuclear cells (PBMC), in which the efflux of fluorescent dye Rhodamine 123 (Rh-123) can be easily measured. In avian species, the isolation of PBMCs is compromised by the presence of thrombocytes having approximately the same size. As an alternative, we validated the use of isolated splenocytes to assess Rhodamine 123 transport in the presence and absence of specific inhibitors for P-gp, MRPs and BCRP. Rh-123 efflux was concentration-dependent with the percentage of efflux that decreased with increasing concentrations. P-gp inhibitors, PSC833 and GF120918, significantly inhibit Rh-123 efflux, whereas inhibitors for MRPs and BCRP, MK571 and Ko-143, respectively, have a limited inhibitory effect. However, the effect of GF120918 was more pronounced as compared to PSC833, suggesting an additional role for BCRP next to P-gp in Rh-123 efflux. Moreover, fluoroquinolones were selected to test the applicability of the described model. None of these fluoroquinolones significantly inhibit P-gp function at concentrations up to 50 microM, with exception of danofloxacin and danofloxacin mesylate that were found to reduce Rh-123 efflux by approximately 15%.     

Pharmacokinetic-pharmacodynamic indices of enrofloxacin in Escherichia coli O78/H12 infected chickens

Aim of the study was to evaluate the effect of quercetin and enrofloxacin with/without quercetin on elimination of pathogen Escherichia coli O78/H12 in infected chickens. Effect of quercetin on disposition of enrofloxacin was investigated and Pharmacokinetic-pharmacodynamic indices were calculated.Enrofloxacin was absorbed after oral administration in infected animals but with large inter-individual variations. Low concentrations of its main metabolite, ciprofloxacin, were found which could be explained with marked reduction of enrofloxacin transformation in infected animals. Quercetin significantly decreased bioavailability of enrofloxacin and its transformation to ciprofloxacin. Lower formation of metabolite was also found in the studied tissues as spleen, heart, lungs and in liver of group treated in combination with quercetin. Results in infected and quercetin (50 mg/kg) treated group shows lower percentage of re-isolates of the pathogen bacteria in comparison to infected and untreated animals, and close to the low dose (10 mg/kg) of enrofloxacin. High dose of enrofloxacin given in a short time in an infection model with high inoculum size, resulted in better eradication of bacteria although re-isolates could be found in spleen. Additional improvement of the outcome of fluoroquinolone therapy could be searched in early start of drug administration according to the terms of metaphylaxis.     

Brain metastases detectability of routine whole body (18)F-FDG PET and low dose CT scanning in 2502 asymptomatic patients with solid extracranial tumors

As fluorine-18-fluorodesoxyglucose positron emission tomography/computed tomography ( (18)F-FDG PET/CT) is gaining wider availability, more and more patients with malignancies undergo whole body PET/CT, mostly to assess tumor spread in the rest of the body, but not in the brain. Brain is a common site of metastatic spread in patients with solid extracranial tumors. Gold standard in the diagnosis of brain metastases remains magnetic resonance imaging (MRI). However MRI is not routinely indicated and is not available for all cancer patients. Fluorine-18-FDG PET is considered as having poor sensitivity in detecting brain metastases, but this may not be true for PET/CT. The aim of our study was to assess the value of (18)F-FDG PET/CT in the detection of brain metastases found by whole body scan including the brain, in patients with solid extracranial neoplasms. A total of 2502 patients with solid extracranial neoplasms were studied. All patients underwent a routine whole body (18)F-FDG PET/CT scan with the whole brain included in the scanned field. Patients with known or suspected brain metastases were preliminary excluded from the study. Hypermetabolic and ring-like brain lesions on the PET scan were considered as metastases. Lesions with CT characteristics of brain metastases were regarded as such irrespective of their metabolic pattern. Lesions in doubt were verified by MRI during first testing or on follow-up or by operation. Our results showed that brain lesions, indicative of and verified to be metastases were detected in 25 out of the 2502 patients (1%), with lung cancer being the most common primary. Twenty three out of these 25 patients had no neurological symptoms by the time of the scan. The detection rate of brain metastases was relatively low, but information was obtained with a minimum increase of radiation burden. In conclusion, whole body (18)F-FDG PET/CT detected brain metastases in 1% of the patients if brain was included in the scanned field. Brain scanning as a part of whole body scan cannot replace routine imaging techniques, but in case of positive findings provides early and crucial information for further patient management, especially in asymptomatic patients.     

Effects of fluoroquinolone treatment on MDR1 and MRP2 mRNA expression in Escherichia coli-infected chickens

Current knowledge about the expression of ABC transport proteins suggests that their expression is regulated by a variety of factors, including pathological conditions, and in particular inflammatory reactions to infection. As ABC transporters are major determinants of absorption, distribution and excretion of many antimicrobials, modulation of their activity may result in increased or decreased tissue levels of drugs, affecting the efficacy of treatment. As fluoroquinolones have been identified as modulators and substrates of a number of drug transporters, we evaluated the effect of danofloxacin mesylate and enrofloxacin treatment on the levels of expression of MDR1 and MRP2 mRNAs in the intestines and livers of broilers with experimentally induced colibacillosis. MDR1 mRNA expression was significantly decreased in infected animals and was partly restored over 5 days of treatment with orally administered danofloxacin mesylate or enrofloxacin. Changes in the level of expression of MRP2 mRNA were less prominent. The study suggests that the treatment of colibacillosis with fluoroquinolones, which resulted in a significant clinical improvement of the animals, also restored the expression of drug transporters. This is of clinical importance as these ABC transporters significantly contribute to the functionality of important biological barriers, protecting the bird and specific tissues from pathogens and bacterial toxins.     

The effect of continuous positive airway pressure on heart rate variability during the night in patients with chronic heart failure and central sleep apnoea

Continuous positive airway pressure (CPAP) improves autonomic activity in patients with chronic heart failure (CHF) and central sleep apnoea (CSA), but its effect on heart rate variability (HRV) during therapy has not been reported. We hypothesized that CPAP may decrease HRV, despite its beneficial effects on sympathetic overactivation, due to the expected stabilization of breathing. Sixty‐seven CHF patients underwent polysomnography (PSG). Ten of them presented with CSA (age 66.1±8.5 years, apnoea‐hypopnea index [AHI]=57.6±23.3, central AHI [cAHI]=41.6±24.6 [mean±SD]) and were subjected to a second PSG with manual CPAP titration. Beat‐to‐beat heart intervals for a 6‐hour period of sleep were extracted from each recording and HRV was analysed. CPAP significantly reduced AHI (AHI=23.1±18.3 P=.004). Standard deviation of normal‐normal interbeat interval (SDNN) (61.5±29.0 vs 49.5±19.3 ms, P=.021), root mean square of successive differences (RMSSD) (21.8±9.2 vs 16.4±7.1 ms, P=.042), total power (lnTP=7.8±1.1 vs 7.4±0.8 ms², P=.037), low frequency power (lnLF=5.5±1.5 vs 5.0±1.4 ms², P=.003) and high frequency power (lnHF=4.6±1.0 vs 4.0±1.0 ms², P=.024) were decreased. There was a strong correlation between the decrease in AHI and the decrease in lnHF (Spearman's ρ=.782). CPAP leads to a decrease in spectral and time domain parameters of HRV during therapy in CHF patients with CSA. These changes are best explained by the effect which CPAP‐influenced breathing pattern and lowered AHI exert on HRV.     

A guide for functional analysis of BRCA1 variants of uncertain significance

Germline mutations in the tumor suppressor gene BRCA1 confer an estimated lifetime risk of 56–80% for breast cancer and 15–60% for ovarian cancer. Since the mid 1990s when BRCA1 was identified, genetic testing has revealed over 1,500 unique germline variants. However, for a significant number of these variants, the effect on protein function is unknown making it difficult to infer the consequences on risks of breast and ovarian cancers. Thus, many individuals undergoing genetic testing for BRCA1 mutations receive test results reporting a variant of uncertain clinical significance (VUS), leading to issues in risk assessment, counseling, and preventive care. Here, we describe functional assays for BRCA1 to directly or indirectly assess the impact of a variant on protein conformation or function and how these results can be used to complement genetic data to classify a VUS as to its clinical significance. Importantly, these methods may provide a framework for genome‐wide pathogenicity assignment. Hum Mutat 33:1526–1537, 2012. © 2012 Wiley Periodicals, Inc.