Isolation of MDCK cells with low expression of mdr1 gene and their use in membrane permeability screening

The Madin-Darby canine kidney (MDCK) cell line is frequently used for permeability screening in drug discovery. It contains endogenous transporters, most prominently canine multidrug resistance P-glycoprotein (Mdr1), which can interfere with studies of P-glycoprotein substrate assessment and permeability measurements. Because MDCK wild type (WT) is genetically heterogeneous, an isolation procedure was investigated in this study to obtain the subclonal line with low P-glycoprotein expression. The best clone obtained had up to 3-fold lower amprenavir efflux and P-glycoprotein expression in comparison to WT. Of 12 standard compounds tested that exhibited active efflux in WT cells, 11 showed a decrease in efflux in the isolated clone. However, the decrease was not below the cut-off value of 2, indicating residual P--glycoprotein activity. Clone isolation via the limiting dilution method, combined with bidirectional amprenavir permeability for clone selection, successfully identified MDCK clones with substantially lower P-glycoprotein efflux and has been demonstrated as a useful tool for assessing passive permeability in early drug discovery.

Expression and Function of ABC Proteins in Fish Intestine

In fish, the intestine is fundamental for digestion, nutrient absorption, and other functions like osmoregulation, acid-base balance, and excretion of some metabolic products. These functions require a large exchange surface area, which, in turn, favors the absorption of natural and anthropogenic foreign substances (xenobiotics) either dissolved in water or contained in the food. According to their chemical nature, nutrients, ions, and water may cross the intestine epithelium cells’ apical and basolateral membranes by passive diffusion or through a wide array of transport proteins and also through endocytosis and exocytosis. In the same way, xenobiotics can cross this barrier by passive diffusion or taking advantage of proteins that transport physiological substrates. The entry of toxic substances is counterbalanced by an active efflux transport mediated by diverse membrane proteins, including the ATP binding cassette (ABC) proteins. Recent advances in structure, molecular properties, and functional studies have shed light on the importance of these proteins in cellular and organismal homeostasis. There is abundant literature on mammalian ABC proteins, while the studies on ABC functions in fish have mainly focused on the liver and, to a minor degree, on the kidney and other organs. Despite their critical importance in normal physiology and as a barrier to prevent xenobiotics incorporation, fish intestine’s ABC transporters have received much less attention. All the ABC subfamilies are present in the fish intestine, although their functionality is still scarcely studied. For example, there are few studies of ABC-mediated transport made with polarized intestinal preparations. Thus, only a few works discriminate apical from basolateral transport activity. We briefly describe the main functions of each ABC subfamily reported for mammals and other fish organs to help understand their roles in the fish intestine. Our study considers immunohistochemical, histological, biochemical, molecular, physiological, and toxicological aspects of fish intestinal ABC proteins. We focus on the most extensively studied fish ABC proteins (subfamilies ABCB, ABCC, and ABCG), considering their apical or basolateral location and distribution along the intestine. We also discuss the implication of fish intestinal ABC proteins in the transport of physiological substrates and aquatic pollutants, such as pesticides, cyanotoxins, metals, hydrocarbons, and pharmaceutical products.

Modulation of the Blood–Brain Barrier for Drug Delivery to Brain

The blood–brain barrier (BBB) precisely controls brain microenvironment and neural activity by regulating substance transport into and out of the brain. However, it severely hinders drug entry into the brain, and the efficiency of various systemic therapies against brain diseases. Modulation of the BBB via opening tight junctions, inhibiting active efflux and/or enhancing transcytosis, possesses the potential to increase BBB permeability and improve intracranial drug concentrations and systemic therapeutic efficiency. Various strategies of BBB modulation have been reported and investigated preclinically and/or clinically. This review describes conventional and emerging BBB modulation strategies and related mechanisms, and safety issues according to BBB structures and functions, to try to give more promising directions for designing more reasonable preclinical and clinical studies.

Characterization of Amino Acid Substitutions in the Two-Component Regulatory System AdeRS Identified in Multidrug-Resistant Acinetobacter baumannii

The active efflux of antimicrobials by bacteria can lead to antimicrobial resistance and persistence and can affect multiple different classes of antimicrobials. Efflux pumps are tightly regulated, and their overexpression can be mediated by changes in their regulators.

Physiologically Based Pharmacokinetic Modeling of Rosuvastatin to Predict Transporter-Mediated Drug-Drug Interactions

Purpose To build a physiologically based pharmacokinetic (PBPK) model of the clinical OATP1B1/OATP1B3/BCRP victim drug rosuvastatin for the investigation and prediction of its transporter-mediated drug-drug interactions (DDIs). Methods The Rosuvastatin model was developed using the open-source PBPK software PK-Sim®, following a middle-out approach. 42 clinical studies (dosing range 0.002–80.0 mg), providing rosuvastatin plasma, urine and feces data, positron emission tomography (PET) measurements of tissue concentrations and 7 different rosuvastatin DDI studies with rifampicin, gemfibrozil and probenecid as the perpetrator drugs, were included to build and qualify the model. Results The carefully developed and thoroughly evaluated model adequately describes the analyzed clinical data, including blood, liver, feces and urine measurements. The processes implemented to describe the rosuvastatin pharmacokinetics and DDIs are active uptake by OATP2B1, OATP1B1/OATP1B3 and OAT3, active efflux by BCRP and Pgp, metabolism by CYP2C9 and passive glomerular filtration. The available clinical rifampicin, gemfibrozil and probenecid DDI studies were modeled using in vitro inhibition constants without adjustments. The good prediction of DDIs was demonstrated by simulated rosuvastatin plasma profiles, DDI AUClast ratios (AUClast during DDI/AUClast without co-administration) and DDI Cmax ratios (Cmax during DDI/Cmax without co-administration), with all simulated DDI ratios within 1.6-fold of the observed values. Conclusions A whole-body PBPK model of rosuvastatin was built and qualified for the prediction of rosuvastatin pharmacokinetics and transporter-mediated DDIs. The model is freely available in the Open Systems Pharmacology model repository, to support future investigations of rosuvastatin pharmacokinetics, rosuvastatin therapy and DDI studies during model-informed drug discovery and development (MID3).

Molecular mechanisms associated with the resistance of Rhizoctonia solani AG-4 isolates to the succinate dehydrogenase inhibitor, thifluzamide

Thifluzamide, a succinate dehydrogenase (SDH) inhibitor, possesses high activity against Rhizoctonia. In this study, 144 R. solani AG-4 (4HGI, 4HGII, and 4HGIII) isolates, the predominate pathogen associated with sugar beet seedling damping-off, were demonstrated to be sensitive to thifluzamide with a calculated mean median effective concentration of 0.0682 ± 0.0025 μg/mL. Thifluzamide-resistant isolates were generated using fungicide-amended media, resulting in four AG-4HGI isolates and eight AG-4HGII isolates with stable resistance and almost no loss in fitness. Evaluation of cross-resistance of the twelve thifluzamide-resistant isolates and their corresponding parental-sensitive isolates revealed a moderately positive correlation between thifluzamide resistance and the level of resistance to eight other fungicides from three groups, the exception being fludioxonil. An active efflux of fungicide through ATP-binding cassette and major facilitator superfamily transporters was found to be correlated to the resistance of R. solani AG-4HGII isolates to thifluzamide based on RNA-sequencing and quantitative reverse transcription-PCR analyses. Sequence analysis of sdhA, sdhB, sdhC, and sdhD revealed replacement of isoleucine by phenylalanine at position 61 in SDHC in nine of the twelve generated thifluzamide-resistant isolates. No other mutations were found in any of the other genes. Collectively, the data indicate that the active efflux of fungicide and a point mutation in sdhC may contribute to the resistance of R. solani AG-4HGI and AG-4HGII isolates to thifluzamide in vitro. This is the first characterization of the potential molecular mechanism associated with the resistance of R. solani AG-4 isolates to thifluzamide, and provides practical guidance for the use of this fungicide.

Evaluation of the Influence of Zhenwu Tang on the Pharmacokinetics of Digoxin in Rats Using HPLC-MS/MS

Digoxin (DIG) is a positive inotropic drug with a narrow therapeutic window that is used in the clinic for heart failure. The active efflux transporter of DIG, P-glycoprotein (P-gp), mediates DIG absorption and excretion in rats and humans. Up to date, several studies have shown that the ginger and Poria extracts in Zhenwu Tang (ZWT) affect P-gp transport activity. This study aimed to explore the effects of ZWT on the tissue distribution and pharmacokinetics of DIG in rats. The deionized water or ZWT (18.75 g/kg) was orally administered to male Sprague–Dawley rats once a day for 14 days as a pretreatment. On day 15, 1 hour after receiving deionized water or ZWT, the rats were given the solution of DIG at 0.045 mg/kg dose, and the collection of blood samples was carried out from the fundus vein or excised tissues at various time points. HPLC-MS/MS was used for the determination of the DIG concentrations in the plasma and the tissues under investigation. The pharmacokinetic interactions between DIG and ZWT after oral coadministration in rats revealed significant reductions in DIG Cmax and AUC0-∞, as well as significant increases in T1/2 and MRT0-∞. When coadministered with ZWT, the DIG concentration in four of the investigated tissues statistically decreased at different time points except for the stomach. This study found that combining DIG with ZWT reduced not only DIG plasma exposure but also DIG accumulation in tissues (heart, liver, lungs, and kidneys). The findings of our study could help to improve the drug's validity and safety in clinical applications and provide a pharmacological basis for the combined use of DIG and ZWT.

Characterization of Integrons and Quinolone Resistance in Clinical Escherichia coli Isolates in Mansoura City, Egypt

Escherichia coli is a common pathogen in both humans and animals. Quinolones are used to treat infections caused by Gram-negative bacteria, but resistance genes emerged. Only scarce studies investigated the association between plasmid-mediated quinolone resistance (PMQR) genes and integrons in clinical isolates of E. coli. The current study investigated the prevalence of quinolone resistance and integrons among 134 clinical E. coli isolates. Eighty (59.70%) isolates were quinolone-resistant, and 60/134 (44.77%) isolates were integron positive with the predominance of class I integrons (98.33%). There was a significant association between quinolone resistance and the presence of integrons ( P < 0.0001 ). Isolates from Urology and Nephrology Center and Gastroenterology Hospital were significantly quinolone-resistant and integron positive ( P ≤ 0.0005 ). Detection of PMQR genes on plasmids of integron-positive isolates showed that the active efflux pump genes oqxAB and qepA had the highest prevalence (72.22%), followed by the aminoglycoside acetyltransferase gene (aac(6′)-Ib-cr, 66.67%) and the quinolone resistance genes (qnr, 61.11%). Amplification and sequencing of integrons’ variable regions illustrated that no quinolone resistance genes were detected, and the most predominant gene cassettes were for trimethoprim and aminoglycoside resistance including dfrA17, dfrB4, and dfrA17-aadA5. In conclusion, this study reported the high prevalence of PMQR genes and integrons among clinical E. coli isolates. Although PMQR genes are not cassette-born, they were associated with integrons’ presence, which contributes to the widespread of quinolone resistance in Egypt.

Antibiotic Susceptibility of Environmental Legionella pneumophila Strains Isolated in Northern Italy

Legionella pneumophila is ubiquitous in aquatic environments and responsible for severe pneumonia in humans through inhalation of aerosol containing Legionella spp. Macrolides and fluoroquinolones are frequently used antimicrobials, but treatment failures are increasingly being reported. As susceptibility testing is not routinely performed, this study aimed to determine the minimum inhibitory concentrations (MICs) on 58 environmental Legionella pneumophila strains (24 of serogroup 1 and 34 of non-serogroup 1) isolated in Northern Italy. MICs of azithromycin, erythromycin, ciprofloxacin, levofloxacin, and rifampicin were determined by the microdilution method using buffered yeast extract broth supplemented with α-ketoglutarate (BYEα). Seventy-five percent of Legionella pneumophila isolates showed MIC values below the tentative highest MICs indicated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST); rifampicin was the most active agent with MIC90 values below 0.008 mg/L. Interestingly, one isolate was tested and found to be PCR-positive for the azithromycin LpeAB active efflux system, further confirmed by the reserpine/resazurin microtiter assay. In conclusion, this study has provided additional susceptibility data for environmental Legionella pneumophila isolates from Northern Italy demonstrating, in general, low MICs values for the tested antimicrobials, although one strain tested was shown to possess the LpeAB resistance determinant, indicating that future surveillance studies are warranted.