Bumetanide is used widely as a tool and off-label treatment to inhibit the Na-K-2Cl cotransporter NKCC1 in the brain and thereby to normalize intra-neuronal chloride levels in several brain disorders. However, following systemic administration, bumetanide only poorly penetrates into the brain parenchyma and does not reach levels sufficient to inhibit NKCC1. The low brain penetration is a consequence of both the high ionization rate and plasma protein binding, which restrict brain entry by passive diffusion, and of brain efflux transport. In previous studies, bumetanide was determined in the whole brain or a few brain regions, such as the hippocampus. However, the blood-brain barrier and its efflux transporters are heterogeneous across brain regions, so it cannot be excluded that bumetanide reaches sufficiently high brain levels for NKCC1 inhibition in some discrete brain areas. Here, bumetanide was determined in 14 brain regions following i.v. administration of 10 mg/kg in rats. Because bumetanide is much more rapidly eliminated by rats than humans, its metabolism was reduced by pretreatment with piperonyl butoxide. Significant, up to 5-fold differences in regional bumetanide levels were determined with the highest levels in the midbrain and olfactory bulb and the lowest levels in the striatum and amygdala. Brain:plasma ratios ranged between 0.004 (amygdala) and 0.022 (olfactory bulb). Regional brain levels were significantly correlated with local cerebral blood flow. However, regional bumetanide levels were far below the IC50 (2.4 μM) determined previously for rat NKCC1. Thus, these data further substantiate that the reported effects of bumetanide in rodent models of brain disorders are not related to NKCC1 inhibition in the brain.

P-glycoprotein (P-gp) limits the oral absorption of drug substances. Potent small molecule P-gp inhibitors (e.g., zosuquidar) and nonionic surfactants (e.g., polysorbate 20) inhibit P-gp by proposedly different mechanisms. Therefore, it was hypothesised that a combination of zosuquidar and polysorbate 20 may potentiate inhibition of P-gp-mediated efflux. P-gp inhibition by zosuquidar and polysorbate 20 in combination was assessed in a calcein-AM assay and in a transcellular etoposide permeability study in MDCKII-MDR1 and Caco-2 cells. Furthermore, solutions of etoposide, zosuquidar, and polysorbate 20 were orally administered to Sprague Dawley rats. Zosuquidar elicited a high level of nonspecific adsorption to various labware, which significantly affected the outcomes of the in vitro studies. Still, at certain zosuquidar and polysorbate 20 concentrations, additive P-gp inhibition was observed in vitro. In vivo, however, oral etoposide bioavailability decreased by coadministration of both zosuquidar and polysorbate 20 when compared to coadministration of etoposide with zosuquidar alone. For future formulation development, the present study provided important and novel knowledge about nonspecific zosuquidar adsorption, as well as insights into combinational P-gp inhibition by a third-generation P-gp inhibitor and a P-gp-inhibiting nonionic surfactant.

Taking tenuazonic acid (TeA) synthetase 1 (TAS1) in Pyricularia oryzae as a reference, the homolog AaTAS1 was first anchored in Alternaria alternata via de novo sequencing. Subsequently, AaMFS1, as a major facilitator superfamily (MFS) protein-encoding gene in the adjacent upstream region, was followed with interest. As hypothesized, AaTAS1 is required for TeA biosynthesis, while AaMFS1 is an efflux pump for the transmembrane transport of TeA. Comparatively, the TeA yield of ΔAaTAS1 and ΔAaMFS1 dropped significantly compared with that of the wild-type strain. Specifically, the A domain of AaTAS1 catalyzed the start of TeA biosynthesis in vitro. Simultaneously, the pathogenicity of ΔAaTAS1 was also significantly decreased. Transcriptome analysis confirmed the abovementioned consistency between the TeA-producing phenotypes and related gene expression. Moreover, the proteins AaTAS1 and AaMFS1 were found present in the cytoplasm, plasma membrane, and intracellular membrane system, respectively, by fluorescence localization. Namely, AaTAS1 was responsible for the biosynthesis of TeA, and AaMFS1 was responsible for the efflux transport of TeA. Certainly, AaTAS1 indirectly regulated the expression of AaMFS1 through the level of synthetic TeA. Overall, data on the novel AaTAS1 and AaMFS1 genes mainly contribute to theoretical advances in mycotoxin biosynthesis and the pathogenicity of phytopathogens to agricultural foods.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

P-glycoprotein inhibitors, like zosuquidar, have widely been used to study the role of P-glycoprotein in oral absorption. Still, systematic studies on the inhibitor dose-response relationship on intestinal drug permeation are lacking. In the present study, we investigated the effect of 0.79 nM-2.5 μM zosuquidar on etoposide permeability across Caco-2 cell monolayers. We also investigated etoposide pharmacokinetics after oral or IV administration to Sprague Dawley rats with co-administration of 0.063-63 mg/kg zosuquidar, as well as the pharmacokinetics of zosuquidar itself. Oral zosuquidar bioavailability was 2.6-4.2%, while oral etoposide bioavailability was 5.5 ± 0.9%, which increased with increasing zosuquidar doses to 35 ± 5%. The intestinal zosuquidar concentration required to induce a half-maximal increase in bioavailability was estimated to 180 μM. In contrast, the IC50 of zosuquidar on etoposide permeability in vitro was only 5-10 nM, and a substantial in vitro-in vivo discrepancy of at least four orders of magnitude was thereby identified. Overall, the present study provides valuable insights for future formulation development that applies fixed dose combinations of P-glycoprotein inhibitors to increase the absorption of poorly permeable P-glycoprotein substrate drugs.

The oral route of drug administration is the most convenient method of drug delivery, but it is associated with variable bioavailability. Food is one of the major factors that affect oral drug absorption by influencing drug properties (e.g., solubility and dissolution rate) and physiological factors (e.g., metabolism and transport across the gastrointestinal tract). The aim of this work was to investigate the effect of food on the high-affinity intestinal efflux transporter substrate drugs. We hypothesized that transport efficiency is higher in the fed state as compared to the fasted state because of the lower intestinal lumen drug concentration due to prolonged gastric emptying time. A systematic analysis of reported clinical food-effect (FE) studies on 311 drugs was performed and the association of the efflux transport efficiency was investigated on the FE magnitude, i.e., changes in maximal plasma concentration and area under the plasma concentration-time profile curve for both solubility and permeability-limited drugs. In total, 124 and 88 drugs showed positive and negative FE, respectively, whereas 99 showed no FE. As expected, the solubility-limited drugs showed positive FE, but interestingly, drugs with a high potential for efflux transport, were associated with negative FE. Moreover, a high-fat diet was associated with a higher magnitude of negative FE for high-affinity efflux transporter substrates as compared to a low-fat diet. To account for changes in drug absorption after food intake, the prolonged gastric emptying time should be considered in the physiologically based pharmacokinetic (PBPK) modeling of orally absorbed efflux transporter substrate drugs.

Brain barrier is both a protective permeability hurdle and a limitation site where therapeutic agents are excluded to enter the target region. Designing drug vehicle to overcome this notorious barrier bottle is challenging. Herein, we construct a stimuli-responsive self-assembled nanovesicle that delivers water-soluble drugs to prevent the efflux transport of brain barriers by responding to the endogenously occurring signals in Alzheimer\'s disease (AD) brain microenvironment. Once stimuli-responsive vesicles are accumulated in intracerebrally, the intrinsically occurring legumain endopeptidase cleaves the Ac-Ala-Ala-Asn-Cys-Asp (AK) short peptide on the drug vesicles to expose the 1,2 thiol amino group to cyclize with the cyano groups on 2-cyano-6-aminobenzothiazole (CABT) of the chaperone vesicles, thus triggering the formation of cross-linked micrometre-scale vesicles. Such a structural alteration completely prevents further brain barriers efflux. The superior neuroprotective capacity of cross-linked vesicles is validated in senescence accelerated mouse prone 8 (SAMP8). This smart multi-drug delivery vesicle is promising to serve as a powerful system for AD treatment and can be adapted for the therapy of other central nervous system (CNS) disorders.

The blood vessels in the central nervous system (CNS) have a series of unique properties, termed the blood-brain barrier (BBB), which stringently regulate the entry of molecules into the brain, thus maintaining proper brain homeostasis. We sought to understand whether neuronal activity could regulate BBB properties. Using both chemogenetics and a volitional behavior paradigm, we identified a core set of brain endothelial genes whose expression is regulated by neuronal activity. In particular, neuronal activity regulates BBB efflux transporter expression and function, which is critical for excluding many small lipophilic molecules from the brain parenchyma. Furthermore, we found that neuronal activity regulates the expression of circadian clock genes within brain endothelial cells, which in turn mediate the activity-dependent control of BBB efflux transport. These results have important clinical implications for CNS drug delivery and clearance of CNS waste products, including Aβ, and for understanding how neuronal activity can modulate diurnal processes.

The efflux pump P-glycoprotein (P-gp) affects drug distribution after absorption in humans and animals. P-gp is encoded by the multidrug resistance gene (MDR1) gene in humans, while rodents (the most common preclinical animal model) express the two isoforms Mdr1a and Mdr1b. Differences in substrate selectivity has also been reported. Our aim was to generate an in vitro cell model with tight barrier properties, expressing functional rat Mdr1a P-gp, as an in vitro tool for investigating species differences. The IPEC-J2 cell line forms extremely tight monolayers and was transfected with a plasmid carrying the rat Mdr1a gene sequence. Expression and P-gp localization at the apical membrane was demonstrated with Western blots and immunocytochemistry. Function of P-gp was shown through digoxin transport experiments in the presence and absence of the P-gp inhibitor zosuquidar. Bidirectional transport experiments across monolayers of the IPEC-J2 rMDR1a cell line and the IPEC-J2 MDR1 cell line, expressing human P-gp, showed comparable magnitude of transport in both the absorptive and efflux direction. We conclude that the newly established IPEC-J2 rMdr1a cell line, in combination with our previously established cell line IPEC-J2 MDR1, has the potential to be a strong in vitro tool to compare P-gp substrate profiles of rat and human P-gp.

Bacterial meningitis is a serious life threatening infection of the CNS. To cause meningitis, blood-borne bacteria need to interact with and penetrate brain endothelial cells (BECs) that comprise the blood-brain barrier. BECs help maintain brain homeostasis and they possess an array of efflux transporters, such as P-glycoprotein (P-gp), that function to efflux potentially harmful compounds from the CNS back into the circulation. Oftentimes, efflux also serves to limit the brain uptake of therapeutic drugs, representing a major hurdle for CNS drug delivery. During meningitis, BEC barrier integrity is compromised; however, little is known about efflux transport perturbations during infection. Thus, understanding the impact of bacterial infection on P-gp function would be important for potential routes of therapeutic intervention. To this end, the meningeal bacterial pathogen, Streptococcus agalactiae, was found to inhibit P-gp activity in human induced pluripotent stem cell-derived BECs, and live bacteria were required for the observed inhibition. This observation was correlated to decreased P-gp expression both in vitro and during infection in vivo using a mouse model of bacterial meningitis. Given the impact of bacterial interactions on P-gp function, it will be important to incorporate these findings into analyses of drug delivery paradigms for bacterial infections of the CNS.

Drug-induced liver injury is an important reason for drug candidate failure. Alterations in the hepatobiliary disposition of bile acids are a proposed mechanism of cholestatic hepatotoxicity. Bile acids are synthesized in the hepatocyte, and excreted into the bile primarily by the bile salt export pump. Therefore, inhibition of the bile salt export pump by drugs has been postulated as a risk factor in the development of cholestatic hepatotoxicity. However, recent publications have shown a lack of correlation between bile salt export pump inhibition potency and drug-induced liver injury incidence. Following inhibition of the bile salt export pump mediated efflux of bile acids, the liver compensates through various mechanisms (adaptive response) including upregulation of basolateral bile acid efflux mediated by the farnesoid X receptor, the master regulator of bile acid homeostasis. The C-DILI™ assay integrates the effects of bile salt export pump inhibition, farnesoid X receptor antagonism, and basolateral efflux inhibition of bile acids to more accurately predict a drug\'s potential to cause cholestatic hepatotoxicity and drug-induced liver injury.