Data demonstrating the challenges of determining the kinetic parameters of P-gp mediated transport of low-water soluble substrates

The presented data are related to the research article entitled “Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates” by Ozgur et al. (2017) [1]. This data report describes the challenges of investigating the concentration-dependent transport of P-glycoprotein (P-gp) substrates with relatively low aqueous solubility. Thus, we provide solubility data on two prototypical P-gp substrates, digoxin and rhodamine 123, representing P-gp substrates with a relatively low- and high-aqueous solubility, respectively. We present a hypothetical Michaelis-Menten curve of the P-gp mediated transport of digoxin to demonstrate that the maximal donor concentration, which can be reached in the experimental transport buffer, is too low to yield transport data in the saturable range of the Michaelis-Menten relationship. Furthermore, we present data on the bidirectional transport of digoxin and rhodamine 123 across cell monolayers of the MDCK II MDR1 cell line and iP-pg cell line in the presence of the selective P-gp inhibitor, zosuquidar/LY335979

A novel derivative of thioridazine shows low toxicity and efficient activity against gram‐positive pathogens

Thioridazine hydrochloride (HCl) has been suggested as a promising antimicrobial helper compound for the treatment of infections with antimicrobial-resistant bacteria. Unfortunately, the therapeutic concentration of thioridazine HCl is generally higher than what can be tolerated clinically, in part due to its toxic side effects on the central nervous system. Therefore, we aimed to synthesize a less toxic thioridazine derivative that would still retain its properties as a helper compound. This resulted in a compound designated 1-methyl-2-(2-(2-(methylthio)-10H-phenothiazin-10-yl)ethyl)-1-pentylpiperidin-1-ium bromide (abbreviated T5), which exhibited low blood–brain barrier permeability. The lowest minimal inhibitory concentration (MIC) against Staphylococcus aureus exposed to the novel compound was reduced 32-fold compared to thioridazine HCl (from 32 µg/mL to 1 µg/mL). The MIC values for T5 against five Gram-positive pathogens ranged from 1 µg/mL to 8 µg/mL. In contrast to thioridazine HCl, T5 does not act synergistically with oxacillin. In silico predictive structure analysis of T5 suggests that an acceptably low toxicity and lack of induced cytotoxicity was demonstrated by a lactate dehydrogenase assay. Conclusively, T5 is suggested as a novel antimicrobial agent against Gram-positive bacteria. However, future pharmacokinetic and pharmacodynamic studies are needed to clarify the clinical potential of this novel discovery

The Pyrazolo[3,4-d]pyrimidine Derivative, SCO-201, Reverses Multidrug Resistance Mediated by ABCG2/BCRP

ATP-binding cassette (ABC) transporters, such as breast cancer resistance protein (BCRP), are key players in resistance to multiple anti-cancer drugs, leading to cancer treatment failure and cancer-related death. Currently, there are no clinically approved drugs for reversal of cancer drug resistance caused by ABC transporters. This study investigated if a novel drug candidate, SCO-201, could inhibit BCRP and reverse BCRP-mediated drug resistance. We applied in vitro cell viability assays in SN-38 (7-Ethyl-10-hydroxycamptothecin)-resistant colon cancer cells and in non-cancer cells with ectopic expression of BCRP. SCO-201 reversed resistance to SN-38 (active metabolite of irinotecan) in both model systems. Dye efflux assays, bidirectional transport assays, and ATPase assays demonstrated that SCO-201 inhibits BCRP. In silico interaction analyses supported the ATPase assay data and suggest that SCO-201 competes with SN-38 for the BCRP drug-binding site. To analyze for inhibition of other transporters or cytochrome P450 (CYP) enzymes, we performed enzyme and transporter assays by in vitro drug metabolism and pharmacokinetics studies, which demonstrated that SCO-201 selectively inhibited BCRP and neither inhibited nor induced CYPs. We conclude that SCO-201 is a specific, potent, and potentially non-toxic drug candidate for the reversal of BCRP-mediated resistance in cancer cells

Insight Into the Anti-staphylococcal Activity of JBC 1847 at Sub-Inhibitory Concentration

Multidrug-resistant pathogens constitute a serious global issue and, therefore, novel antimicrobials with new modes of action are urgently needed. Here, we investigated the effect of a phenothiazine derivative (JBC 1847) with high antimicrobial activity on Staphylococcus aureus, using a wide range of in vitro assays, flow cytometry, and RNA transcriptomics. The flow cytometry results showed that JBC 1847 rapidly caused depolarization of the cell membrane, while the macromolecule synthesis inhibition assay showed that the synthesis rates of DNA, RNA, cell wall, and proteins, respectively, were strongly decreased. Transcriptome analysis of S. aureus exposed to sub-inhibitory concentrations of JBC 1847 identified a total of 78 downregulated genes, whereas not a single gene was found to be significantly upregulated. Most importantly, there was downregulation of genes involved in adenosintrifosfat (ATP)-dependent pathways, including histidine biosynthesis, which is likely to correlate with the observed lower level of intracellular ATP in JBC 1847–treated cells. Furthermore, we showed that JBC 1847 is bactericidal against both exponentially growing cells and cells in a stationary growth phase. In conclusion, our results showed that the antimicrobial properties of JBC 1847 were primarily caused by depolarization of the cell membrane resulting in dissipation of the proton motive force (PMF), whereby many essential bacterial processes are affected. JBC 1847 resulted in lowered intracellular levels of ATP followed by decreased macromolecule synthesis rate and downregulation of genes essential for the amino acid metabolism in S. aureus. Bacterial compensatory mechanisms for this proposed multi-target activity of JBC 1847 seem to be limited based on the observed very low frequency of resistance toward the compound

IPEC-J2 rMdr1a, a New Cell Line with Functional Expression of Rat P-glycoprotein Encoded by Rat Mdr1a for Drug Screening Purposes

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