Transporters of the SLC22 family, such as organic cation transporter 1 (OCT1), possess very broad substrate specificity. It is unclear to what extent the inhibitory potencies of OCT1 depend on the substrate used. Here, we describe a multisubstrate drug cocktail that allows for the simultaneous testing of drug-drug interactions using 8 different victim drugs: fenoterol, salbutamol, sumatriptan, zolmitriptan, ipratropium, trospium, methylnaltrexone, and metformin. There were no significant differences in Michaelis constant (K_M) and v_max of the OCT1-mediated uptake of the substrates alone or in the cocktail. Depending on the victim drug analyzed, we observed 6.7-fold differences in the inhibitory potency of fenoterol (IC₅₀ of 0.75 μM for metformin and 5.1 μM for sumatriptan). Similarly, the inhibitory potency of verapamil varied 6.7-fold (IC₅₀ of 1.3 μM for zolmitriptan and 8.7 μM for ipratropium). Two groups of inhibitors showed strong correlations in their victim-dependent inhibitory potencies. Group 1 comprised verapamil, quinidine, fenoterol, and ipratropium, and group 2 comprised metformin, sumatriptan, and trimethoprim. By comparing OCT1 paralogs and orthologs, the broadest substrate spectra were observed for OCT1 and multidrug and toxin extrusion 1, followed by OCT2, multidrug and toxin extrusion 2-K, and OCT3. In contrast, organic cation transporters novel 1 and organic cation transporters novel 2 exhibited very narrow substrate specificity, transporting only L-carnitine and L-ergothioneine, respectively. In conclusion, OCT1 demonstrates substantial differences in inhibitory potencies, depending on the victim drug used. We developed a cocktail approach that enables rapid screening for such differences, facilitating the identification of drug-drug interactions at the early stages of drug development. This approach can be extended to other transporters with broad substrate specificity. SIGNIFICANCE STATEMENT: Polyspecific transporters have a broad substrate-binding cavity with no defined single binding position. Consequently, inhibitors may exhibit different inhibitory potencies depending on the victim drug used for testing. Here, we demonstrate this for organic cation transporter 1 (OCT1, SLC22A1) and presents a drug cocktail designed to identify varying inhibitory potencies in vitro and prevent false-negative drug-drug interaction results during early drug development. This approach can be extended to other polyspecific drug transporters.

Spray-dried amorphous solid dispersions of new chemical entities and pH-dependent soluble polymer hydroxypropyl methylcellulose acetate succinate (HPMC-AS) were found to form solid agglomerates in the gastrointestinal tract of rodents after oral administration. These agglomerates, referring to descriptions of intra-gastrointestinal aggregated oral dosage forms termed pharmacobezoars, represent a potential risk for animal welfare. Previously, we introduced an in vitro model to assess the agglomeration potential of amorphous solid dispersions from suspensions and how it can be reduced. In this work, we investigated if the in vitro effective approach of viscosity enhancement of the vehicle used to prepare suspensions of amorphous solid dispersions could reduce the pharmacobezoar formation potential following repeated daily oral dosing to rats as well. The dose level of 2400 mg/kg/day used in the main study was determined in a dose finding study carried out in advance. In the dose finding study, MRI investigations were carried out at short time intervals to gain insights into the process of pharmacobezoar formation. Whereas MRI investigations underlined the importance of the forestomach for the formation of pharmacobezoars, viscosity enhancement of the vehicle reduced the incidence of pharmacobezoars, delayed the onset of pharmacobezoar formation and reduced the overall mass of pharmacobezoars found at necropsy.

Primary hepatocytes are widely used in preclinical drug development, with their transporter expression being well-characterized. However, less is known about non-parenchymal liver cells (NPCs), which constitute 40% of the liver’s cell population and include sinusoidal endothelial cells and Kupffer cells. This study aimed to characterize transporter expression in murine NPCs compared to hepatocytes. Cell fractions were isolated using collagenase perfusion, density gradient centrifugation, and magnetic-activated cell sorting (MACS) with F4/80 and CD146 antibodies. Transporter expression and separation quality were analyzed via RT-qPCR. Results showed NPC-specific genes were significantly lower in hepatocytes and vice versa. Importantly, NPCs exhibited higher expression of several transporters: Abcc1/Mrp1 (87-fold), Abcc4/Mrp4 (4-fold), Abcc5/Mrp5 (40-fold), as well as Slc15a2/PepT2 (16-fold), Slc28a2/Cnt2 (20-fold), Slco3a1/Oatp3a1 (15-fold), and Slco4a1/Oatp4a1 (13-fold), compared to hepatocytes. Hepatocytes showed dominant expression of Abcc2/Mrp2, Abcg2/Bcrp, Slc22a1/Oct1, and others. Minimal differences in transporter expression were found between Kupffer and endothelial cells. In conclusion, the efflux transporters Abcc1/Mrp1 and Abcc5/Mrp5 are predominantly expressed in NPCs. This suggests that NPCs are potentially relevant for the transport of certain drugs and should be included in in vitro preclinical testing.