Application of the Fluorescent Probe 1-Anilinonaphthalene-8-Sulfonate to the Measurement of the Nonspecific Binding of Drugs to Human Liver Microsomes

McLure, James Alexander; Birkett, Donald; Elliot, David J.; Williams, John A.; Rowland, Andrew; Miners, John O.

doi:10.1124/dmd.111.039354

Cited by 7 publications

(13 citation statements)

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“…Cell uptake of chloroquine was 2.3 fold higher in P-MDCK cells than wild type MDCK cells (p value = 1.598E −05 , n = 6); whereas, there was no significant difference in cell uptake of salicylic acid between transfected and wild type cells (Figure 5). The cell uptake of chloroquine was significantly higher than salicylic acid in both wild type as well as transfected MDCK cells, which is due to higher lipophilicity of chloroquine 24 as compared to salicylic acid 25 .…”

Section: Resultsmentioning

confidence: 93%

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Pigmented-MDCK (P-MDCK) Cell Line with Tunable Melanin Expression: An in Vitro Model for the Outer Blood–Retinal Barrier

2012

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Purpose Retinal pigment epithelium, which forms the outer blood-retinal-barrier, is a critical barrier for transport of drugs to the retina. The purpose of this study was to develop a pigmented MDCK (P-MDCK) cell line as a rapidly established in vitro model for the outer blood-retinal-barrier to assess the influence of melanin pigment on solute permeability. Methods A melanin synthesizing P-MDCK cell line was developed by lentiviral transduction of human tyrosinase and p-protein genes in MDCK (NBL-2) cells. Melanin content, tyrosinase activity (conversion of L-dopa to dopachrome), and transepithelial electrical resistance (TEER) were measured. Expression of tyrosinase protein and p-protein in P-MDCK cells was confirmed by confocal microscopy. Effect of L-tyrosine (0 to 2 mM) in culture medium on melanin synthesis in P-MDCK cells was evaluated. Cell uptake and transepithelial transport of pigment-binding chloroquine (Log D = 1.59) and a negative control salicylic acid (Log D = −1.14) were investigated. Results P-MDCK cells expressed tyrosinase and p-protein. Tyrosinase activity was 4.5 fold higher in P-MDCK cells as compared to wild-type MDCK cells. The transepithelial electrical resistance stabilized by day 4 in both cell types, with the TEER being 871 ± 30 and 876 ± 53 Ω.cm2 for P-MDCK and wild-type cells, respectively. Melanin content in P-MDCK cells depended on the concentration of L-tyrosine in culture medium, and increased from 3 to 54 µg/mg protein with an increase in L-tyrosine content from 0 to 2 mM. When the cells were grown in 2 mM L-tyrosine, uptake of chloroquine was 2.3 fold higher and the transepithelial transport was 2.2 fold lower in P-MDCK cells when compared to wild-type MDCK cells. No significant difference was observed for both cell uptake and transport of salicylic acid. Conclusions We developed a P-MDCK cell line with tunable melanin synthesis as a rapidly developing surrogate for retinal pigment epithelium.

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Section: Resultsmentioning

confidence: 93%

“…Chloroquine, a positively charged. lipophilic drug molecule 24 has high affinity for melanin pigment and is commonly used as a positive control for melanin binding 37 . In contrast, salicylic acid.…”

Section: Discussionmentioning

confidence: 99%

Pigmented-MDCK (P-MDCK) Cell Line with Tunable Melanin Expression: An in Vitro Model for the Outer Blood–Retinal Barrier

2012

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“…Thus, log P is clearly not the sole determinant of extensive NSB of TKIs to HLM. We and others have shown previously that NSB is generally highest for lipophilic organic bases with molecular masses .200 Da (Austin et al, 2002;Hallifax and Houston, 2006;Sykes et al, 2006;McLure et al, 2011). However, other physicochemical properties also influence the degree of NSB.…”

Section: Resultsmentioning

confidence: 99%

“…More generally, the choice of log P algorithm used for the prediction of NSB and other parameters warrants further investigation. Available evidence demonstrates that NSB, particularly of lipophilic basic drugs, arises largely from the partitioning of drugs into phospholipid membranes (Margolis and Obach, 2003;Nussio et al, 2007;McLure et al, 2011;Nagar and Korzekwa, 2012). Neutron diffraction studies with the charged lipophilic base propranolol showed that that the lipophilic naphthalene moiety partitions into the lipid bilayer, whereas the charged amine group apparently associates with the phospholipid head group (Herbette et al, 1983).…”

Section: Resultsmentioning

confidence: 99%

“…However, other physicochemical properties also influence the degree of NSB. These include the presence of halogen atoms (especially the trifluoromethyl group), polar surface area, charge distribution, and number of hydrogen bond acceptors/donors [summarized by McLure et al (2011)]. Not only does the addition of fluorine or the trifluoromethyl groups to an aryl ring or adjacent to a heteroatomcontaining functional group increase lipophilicity (Hagmann, 2008), but halogenation of drugs is more generally known to enhance drug membrane binding and permeation.…”

Section: Resultsmentioning

confidence: 99%

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The Nonspecific Binding of Tyrosine Kinase Inhibitors to Human Liver Microsomes

et al. 2015

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Drugs and other chemicals frequently bind nonspecifically to the constituents of an in vitro incubation mixture, particularly the enzyme source [e.g., human liver microsomes (HLM)]. Correction for nonspecific binding (NSB) is essential for the accurate calculation of the kinetic parameters K m , Cl int , and K i . Many tyrosine kinase inhibitors (TKIs) are lipophilic organic bases that are nonionized at physiologic pH. Attempts to measure the NSB of several TKIs to HLM by equilibrium dialysis proved unsuccessful, presumably due to the limited aqueous solubility of these compounds. Thus, the addition of detergents to equilibrium dialysis samples was investigated as an approach to measure the NSB of TKIs. The binding of six validation set nonionized lipophilic bases (felodipine, isradipine, loratidine, midazolam, nifedipine, and pazopanib) to HLM (0.25 mg/ml) was shown to be unaffected by the addition of CHAPS (6 mM) to the dialysis medium. This approach was subsequently applied to measurement of the binding of axitinib, dabrafenib, erlotinib, gefitinib, ibrutinib, lapatinib, nilotinib, nintedanib, regorafenib, sorafenib, and trametinib to HLM (0.25 mg/ml). As with the validation set drugs, attainment of equilibrium was demonstrated in HLM-HLM and buffer-buffer control dialysis experiments. Values of the fraction unbound to HLM ranged from 0.14 (regorafenib and sorafenib) to 0.93 (nintedanib), and were generally consistent with the known physicochemical determinants of drug NSB. The extensive NSB of many TKIs to HLM underscores the importance of correction for TKI binding to HLM and, presumably, other enzyme sources present in in vitro incubation mixtures. IntroductionIn vitro approaches, using human liver microsomes (HLMs), human hepatocytes, or recombinant proteins as the enzyme source are used widely to characterize the kinetics of drug metabolism and drug-drug interaction potential (Houston, 1994;Obach, 1999;Rostami-Hodjegan and Tucker, 2007;Miners et al., 2010). However, numerous drugs, especially lipophilic organic bases and neutral compounds, bind nonspecifically to the microsomal membrane. Importantly, nonspecific binding (NSB) reduces the concentration of the unbound drug in the incubation medium, resulting in overestimation of the measured kinetic constants K m (or S 50 ) and the inhibitor constant K i . Consequently, in vitro intrinsic clearance (determined as V max /K m ) and inhibitory drug-drug interaction potential (assessed as [I]/K i , where [I] is the inhibitor concentration) are both underpredicted (Obach, 1999;McLure et al., 2000;Margolis and Obach, 2003;Grime and Riley, 2006;Miners et al., 2006Miners et al., , 2010. Thus, it is now widely accepted that NSB should be accounted for when kinetic parameters are calculated from in vitro metabolism and inhibition studies.Tyrosine kinase inhibitors (TKIs) are an emerging group of drugs used in the treatment of many forms of cancer and some other diseases (e.g., idiopathic pulmonary fibrosis). Typically, TKIs are lipophilic (log P values . 2....

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Evidence-based strategies for the characterisation of human drug and chemical glucuronidation in vitro and UDP-glucuronosyltransferase reaction phenotyping

Miners

Rowland

Novak

et al. 2021

Pharmacology & Therapeutics

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Application of the Fluorescent Probe 1-Anilinonaphthalene-8-Sulfonate to the Measurement of the Nonspecific Binding of Drugs to Human Liver Microsomes

Abstract: ABSTRACT:The

Cited by 7 publications

References 27 publications

Pigmented-MDCK (P-MDCK) Cell Line with Tunable Melanin Expression: An in Vitro Model for the Outer Blood–Retinal Barrier

Pigmented-MDCK (P-MDCK) Cell Line with Tunable Melanin Expression: An in Vitro Model for the Outer Blood–Retinal Barrier

The Nonspecific Binding of Tyrosine Kinase Inhibitors to Human Liver Microsomes

Evidence-based strategies for the characterisation of human drug and chemical glucuronidation in vitro and UDP-glucuronosyltransferase reaction phenotyping

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