title: The Nonspecific Binding of Tyrosine Kinase Inhibitors to Human Liver Microsomes.
abstract: 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 Km, Clint, and Ki. 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.
authors.affiliation: Department of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, School of Medicine, Flinders University, Adelaide, Australia.
authors.forename: Kushari
authors.initials: K
authors.lastname: Burns
authors.affiliation: Department of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, School of Medicine, Flinders University, Adelaide, Australia.
authors.forename: Pramod C
authors.initials: PC
authors.lastname: Nair
authors.affiliation: Department of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, School of Medicine, Flinders University, Adelaide, Australia.
authors.forename: Andrew
authors.initials: A
authors.lastname: Rowland
authors.affiliation: Department of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, School of Medicine, Flinders University, Adelaide, Australia.
authors.forename: Peter I
authors.initials: PI
authors.lastname: Mackenzie
authors.affiliation: Department of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, School of Medicine, Flinders University, Adelaide, Australia.
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Knights
authors.affiliation: Department of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, School of Medicine, Flinders University, Adelaide, Australia john.miners@flinders.edu.au.
authors.forename: John O
authors.initials: JO
authors.lastname: Miners
mesh_terms: D006801:Humans; D007191:Indazoles; D008862:Microsomes, Liver / Q000378:metabolism*; D009536:Niacinamide / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000077157:Sorafenib; D013449:Sulfonamides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
chemical_list: D007191:Indazoles; D010671:Phenylurea Compounds; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011743:Pyrimidines; D013449:Sulfonamides; C559147:regorafenib; D009536:Niacinamide; C516667:pazopanib; D000077157:Sorafenib; D011505:Protein-Tyrosine Kinases