Pharmacokinetics and disposition of anlotinib, an oral tyrosine kinase inhibitor, in experimental animal species

Abstract

Anlotinib is a new oral tyrosine kinase inhibitor; this study was designed to characterize its pharmacokinetics and disposition. Anlotinib was evaluated in rats, tumor-bearing mice, and dogs and also assessed in vitro to characterize its pharmacokinetics and disposition and drug interaction potential. Samples were analyzed by liquid chromatography/mass spectrometry. Anlotinib, having good membrane permeability, was rapidly absorbed with oral bioavailability of 28%-58% in rats and 41%-77% in dogs. Terminal half-life of anlotinib in dogs (22.8±11.0 h) was longer than that in rats (5.1±1.6 h). This difference appeared to be mainly associated with an interspecies difference in total plasma clearance (rats, 5.35±1.31 L·h^-1·kg^-1; dogs, 0.40±0.06 L·h^-1/kg^-1). Cytochrome P450-mediated metabolism was probably the major elimination pathway. Human CYP3A had the greatest metabolic capability with other human P450s playing minor roles. Anlotinib exhibited large apparent volumes of distribution in rats (27.6±3.1 L/kg) and dogs (6.6±2.5 L/kg) and was highly bound in rat (97%), dog (96%), and human plasma (93%). In human plasma, anlotinib was predominantly bound to albumin and lipoproteins, rather than to α₁-acid glycoprotein or γ-globulins. Concentrations of anlotinib in various tissue homogenates of rat and in those of tumor-bearing mouse were significantly higher than the associated plasma concentrations. Anlotinib exhibited limited in vitro potency to inhibit many human P450s, UDP-glucuronosyltransferases, and transporters, except for CYP3A4 and CYP2C9 (in vitro half maximum inhibitory concentrations, <1 μmol/L). Based on early reported human pharmacokinetics, drug interaction indices were 0.16 for CYP3A4 and 0.02 for CYP2C9, suggesting that anlotinib had a low propensity to precipitate drug interactions on these enzymes. Anlotinib exhibits many pharmacokinetic characteristics similar to other tyrosine kinase inhibitors, except for terminal half-life, interactions with drug metabolizing enzymes and transporters, and plasma protein binding.

Figure 1

Mean plasma concentrations of anlotinib over time in male rats (A), female rats (B), male dogs (C), and female dogs (D) after an oral or intravenous dose of anlotinib.

Figure 2

Tissue distribution of anlotinib in male rats (A), female rats (B), and female tumor-bearing mice (C–E) after an oral dose of anlotinib (3 mg/kg for rats, 0.75, 1.5, and 3 mg/kg for tumor-bearing mice).

Figure 3

Proposed metabolic pathways of anlotinib. P450, cytochrome P450 enzymes; NADPH, reduced β-nicotinamide adenine dinucleotiden phosphate; NADP, β-nicotinamide adenine dinucleotide phosphate; UGT, uridine 5′-diphosphoglucuronosyltransferase; UDPGA, uridine 5′-diphosphoglucuronic acid; UDP, uridine 5′-diphosphate; SULT, sulfotransferase; PAPS, 3′-phosphoadenosine-5′-phosphosulphate; PAP, 3′-phosphoadenosine-5′-phosphate; Glu, glucuronosyl.

Figure 4

Human cytochrome P450 enzymes mediating in vitro oxidation of anlotinib (A) and comparative metabolic capabilities of liver microsomes of different species in mediating the metabolism (B–E). For the enzyme identification using cDNA-expressed human P450 enzymes, the substrate anlotinib and enzyme concentration were 2 μmol/L and 50 pmol P450/mL, respectively, with an incubation time of 60 min. The metabolites formed were expressed as relative abundance, with the most abundant M10 being 100% after incubation with CYP3A4. For the metabolic capability comparison using rat liver microsomes (B), dog liver microsomes (C), and human liver microsomes (D), the substrate anlotinib and enzyme concentration were 2 μmol/L and 0.5 mg protein/mL, respectively, with incubation times of 3, 7.5, 15, 30, and 60 min. The metabolites formed were expressed as relative abundance, with the most abundant M10 being 100% after incubation with rat liver microsomes for 30 min. Panel (E) shows comparative relative total abundance of M4, M5, M8, M9, M10, and M11 formed from anlotinib after incubation with NADPH-fortified rat liver microsomes, dog liver microsomes, and human liver microsomes. The chemical structures of formed metabolites were shown in Figure 3. Quantification of the oxidized metabolites was achieved using the calibration curve of anlotinib.