Article Figures & Data
Figures
- Fig. 1.
Chemical structure of the radiolabeled icenticaftor.
- Fig. 2.
Arithmetic mean (S.D.) plasma concentrations of total radioactivity and icenticaftor. (A) Linear view 0–12 hours after the administration of the [14C]icenticaftor dose. (B) Linear view 0–96 hours after the [14C]icenticaftor dose. The figures show a combination of radioactive (liquid scintillation counting) and nonradioactive (LC-MS/MS) methods. The radiolabeled drug-related compounds (total 14C) display single-dose PK, whereas the icenticaftor concentrations reflect steady state PK.
- Fig. 3.
Representative metabolite profile in plasma at 4 and 48 hours postdose. (A) Plasma metabolite profile at 4 hours (Tmax). (B) Plasma metabolite profile at 48 hours. The profiles were obtained using HPLC analysis after direct injection of the plasma extract into the HPLC system and subsequent detection of radioactivity in 96-well plates.
- Fig. 4.
Concentration-time course of icenticaftor and its main metabolites in plasma at 0–48 hours postdose. Metabolite profiles in plasma were determined from plasma pools of six subjects using HPLC analysis, with radioactivity detection at time points 1, 3, 4, 8, 12, 24, and 48 hours.
- Fig. 5.
Representative metabolite profiles in (A) urine and (B) feces. The urine fraction pool (0–96 hours; N = 6), containing 90.4% of the total excreted dose, and the feces fraction pool (0–120 hours; N = 6) were evaluated using HPLC analysis, with subsequent radioactivity detection in 96-well plates.
- Fig. 6.
Electrospray mass spectrum of icenticaftor. (A) Key fragments used for the elucidation of the structure of metabolites. (B) Electrospray ionization, positive ion mode, sample cone voltage of 30 V, trap collision energy ramp of 10–40 eV. The accurate mass measurements were in agreement with the proposed fragmentation. The difference between the calculated and measured masses was ≤0.5 mDa for all indicated fragment ions.
- Fig. 7.
Scheme of the major biotransformation pathway of icenticaftor in humans (Pathway I). The metabolites were detected in plasma, urine, and/or feces. f, feces; p, plasma; u, urine.
- Fig. 8.
Cumulative excretion of radioactivity in urine and feces.
Tables
- TABLE 1
Primary pharmacokinetic parameters of radioactivity and icenticaftor in blood and plasma
PK parameter (units)a Total blood radioactivity Total plasma radioactivity Plasma icenticaftor Tmax (h), median (range) 4 (1–4) 4 (1–4) 4 (1–4) Cmax (ng/ml) 2670 (509) 4280 (838) 1680 (568) Tlast (h), range 24–48 48–96 12 AUClast (ng*h/ml) 24700 (5290) 45600 (7830) 8170 (3270) T1/2 (h) 15.4 (5.79) 20.6 (9.55) – Typical time interval (h), rangeb 8–48 24–96 – AUCinf (ng*h/ml) 27900 (4610) 48300 (8120) – Percentage of 14[C]-AUCinf plasma 57.9 (2.91) – 16.6 (5.06) AUC%extrap 12.2 (6.21) 5.74 (2.53) N/A Vz/F (L) – – 335 (161) CLss/F (L/h) – – 56.8 (25.6) –, not calculated; AUC%extrap, percentage of AUCinf extrapolated; N/A, not applicable; Tlast, time of last measured concentration.
↵aMean (S.D.) unless otherwise stated.
↵bThe range shows the time interval for the elimination half-life, which highlights the differences between blood and plasma. A shorter half-life in blood is likely to be caused by a higher lower limit of quantification than that in plasma.
- TABLE 2
AUC0–48h, AUCinf, and T1/2 of icenticaftor and its metabolites in plasma based on metabolite pattern analysis
Peaka Compound/
metaboliteAUC0–48h
(nmol*h/L)Percentage
AUC0–48hAUCinf
(nmol*h/L)Percentage AUCinf T1/2
(h)M5 O-demethylation, glucuronidation 12800 10.7 13800 10.7 12.1 M8 N-glucuronidation 42200 35.3 44100 33.9 11.2 M9 O-glucuronidation 17300 14.5 18100 13.9 11.6 M10 C-hydroxylation 2310 1.93 2840 2.19 – M14 O-demethylation 1790 1.50 2000 1.54 16.8 M17 N-dealkylation 3630 3.04 3840 2.96 10.7 Icenticaftor Parent drug 22100 18.5 22700b 17.5 11.2 Sum of minor identified metabolites (each ≤1%) 7930 6.63 – – – Sum of unknown trace metabolites (each ≤0.30%) 515 0.430 – – – Lost during sample processing and HPLC 8870 7.42 – – – Total 14C (total of radiolabeled components) 120000 100 130000 100 14.2 Enzyme Metabolite Km,u (μM)a Vmax (pmol/min/mg)b CLint,u (μl/min/mg) fm (%) CYP1A2 M14, M16, M17, M18, M19 4.61 14.5 3.14 11.6 CYP3A4 M14, M16, M17, M18, M19 13.0 68.1 5.24 19.4 UGT1A9c M8 0.34 5.32 15.2 56.0 UGT2B7 M9 4.64 17.0 3.67 13.0 CLint,u, unbound intrinsic clearance; Vmax, maximum velocity (reaction velocity at saturating substrate concentration).
↵aBase fraction of the unbound test substance in microsomes = 0.2571 × (mg of microsomal protein)2 − 0.5798 × (mg of microsomal protein) + 0.7995 (see Supplemental Material [Section 8], Supplemental Table 8, and Supplemental Fig. 10).
↵bBased on liver enzyme abundance of 52.0, 137, 31, and 71 pmol/mg for CYP1A2, CYP3A4, UGT1A9, and UGT2B7, respectively.
↵cUGT1A3 and UGT1A4 were also identified as minor metabolizing enzymes.
Data Supplement
- Supplemental Data -
Supplemental Data. Inclusion and Exclusion Criteria.
Supplemental Data. Sample Collection and Preparation.
Supplemental Data. Analysis of Total Radioactivity.
Supplemental Data. Determination of Plasma Icenticaftor Concentrations.
Supplemental Data. Determination of Metabolite Profiles in Plasma, Urine, and Feces.
Supplemental Data. Synthesis of Reference Standards.
Supplemental Data. In Vitro Phenotyping of Cytochrome P450 (CYP) Enzymes.
Supplemental Data. In Vitro Phenotyping of Uridine Diphosphate Glucuronosyltransferase (UGT) Isozymes.
Supplemental Data. In Vitro Biotransformation in Mouse, Rat, Monkey, and Human Hepatocytes.
Supplemental Data. Rat ADME Study.
Supplemental Table S1. Concentration of icenticaftor and its metabolites in human plasma following the administration of a single oral dose of 400 mg [14C]icenticaftor at steady state.
Supplemental Table S2. Percentage of icenticaftor and its metabolites in human urine and feces.
Supplemental Table S3. Mass spectral biotransformation data (human).
Supplemental Table S4. Species comparison of biotransformation data of major metabolites following hepatocyte incubations.
Supplemental Table S5. Profiles of icenticaftor and its metabolites in bile duct-cannulated rats after the administration of a single intravenous dose of 3 mg/kg.
Supplemental Table S6. Profiles of icenticaftor and its metabolites in intact rats after the administration of a single oral dose of 10 mg/kg.
Supplemental Table S7. Kinetic parameters for icenticaftor glucuronidation in HLMs.
Supplemental Table S8. Identification of human CYP and UGT isoenzymes involved in the metabolism of icenticaftor.
Supplemental Figure S1. Dose nonlinearity after the administration of single and multiple-dose icenticaftor.
Supplemental Figure S2. Study design.
Supplemental Figure S3. Characterization of reference standards by nuclear magnetic resonance spectroscopy.
Supplemental Figure S4. Total radioactivity concentrations in blood and plasma (semilogarithmic view).
Supplemental Figure S5. Scheme of the minor biotransformation pathways of icenticaftor in humans (Pathway II).
Supplemental Figure S6. Metabolite profiles of [14C]icenticaftor in human hepatocytes at concentrations of (A) 1 μM, (B) 10 μM, and (C) 30 μM at 24 hours.
Supplemental Figure S7. In vitro metabolic pathways across species.
Supplemental Figure S8. Radiochromatogram of [14C]icenticaftor after incubation for 20 minutes in HLMs in the presence of UDPGA.
Supplemental Figure S9. Radiochromatogram of icenticaftor after incubation in HLMs in the presence of NADPH.
Supplemental Figure S10. Unbound fraction (fu,mic) of icenticaftor in HLMs.
- Supplemental Data -
