Article Figures & Data
Figures
- Fig. 1.
Dose-normalized log mean (plus or minus standard deviation) plasma concentration-time profiles (linear inset) for abrocitinib (○ = 200 mg oral and ■ = 100 μg i.v.) and total radioactivity (Δ = 80 μg oral) following single dose administration. ng-eq, nanogram-equivalent.
- Fig. 2.
Cumulative mean (plus or minus standard deviation) recovery of administered radioactivity in urine (85.0% ± 6.0%) and feces (9.5% ± 0.5%) following a single oral dose of 200 mg 14C-abrocitinib (n = 6). Total recovery was 94.5% ± 5.9%.
- Fig. 3.
Representative HPLC radiochromatograms of plasma (A), urine (B), and feces (C) following a single 200 mg dose of 14C-abrocitinib.
- Fig. 4.
Proposed metabolic pathways of abrocitinib following oral administration.
- Fig. 5.
Proposed mass balance model for abrocitinib following oral administration. CLp, plasma clearance; CLr, renal clearance; F, bioavailability; fafg, fraction absorbed × fraction surviving gut metabolism; Vss, steady state volume of distribution.
Tables
- TABLE 1
Mean pharmacokinetic parameters for abrocitinib following intravenous and oral administration
Plasma Pharmacokinetics Parameter 14C-Abrocitinib,aIntravenous Abrocitinib, Oral Period B B Dose, mg 0.1 200 Body weight, kg (S.D.) 83.1 (13.7) 83.1 (13.7) AUCinf, ng* h/mL 1.56 (15) 1850 (35) Cmax, ng/mL 778 (45) Tmax, h (range) 0.50 (0.5–2.0) t1/2, h (S.D.) 1.45 (0.08) 1.52 (0.09) CL, L/h 64.2 (14) Vss, L 100 (19) %Fb (90% CI) 59.8 (45.9–77.8) Urine Pharmacokinetics Parameter Total 14C PO Total 14C Intravenous Period A B % of 14C dose in urine 85.1 (7) 93.3 (1) %fafgc 91.3 Plasma pharmacokinetics were assessed during Period B using noncompartmental analysis of concentration–time data. Geometric mean values (% coefficient of variation) except mean body weight and t1/2 (plus or minus standard deviation) and median Tmax (range).
aPeriod B dosing: abrocitinib, 200 mg, orally, followed by an intravenous dose of 14C-abrocitinib (5 minutes infusion) at ≈1 hour after oral dose.
b%F = 100% × (AUCpo/AUCiv) × (Doseiv/Dosepo).
c%fafg = 100% × (Total 14Cpo/Total 14Civ).
CI, confidence interval; CL, clearance; Vss, steady-state volume of distribution.
- TABLE 2
Abrocitinib and metabolites in pooled plasma, urine, and feces samples following a single oral dose of 200 mg 14C-abrocitinib (period A, n = 6)
Analyte Plasma
(% of Total Radioactivity)Urineb
(% of Dose)Fecesb
(% of Dose)Total Urine/Feces
(% of Dose)Abrocitinib 25.8 0.6 0.3 0.9 M4 13.8 15.4 0.3 15.7 M2a 12.4 13.5 0.5 14.0 M1 11.3 16.2 1.7 17.9 M3a 4.8 4.5 0.3 4.8 M7 4.6 5.6 1.5 7.1 M6 3.4 12.2 0.8 13.0 372-1 2.5 0.7 0.2 0.9 358-1 2.0 1.1 0.1 1.2 356-1a 1.5 1.7 0.2 1.9 356-2 1.5 3.3 0.6 3.9 M8 1.4 1.3 nd 1.3 340-5 1.0 0.4 0.2 0.6 M5 0.4 0.3 nd 0.3 Identified analytes ordered in relationship to prevalence in plasma.
nd, not detected.
aM2 and M3 are stereoisomers.
bOnly percentages of identified metabolites are reported.
- TABLE 3
Molecular ions and characteristic fragment ions of abrocitinib and metabolites detected in plasma and excreta
ID Standard ID Retention Time (min) [M+H]+
m/zParent Fragments
m/zDiagnostic Fragments
m/zM6 PF-07095462 3.4 370.1179 70 217, 165 340-5 4.9 340.1074 201, 176, 175, 149, 70 296 372-1 5.0 372.1334 176, 70 M7 PF-06737821 5.2 354.1230 201, 175, 149, 70 218 M8 PF-07055039 5.8 356.1387 70 217, 165 358-1 6.1 358.1543 176, 70 235, 217, 183, 165 356-1a 6.4 356.1386 70 217, 192, 191, 165 356-2 6.5 356.1384 176, 70 233, 207, 181 M1 PF-06471658 7.6 340.1438 201, 175, 149, 70 218 M2 PF-07055087 8.5a 340.1438 201, 175, 149, 70 218 M3 PF-07055090 8.5a 340.1438 201, 175, 149, 70 218 M4 PF-07054874 10.0b 340.1438 176, 70 234, 217, 191, 165 M5 PF-07054926 10.0b 310.1332 176, 70 204, 187, 161, 135 Abrocitinib PF-04965842 11.5 324.1490 201, 176, 175, 149, 70 aCoeluting enantiomers followed by a second chromatographic separation (see Methods).
bCoeluting peaks with subsequent identification from urine isolation and nuclear magnetic resonance analyses (see Methods).
Parameter M1 M7 M2/M3 M4 358-1 372-1 149 Abrocitinib % radioactivity 5.6 0.35 4.2 3.6 0.16 0.16 0.42 85 Metabolite fCL 0.39 0.024 0.29 0.25 0.011 0.011 0.029 — Final fCL 0.41 0.29 0.27 0.029 — - TABLE 5
Enzyme kinetic parameters of abrocitinib metabolism in human liver microsomes using Michaelis-Menten substrate inhibition modeling
Kinetic Parameter M1 M2/M3 M4 Total Km, µM 143 ± 20 251 ± 26 519 ± 100 — Vmax, pmol/min per mg 26.7 ± 2.2 73.8 ± 5.0 881 ± 125 — Ki, µM 1480 ± 316 1680 ± 320 1340 ± 430 — CLint, µL/min per mg 0.187 0.294 1.7 2.18 CLint,app,sc, mL/min per kg 0.176 0.278 1.6 2.05 Fractional proportion CLint,app,sc 0.086 0.136 0.780 Data expressed as the mean plus or minus standard error of mean from triplicate replicates; unbound fraction (fu,inc) of abrocitinib in human liver microsomes (0.3 mg/mL) was 1.
fu,inc, unbound fraction; Ki, inhibition constant for substrates exhibiting substrate inhibition kinetics; Vmax, maximum rate of metabolism.
- TABLE 6
Selective P450 chemical inhibition of abrocitinib metabolism in pooled human hepatocytes and overall fractional P450 pathway assignment (fm)
Metabolite fCL % P450 Inhibition
(Scaled)P450 Pathway fm 2B6 2C9 2C19 3A4 2B6 2C9 2C19 3A4 M1 (M7) 0.41 16 24 52 8.0 0.066 0.10 0.22 0.033 M2/M3 0.29 44 55 0.13 0.16 M4 (358-1, 372-1) 0.27 25 57 18 0.067 0.15 0.048 149 0.029 100 0.030 Overall fm 0.066 0.30 0.53 0.11 Nonstatistical contributions by the remaining P450 isoforms for M1 (1A2, 2C8, 2D6), M2/M3 (1A2, 2B6, 2C8, 2D6, 3A), M4 (1A2, 2B6, 2C8, 2D6), and 149 (1A2, 2B6, 2C8, 2C9, 2C19, 2D6) were observed and excluded from fraction metabolized determinations. Fraction metabolized was calculated as the product of fCL and fraction inhibited (scaled) as described in eq. 7.
JAK Assay IC50 (nM)a Abrocitinib M1 M2 M4 JAK1 29.2 43.4 17.9 >10,000 JAK2 803 1140 886 >10,000 JAK3 >10,000 >10,000 >10,000 >10,000 TYK2 1250 3190 1210 >10,000 aIC50 values obtained at 1 mM adenosine triphosphate.
Cytokine-Induced pSTAT Assay IC50/IC50,ua (nM) Cytokine JAK Pair pSTAT Cell Assay Abrocitinib M1 M2 IL-4 JAK1/JAK2 pSTAT6 Human keratinocytes 77.0 433 134 IL-13 JAK1/JAK2 pSTAT6 Human keratinocytes 81.9 236 84.1 IL-22 JAK1/TYK2 pSTAT3 Human keratinocytes 420 703 198 IL-31 JAK1/JAK2 pSTAT3 IFNγ-primed THP-1 40.0 79.6 56.0 TSLP JAK1/JAK2 pSTAT3 hWB (CD3+ cells) 1020/343 785/438 271/152 IFNα JAK1/TYK2 pSTAT3 hWB (lymphocytes) 174/58.5 296/165 90.5/50.6 IFNγ JAK1/JAK2 pSTAT1 hWB (CD14+ cells) 1690/569 1950/1090 2160/1210 IL-6 JAK1/JAK2 pSTAT1 hWB (CD3+ cells) 343/115 171/95.3 136/76.0 IL-12 JAK2/TYK2 pSTAT4 hWB (lymphocytes) 9730/3270 33,400/18,600 5170/2890 IL-15 JAK1/JAK3 pSTAT5 hWB (lymphocytes) 537/181 558/311 353/197 IL-21 JAK1/JAK3 pSTAT3 hWB (lymphocytes) 516/174 844/471 487/272 IL-23 JAK2/TYK2 pSTAT3 hWB (lymphocytes) >16,500>5550 26,200/14,600 6210/3470 IL-27 JAK1/JAK2 pSTAT3 hWB (lymphocytes) 228/76.7 382/213 234/131 EPO JAK2/JAK2 pSTAT5 hWB (spiked CD34+ cells) 7180/2420 9750/5440 9470/5290 B/P, blood/plasma ratio; EPO, erythropoietin; hWB, human whole blood; IC50,u, unbound IC50; pSTAT, phospho-signal transducer and activator of transcription; TSLP, thymic stromal lymphopoietin.
ahWB IC50/IC50,u: total IC50/unbound IC50; unbound IC50 = hWB IC50 × (fu/B/P ratio); abrocitinib fu = 0.36; M1 fu = 0.63; M2 fu = 0.71; abrocitinib B/P = 1.07; M1 B/P = 1.13; M2 B/P = 1.27; mean IC50 from at least two experiments.
Data Supplement
- Supplemental Data -
Supplemental Methods
Supplemental Table S1. LC-MS/MS Conditions for Metabolite Profiling and 14C-Abrocitinib Quantitation.
Supplemental Table S2. LC-MS/MS Conditions for Isolation of Metabolites M6 and M7 - Initial Assessment.
Supplemental Table S3. LC-MS/MS Conditions for Isolation of Metabolites M6 and M7 - Initial Fractionation.
Supplemental Table S4. LC-MS/MS Conditions for Isolation of Metabolites M6 and M7 - Analysis of Fractions.
Supplemental Table S5. LC-MS/MS Conditions for Isolation of Metabolites M6 and M7 – Final Fractionation.
Supplemental Table S6. LC-MS/MS Conditions for Abrocitinib in Plasma.
Supplemental Table S7. LC-MS/MS Conditions for In Vitro Hepatocyte Fractional Metabolism
Supplemental Table S8. LC-MS/MS conditions for in vitro CYP450 assignment (Enzyme kinetics and chemical inhibition)
Supplemental Table S9. LC-MS/MS conditions for abrocitinib in plasma protein binding and blood-to-plasma ratio studies.
Supplemental Table S10. LC-MS/MS conditions for M1 in plasma protein binding and blood-to-plasma ratio studies.
Supplemental Table S11. LC-MS/MS conditions for M2 in plasma protein binding and blood-to-plasma ratio studies.
Supplemental Table S12. Incidence of treatment-emergent mild adverse events.
Supplemental Table S13. Diagnostic MS product ions used for abrocitinib and proposed metabolite structures.
Supplemental Figure S1. 1H13C HMBC spectrum (left) and 1H1H COSY spectrum (right) of M6 (PF-07095462, 370, m/z 370) metabolite isolated from pooled human urine.
Supplemental Figure S2. 1H13C HMBC spectrum (left) and 1H1H COSY spectrum (right) of M7 (PF-06737821, 354-1, m/z 354) metabolite isolated from pooled human urine.
Supplemental Figure S3. Radiochromatogram following 30-minute incubation of 1 µM 14C-abroctinib (PF04965842) in human hepatocytes (0.75 million cells/mL)
Supplemental Figure S4. Enzyme kinetic plots for primary metabolites M1 (A), M2/M3 (B), M4 (C), and 149 (D) and secondary metabolites M7 (E), 358-1 (F), and 372-1 (G) from abrocitinib metabolism.
- Supplemental Data -
