Effect of N-methyl deuteration on metabolism and pharmacokinetics of enzalutamide

Figures & data

Figure 1 Mechanistic pathway for CYP450-catalyzed demethylation of alkylamines.

Abbreviation: CYP450, cytochrome P450.

Figure 2 Chemical structure of (A) ENT and (B) d₃-ENT.

Abbreviations: ENT, enzalutamide; d₃-ENT, deuterated enzalutamide.

Table 1 UPLC/Q-TOF MS data for ENT and d₃-ENT metabolites detected in HLMs

No	Description	m/z		Error (ppm)	RT (minutes)	Formula	Fragment ions
		[M + HCOO]^−
M0	ENT	509.091	−1.4		11.80	C21H16F4N4O2S	406.065, 336.076, 253.060, 158.063
M1	Amide hydrolysis	450.054a	−1.9		11.90	C20H13F4N3O3S	406.048, 253.047, 158.054
M2	N-demethylation	495.075	−2.1		11.05	C20H14F4N4O2S	322.061, 223.089
M6	Oxidation	525.086	1.4		10.66	C21H16F4N4O3S	479.068, 406.046, 322.052, 253.049
M0	d3-EnT	512.109	1.8		11.80	C21H13D3F4N4O2S	406.066, 339.097, 253.061, 158.064
M7	Oxidation	527.086	3.1		10.66	C21H14D2F4N4O3S	481.077, 406.051, 322.062, 253.051

Note:

a m/z [M – H]⁻.

Abbreviations: UPLC/Q-TOF MS, ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry; ENT, enzalutamide; d₃-ENT, deuterated enzalutamide; HLM, human liver microsomes; RT, retention time.

Figure 3 Relative metabolite formation of M6 and M7 after incubation for 4 hours with liver microsomes from different species.

Notes: M6 represents the monooxidation product of ENT (OH–ENT), and M7 represents the monooxidation product of d₃-ENT (OH–d₂–ENT). Data were acquired by the UV absorption spectrum of UPLC/Q-TOF MS. **Significant differences (Student’s t-test) versus values for ENT at the P<0.01 level.
Abbreviations: ENT, enzalutamide; d₃-ENT, deuterated enzalutamide; HLM, human liver microsomes; cyLM, monkey liver microsomes; DLM, dog liver microsomes; RLM, rat liver microsomes; MLM, mouse liver microsomes; UV, ultraviolet; UPLC/Q-TOF MS, ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Figure 4 Relative parent remaining of ENT, d₃-ENT, and M2 (concentration: 1 μM each) in rat plasma after incubation for up to 6 hours.

Note: Data are the mean of duplicate samples.
Abbreviations: ENT, enzalutamide; d₃-ENT, deuterated enzalutamide; M2, N-demethyl metabolite of ENT.

Figure 5 Plasma concentration–time profiles for M1 and M2 after intravenous administration of 5 mg/kg M2 (dissolved in PEG200/Tween 80/saline solution 1/1/100, v/v) in male Sprague Dawley rats.

Note: Data are the mean of results from three rats.
Abbreviations: M1, amide hydrolysis metabolite of enzalutamide; M2, N-demethyl metabolite of enzalutamide; PEG, polyethylene glycol.0

Table 2 Intrinsic clearance values for ENT and d₃-ENT in liver microsomes

Substrate	RLMs			HLMs
	Km(μM)	Vmax (pmol/min/mg protein)	CLint, in vitro (μL/min/mg protein)	Km(μM)	Vmax (pmol/min/mg protein)	CLint, in vitro (μL/min/mg protein)
ENT	71.5	169.9	2.38	36.7	32.8	0.894
D3-ENT	79.6	126.4	1.59	12.6	6.51	0.517
KH/KD			1.50			1.73

Notes: Intrinsic clearance values were determined by substrate depletion in mixed-sex male Sprague Dawley rat or human liver microsomes. The microsomal protein concentration was 1.0 mg/mL. The cofactor NADPH was included to activate cytochrome P450 enzymes. Data are the mean of duplicate determinations. K_m, Michaelis constant; V_max, maximum velocity; CL_int, intrinsic clearance; K_H/K_D, intrinsic primary isotope effect.

Abbreviations: ENT, enzalutamide; d₃-ENT, deuterated enzalutamide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; RLM, rat liver microsomes; HLM, human liver microsomes.

Figure 6 Kinetic profiles for the substrate depletion of ENT (left) and d₃-ENT (right) in (A) RLM and (B) HLM.

Notes: Eadie–Hofstee plots (V/S versus V) are shown as insets. Data points represent the mean of duplicate determinations. V represents the reaction rate, and S represents the substrate concentration.
Abbreviations: ENT, enzalutamide; d₃-ENT, deuterated enzalutamide; RLM, rat liver microsomes; HLM, human liver microsomes.

Figure 7 Proposed metabolic pathways of d₃-ENT in rat.

Notes: d₃-ENT is extensively metabolized in vivo by oxidation, N-demethylation, and amide hydrolysis. The major metabolites are the N-demethyl metabolite M2 and the amide hydrolytic metabolite M1. M4 and M5 are the S-substituted-to-O metabolite and amide hydrolysis-and-further metabolite, respectively. M7 is the N-CD₂OH metabolite of d₃-ENT.
Abbreviation: d₃-ENT, deuterated enzalutamide.

Figure 8 Plasma concentration–time profiles for (A) M1 and (B) M2 after oral administration of 20 mg/kg ENT and d₃-ENT to male Sprague Dawley rats, respectively. Notes: Data are the mean ± SD of four rats. M1 and M2 are the amide hydrolysis metabolite and N-demethylation metabolite, respectively.

Abbreviations: ENT, enzalutamide; d₃-ENT, deuterated enzalutamide; SD, standard deviation.

Table 3 Pharmacokinetic parameters in male rats after simultaneous administration of a 1:1 formulation of ENT and d₃-ENT (10/10 mg/kg)

Parameters	ENT	D3-ENT
Dose (mg/kg)	10	10
Tmax (hours)	7.7±3.7	18.1±10.0
t½ (hours)	11.9±2.4	18.4±2.2*
Cmax (ng/mL)	2,258±193	3,055±229**
AUC0–t (h⋅ng/mL)	51,483±2,904	104,401±6,393**
MRT (hours)	18.1±0.5	28.6±3.7**

Notes: Data are presented as the mean ± SD of values obtained from three male Sprague Dawley rats. T_max, time to C_max; t_½, elimination half-life; C_max, maximum observed plasma concentration; AUC_0–t, area under the plasma concentration–time curve from time zero to the last measurable sampling time point.

* Significant differences (Student’s t-test) versus values for ENT at the P<0.05 level.

** Significant differences (Student’s t-test) versus values for ENT at the P<0.01 level.

Abbreviations: ENT, enzalutamide; d₃-ENT, deuterated enzalutamide; MRT, mean residence time.

Figure 9 Plasma concentration–time profiles for ENT and d₃-ENT after simultaneous oral administration of a 1:1 formulation of ENT and d₃-ENT (10/10 mg/kg) in male Sprague Dawley rats.

Note: Data are the mean ± SD of three rats.
Abbreviations: ENT, enzalutamide; d₃-ENT, deuterated enzalutamide; SD, standard deviation.