Preclinical metabolism and the disposition of vornorexant/TS‐142, a novel dual orexin 1/2 receptor antagonist for the treatment of insomnia

Abstract

We investigated the metabolism and disposition of vornorexant, a novel dual orexin receptor antagonist, in rats and dogs, and clarified in vitro metabolite profiles in humans. Furthermore, we investigated the pharmacokinetics of active metabolites in rats and dogs and their CNS distribution in rats to elucidate its contribution to drug efficacy. [¹⁴C]vornorexant was rapidly and mostly absorbed after the oral administration in rats and dogs. The drug‐derived radioactivity, including metabolites, was distributed to major organs such as the liver, kidneys in rats, and was almost eliminated within 24 h post‐dose in both species. Metabolite profiling revealed that main clearance mechanism of vornorexant was metabolism via multiple pathways by oxidation. The major circulating components were the cleaved metabolites (M10, M12) in rats, and the unchanged form in dogs, followed by M1, and then M3. Incubation with human hepatocytes resulted in formation of metabolites, including M1, M3, M10, and M12. The metabolic pathways were similar in all tested species. Resulting from the PK and CNS distribution of active metabolites (M1 and M3) with weaker pharmacological activity, the concentration of the unchanged form was higher than that of active metabolites in rat CSF and dog plasma, suggesting that the unchanged form mainly contributed to the drug efficacy. These findings demonstrate that vornorexant is absorbed immediately after administration, and vornorexant and its metabolites are rapidly and completely eliminated in rats and dogs. Thus, vornorexant may have favorable pharmacokinetic profiles as a hypnotic drug to provide rapid onset of action and minimal next‐day residual effects in humans.

Proposed metabolic pathways of vornorexant are presented. Vornorexant is rapidly absorbed and undergoes extensive metabolism. Vornorexant and its metabolites are rapidly eliminated without tissue residue. The unchanged form mainly contributes to the drug efficacy.

Abbreviations

AUC_0–t

area under the concentration–time curve from time 0 to t

AUC_0–∞

area under the concentration–time curve from time 0 to infinity

BDC

bile duct cannulated

BLQ

below quantitation limit

CL_total

total plasma clearance

C _max

maximal concentration

CNS

central nervous system

CSF

cerebrospinal fluid

HPLC

high‐performance liquid chromatography

LC–MS

liquid chromatography–mass spectrometry

LC–MS/MS

liquid chromatography–tandem mass spectrometry

M

metabolite number

OX₁ receptor

orexin‐1 receptor

OX₂ receptor

orexin‐2 receptor

PAMPA

parallel artificial membrane permeability assay

P‐gp

P‐glycoprotein

PK

pharmacokinetic

QWBA

quantitative whole‐body autoradiography

RI‐HPLC

high‐performance liquid chromatography equipped with radiochemical flow detection

SD

Sprague–Dawley

t _1/2

terminal half‐life

t _max

time to peak concentration

V _dss

volume of distribution at steady‐state

1. INTRODUCTION

Insomnia is a common sleep disorder that negatively affects daily functioning and decreases the quality of life. ¹ , ² , ³ , ⁴ The symptoms are characterized by difficulties in falling asleep, maintaining sleep, and/or waking up too early. Epidemiological investigations have indicated that insomnia is a risk factor for several physical and psychiatric disorders, including cardiovascular diseases, type 2 diabetes, depression, and others. ⁵ , ⁶ , ⁷

Benzodiazepine‐like medications have been widely prescribed for insomnia. ⁸ These drugs improve sleeping difficulties, but their long‐term use has been reported to be associated with an increased risk of falls, drug abuse, and next‐day cognitive impairment, potentially leading to driving accidents. ⁹ To reduce such next‐day effects, non‐benzodiazepine hypnotics (Z‐drugs) with shorter elimination half‐lives have been developed and are widely used as first‐line therapy. However, the U.S. Food and Drug Administration (FDA) has announced additional caution on prescribing Z‐drugs that have a risk of causing serious injuries more frequently than other hypnotics, due to sleep behaviors such as sleep walking or driving while not fully awake. ¹⁰ Thus, there are unmet needs for safer hypnotic drugs.

To meet the clinical needs, orexin receptor antagonists (ORAs) have been developed. ¹¹ Orexin neuropeptides (Orexin‐A and ‐B) potently regulate sleep and wakefulness via their receptors, the OX₁ and OX₂ receptors. ¹² , ¹³ ORAs exert relatively weaker muscle relaxant effects compared with benzodiazepine receptor agonists because of their different mechanism of action, however, the long elimination half‐life of even ORA is potentially associated with a higher risk of reduced daytime functioning, such as cognitive functioning and driving performance. ¹⁴ , ¹⁵ We considered that safer and more useful hypnotic drugs must be rapidly absorbed and eliminated, with a suitable half‐life, induce rapid sleep onset and allow sleep maintenance throughout the night, and have no next‐day residual effects.

ORAs such as suvorexant, lemborexant, and daridorexant have received approval from the FDA. These drugs are well‐absorbed and promote sleep in humans, ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ , ²¹ but these drugs appear to have relatively long half‐lives (approximately 12 h for suvorexant, 17–19 h effective half‐life for lemborexant, and approximately 8 h for daridorexant) ²² , ²³ , ²⁴ as compared with the Z‐drugs, which have short half‐lives (less than 5 h). ²⁵ , ²⁶ In a regulatory review, 20 mg per night was approved as the maximum dose of suvorexant, but not 40 mg, due to concern about next‐day residual effects, including impaired driving performance. ²⁷ , ²⁸

Therefore, we explored potent ORAs with favorable PK properties, including rapid absorption and relatively short t _1/2, and discovered vornorexant. ²⁹ To achieve a short t _1/2, vornorexant (the active ingredient of TS‐142) was designed to have low lipophilicity and small volume of distribution, but not high clearance, because the bioavailability becomes attenuated with high clearance. We found that vornorexant was rapidly absorbed (T _max <1 h) and eliminated with a short t _1/2 of approximately 1.0 and 2.5 h after the oral administration in rats and dogs, respectively. The predicted human t _1/2, based on the preclinical data, was 0.9–2.0 h. Indeed, vornorexant exhibited the expected pharmacokinetics with rapid absorption (T _max <1 h) and short t _1/2 of 1.32–3.25 h at doses of 1–30 mg in healthy volunteers. ³⁰ Vornorexant has exhibited the potent efficacy for inducing sleep onset and maintaining sleep, and also well tolerability in clinical studies in patients with insomnia. ³¹

Vornorexant seems to have desirable pharmacokinetics, with rapid absorption and relatively short half‐life. However, the detailed pharmacokinetics, such as the clearance mechanism, tissue distribution, mass balance, and metabolite profiles of vornorexant remained unclear. In addition, the presence of active metabolites of vornorexant has not yet been clarified. We considered that elucidation of the metabolism and disposition of vornorexant and its metabolites, including active metabolites, would contribute to effective and safe uses of vornorexant in clinical situations.

Herein, we investigated the metabolism and disposition of vornorexant and its metabolites in rats and dogs after oral or intravenous administration of ¹⁴C‐labeled vornorexant, and clarified the metabolite profiles resulting from in vitro metabolism in rats, dogs, and humans. Furthermore, we investigated the PK of vornorexant and its active metabolites in rats and dogs, and their distribution into CNS in rats, to elucidate the contribution of the active metabolites to drug efficacy. From the present findings, we considered whether vornorexant might exhibit the ideal pharmacokinetics as a hypnotic drug.

2. MATERIALS AND METHODS

2.1. Materials

Vornorexant and authentic metabolite standards were synthesized at Taisho Pharmaceutical (Saitama, Japan). Since vornorexant could be cleaved at the oxazinane ring which is located in the middle of the molecule, we synthesized two types of [¹⁴C]vornorexant, [pyrazole‐¹⁴C]vornorexant and [carbonyl‐¹⁴C]vornorexant, with different labelling positions of ¹⁴C to trace the cleaved metabolites (Figure 1). [carbonyl‐¹⁴C]vornorexant (specific activity, 4.87 MBq/mg; radiochemical purity, >98%) and [pyrazole ring‐¹⁴C]vornorexant (specific activity, 4.29 MBq/mg; radiochemical purity, >98%) were synthesized at Sekisui Medical (Ibaraki, Japan). Cryopreserved hepatocytes from male Sprague–Dawley (SD) rats and male beagle dogs were purchased from Sekisui XenoTech (Kansas City, KS). Cryopreserved hepatocytes from humans were purchased from BioIVT (Westbury, NY). All other chemicals and reagents that were used were commercially available.

FIGURE 1.

Chemical structures of [pyrazole ring‐¹⁴C]vornorexant and [carbonyl‐¹⁴C]vornorexant. Asterisk donates the position of the ¹⁴C label.

2.2. Animals

Male SD and Wistar rats were purchased from Jackson Laboratory Japan (Kanagawa, Japan). Male Long Evans rats were purchased from the Institute for Animal Reproduction (Ibaraki, Japan). Male beagle dogs were purchased from Marshall BioResources Japan (Ibaraki, Japan). The animals were maintained under controlled temperature (24 ± 4°C) and humidity (50 ± 20%) conditions under a 12‐h light/dark cycle. Food and water were provided ad libitum. All animal studies were conducted after the experimental protocols were approved by the Institutional Animal Care and Use Committee.

2.3. Absorption and excretion

The absorption and excretion studies of [¹⁴C]vornorexant were performed in fasting SD rats and dogs (n = 3). Each [¹⁴C]vornorexant with different ¹⁴C‐labeling positions ([carbonyl‐¹⁴C]vornorexant and [pyrazole ring‐¹⁴C]vornorexant) was administered to rats. Since the absorption and excretion of both types of [¹⁴C]vornorexant were similar in rats, only [carbonyl‐¹⁴C]vornorexant was administered to dogs. For intravenous administration, [¹⁴C]vornorexant (3.7–4.87 MBq/mg) was dissolved in polyethylene glycol 400, and administered to rats (1 mg/kg) or dogs (0.5 mg/kg). For oral administration, [¹⁴C]vornorexant (1.23–2.47 MBq/mg) was suspended in 0.5 w/v% methylcellulose 400, and administered to both animals at a dose of 3 mg/kg. Blood samples were collected at 0.083 (only intravenous), 0.25, 0.5, 1, 2, 4, 6, 8, 24, 48, 72, 96, 120, 144, and 168 h post‐dose, and centrifuged to obtain plasma. Urine samples were collected at 0–8, 8–24, 24–48, 48–72, 72–96, 96–120, 120–144, and 144–168 h post‐dose. Fecal samples were collected at 24‐h intervals until 168 h post‐dose, mixed with 4 times the sample volume of distilled water, and homogenized. To obtain bile samples up to 48 h post‐dose, each type of [¹⁴C]vornorexant was administered to bile‐duct cannulated (BDC) rats.

2.4. Quantitative whole‐body autoradiography

After each type of [¹⁴C]vornorexant (1.23 MBq/mg) was administered orally at a single dose of 3 mg/kg to fasting Long Evans rats, the distribution of radioactivity was determined by quantitative whole‐body autoradiography (QWBA). The rats were euthanized at 0.5, 2, 6, 24, 72, and 168 h post‐dose. The experimental conditions are described in Table S1.

2.5. Measurement of radioactivity

All samples were mixed with a liquid scintillation cocktail and the radioactivity levels measured using a liquid scintillation counter (LSC, PerkinElmer) for 2 min. Where necessary, the samples were solubilized with Soluene‐350 (PerkinElmer, Waltham, MA, USA) and decolorized.

2.6. In vitro metabolite profiling

Hepatocyte suspensions from rats, dogs, and humans were prepared using a thawing medium, the hepatocyte isolation kit (XenoTech) for rats and dogs or InVitroGRO HT Medium (Bioreclamation IVT) for humans. A 50‐μL aliquot of [pyrazole ring‐¹⁴C]vornorexant or [carbonyl‐¹⁴C]vornorexant (final 25 μmol/L) in Leibovitz's medium was added to a 50‐μL aliquot of the hepatocyte suspensions (final 0.5 × 10⁶ cells/mL). After 4‐h incubation at 37°C, 200 μL of acetonitrile/methanol (9:1, v/v) was added to terminate the reactions. After the mixtures were centrifuged (2607g, 4°C, 10 min), the supernatants were injected into an HPLC system equipped with a radioisotope detector (RI‐HPLC) and the liquid chromatography‐mass spectrometry (LC–MS) system to elucidate the in vitro metabolite profiles of vornorexant (see Table S2A).

2.7. In vivo metabolite profiling

[¹⁴C]vornorexant (final 1.23–2.47 MBq/mg) was administered orally at a single dose of 3 mg/kg to fasting SD rats and dogs. Plasma (0.5 h), urine (0–8 h), feces (0–24 h) from rats, and plasma (1 and 4 h), urine (0–24 h), feces (0–48 h) from dogs were collected using the same methods as described in absorption and excretion studies. Equal volumes of plasma samples were pooled, and urine and fecal homogenate samples were pooled based on the sample weights (n = 3). The sample processing and the analysis conditions are described in Table S2A.

2.8. Parallel artificial membrane permeability assay

The membrane permeability of vornorexant (10 μmol/L) was measured using a parallel artificial membrane permeability assay (PAMPA) Evolution instrument (Pion, Billerica, MA), as described in previous report. ³²

2.9. PK in rats and dogs

The PK of vornorexant, M1, and M3 in fasting SD rats and dogs were determined following single intravenous or the oral administration of vornorexant (n = 3). Vornorexant was dissolved in polyethylene glycol 400, and administered intravenously to rats (1 mg/kg) or dogs (0.5 mg/kg). Vornorexant was suspended in 0.5 w/v% methylcellulose 400, and administered orally to rats and dogs at a dose of 3 mg/kg. Blood samples were collected in tubes containing EDTA‐2K at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h post‐dose. Plasma were obtained by centrifugation (8000g, 4°C, 5 min), and treated with acetonitrile/methanol (9:1, v/v) containing the internal standard (IS). After centrifugation, the supernatants were subjected to the validated high‐performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis (see Table S2B).

The distributions of vornorexant, M1, and M3 into the CNS in Wistar rats were investigated after single oral administration of vornorexant (3 mg/kg). Blood samples were collected at 0.25, 0.5, 1, 2, 3, and 4 h post‐dose, and plasma was obtained by centrifugation. The brain and cerebrospinal fluid (CSF) were excised at the same time as the blood. The brain and CSF samples were treated with acetonitrile/methanol (9:1, v/v) containing the IS. After the centrifugation, the supernatants were subjected to the validated LC–MS/MS analysis (see Table S2B).

2.10. PK analysis

The plasma concentrations versus time data were analyzed by non‐compartmental analysis using the PK analysis software WinNonlin (Certara, Princeton, NJ). The oral BA and fraction absorbed of radioactivity were calculated using the following equation:

$$\begin{array}{c}\mathrm{BA}\text{or fraction absorbed}\left(\%\right)=\text{Dose}-\text{normalized}{\mathrm{AUC}}_{\text{oral},0–\infty }/\hfill \\ \text{Dose}-\text{normalized}{\mathrm{AUC}}_{\mathrm{i}.\mathrm{v}.,0–\infty }\times 100\hfill \end{array}$$

2.11. Plasma protein binding

Plasma protein binding ratios of vornorexant, M1, and M3 were examined using an equilibrium dialysis method. The experimental condition is described in Table S3A.

2.12. Evaluation of the substrate for P‐glycoprotein (P‐gp)

The transport studies of vornorexant, M1, and M3 were conducted in human P‐gp‐expressing LLC‐PK1 cells. The experimental conditions are described in Table S3B.

3. RESULTS

3.1. Absorption and excretion of [ ¹⁴C]vornorexant in rats and dogs

The plasma radioactivity levels after a single intravenous or the oral administration of [¹⁴C]vornorexant to rats and dogs were evaluated, and the calculated PK parameters are summarized in Table 1. In rats, after the oral administration (3 mg/kg) of [pyrazole ring‐¹⁴C]vornorexant or [carbonyl‐¹⁴C]vornorexant, the plasma radioactivity levels increased rapidly and reached the C _max of 2900 or 1780 ng eq./mL, respectively, within 0.25 h post‐dose. The AUC (3890, 4480 h·ng eq./mL) of the radioactivity were more than 35‐fold higher than the AUC (107 h·ng/mL as shown in Table 4) of vornorexant after the oral administration at same dose, indicating that the plasma radioactivity included high level of metabolites. In dogs, after the oral administration of [carbonyl‐¹⁴C]vornorexant (3 mg/kg), the plasma radioactivity level increased rapidly and reached the C _max of 5950 ng eq./mL within 1 h post‐dose. The AUC (54 800 h·ng eq./mL) of the radioactivity was more than 5‐fold higher than the AUC (10 700 h·ng/mL as shown in Table 4) of vornorexant after the oral administration at same dose. The oral absorption of drug‐derived radioactivity after the oral administration of [¹⁴C]vornorexant was more than 74.9% in rats and dogs.

TABLE 1.

Pharmacokinetic parameters of the radioactivity in the plasma of rats and dogs after a single intravenous and oral administration [¹⁴C]vornorexant.

Species	Dosed compound	Route	C max (ng eq./mL)	t max (h)	AUC0–∞ (h·ng eq./mL)	Absorption (%)
Rat	[pyrazole ring‐14C]vornorexant	i.v.	–	–	1550 ± 30	–
		Oral	2900 ± 250	0.25 ± 0.00	3890 ± 460	83.7
	[carbonyl‐14C]vornorexant	i.v.	–	–	1900 ± 110	–
		Oral	1780 ± 820	0.25 ± 0.00	4480 ± 1370	78.6
Dog	[carbonyl‐14C]vornorexant	i.v.	–	–	12 200 ± 4000	–
		Oral	5950 ± 370	1.0 ± 0.0	54 800 ± 12 400	74.9

Note: Intravenous dose: 1 and 0.5 mg/kg in rats and dogs, respectively, Oral dose: 3 mg/kg in rats and dogs. Data are presented as the mean ± S.D. (n = 3).

Abbreviations: –, not applicable; i.v., intravenous.

TABLE 4.

Plasma pharmacokinetic parameters of vornorexant, M1, and M3 after a single intravenous or oral administration of vornorexant to male rats and dogs.

Species	Rats				Dogs
Route	Intravenous	Oral			Intravenous	Oral
Monitored compounds	Vornorexant	Vornorexant	M1	M3	Vornorexant	Vornorexant	M1	M3
CLtotal (mL/h/kg)	2150 ± 160	–	–	–	163 ± 18	–	–	–
V dss (mL/kg)	726 ± 96	–	–	–	316 ± 31	–	–	–
t 1/2 (h)	0.37 ± 0.07	0.96 ± 0.43	1.1 ± 0.2	1.2 ± NC	1.3 ± 0.4	2.7 ± 0.8	4.6 ± 0.6	5.2 ± 2.5
C max (ng/mL)	–	55.4 ± 36.5	82.5 ± 46.5	30.9 ± 14.9	–	2360 ± 460	491 ± 77	134 ± 33
t max (h)	–	0.67 ± 0.29	0.83 ± 0.29	1.0 ± 0.0	–	1.3 ± 0.6	2.0 ± 0.0	2.0 ± 0.0
AUC0–∞ (h·ng/mL)	467 ± 34	107 ± 66	176 ± 100	86.1 ± NC	3100 ± 380	10 700 ± 1800	3500 ± 340	1250 ± 370
BA a (%)	–	7.6	–	–	–	58.0	–	–

Note: Intravenous dose: 1 mg/kg and 0.5 mg/kg in rats and dogs, respectively, Oral dose: 3 mg/kg in rats and dogs. Data are presented as the mean ± S.D. (n = 3) except for bioavailability which represents as the mean value of three animals.

Abbreviations: –, not applicable; NC, not calculated.

^a Bioavailability = (dose‐normalized AUC_0–∞ following the oral administration)/(dose‐normalized AUC_0–∞ following the intravenous administration) × 100.

The cumulative excretions of radioactivity in rats and dogs after a single oral administration of [¹⁴C]vornorexant (3 mg/kg) are shown in Figure 2. After the oral administration of [pyrazole ring‐¹⁴C]vornorexant to rats, 56.9% and 41.8% of dose were excreted in the urine and feces, respectively. After the oral administration of [carbonyl‐¹⁴C]vornorexant to rats, 35.1% and 58.9% of dose were excreted in the urine and feces, respectively. After the oral administration of [carbonyl‐¹⁴C]vornorexant to dogs, 51.1% and 45.3% of dose were excreted in urine and feces, respectively. In rats and dogs, more than 82.7% of dose was rapidly excreted within 24 h post‐dose, and more than 94.1% of dose was excreted by 168 h post‐dose. The same pattern of excretion as that after the oral administration was observed after a single intravenous administration of [¹⁴C]vornorexant to rats and dogs (see Table S4).

FIGURE 2.

Cumulative excretion of radioactivity after a single oral administration of (A) [pyrazole ring‐¹⁴C]vornorexant in rats, (B) [carbonyl‐¹⁴C]vornorexant in rats, and (C) [carbonyl‐¹⁴C]vornorexant in dogs. The drug was administered at a dose of 3 mg/kg in each case. Data are represented as the means ± SD (n = 3).

After a single oral administration of [pyrazole ring‐¹⁴C]vornorexant and [carbonyl‐¹⁴C]vornorexant to BDC rats, 42.1% and 59.8% of dose, respectively, were excreted in bile, 50.5% and 31.0% of dose, respectively, were excreted in urine, and 7.8% and 6.4% of dose, respectively, were excreted in feces (see Table S4). The excreted radioactivity in bile of BDC rats was similar to that in feces of intact rats, suggesting that most of the radioactivity detected in feces was derived from biliary excretion.

3.2. Distribution in the rats

After a single oral administration of [pyrazole ring‐¹⁴C]vornorexant or [carbonyl‐¹⁴C]vornorexant to rats at a dose of 3 mg/kg, the distribution of radioactivity was investigated by QWBA (Table 2). At 0.5 h post‐dose, the liver, kidneys, stomach, or small intestine exhibited more than 3‐folds higher radioactivity concentrations than the blood. The radioactivity levels in cerebellum, cerebrum, eyeballs, medulla oblongata, and spinal cord were less than about one‐fifth of the blood. At 24 h post‐dose, radioactivity was detectable in the liver, kidneys, and pigmented skin, but in none of the other tissues. At 72 h post‐dose, radioactivity was absent or less than 1% of the respective C _max values, even in the liver, kidneys, and pigmented skin, indicating that no significant radioactivity remained in any tissue.

TABLE 2.

Tissue concentrations of radioactivity in rats after a single oral administration of [¹⁴C]vornorexant (3 mg/kg).

Tissue	Concentration of radioactivity (ng eq./mL or g)
	[pyrazole ring‐14C]vornorexant						[carbonyl‐14C]vornorexant
	0.5 h	2 h	6 h	24 h	72 h	168 h	0.5 h	2 h	6 h	24 h	72 h	168 h
Adrenal gland	1350	276	27.0	ND	ND	ND	1180	186	46.6	ND	ND	ND
Aorta	973	307	ND	ND	ND	ND	578	116	ND	ND	ND	ND
Blood	974	466	59.3	ND	ND	ND	1340	293	60.3	ND	ND	ND
Bone	77.9	41.7	ND	ND	ND	ND	323	29.6	ND	ND	ND	ND
Bone marrow	467	135	ND	ND	ND	ND	341	21.5	ND	ND	ND	ND
Brown fat	813	119	ND	ND	ND	ND	796	152	50.7	ND	ND	ND
Cecum	732	ND	694	ND	ND	ND	1570	737	764	ND	ND	ND
Cerebellum	92.7	ND	ND	ND	ND	ND	127	30.9	29.4	ND	ND	ND
Cerebrum	84.5	ND	ND	ND	ND	ND	122	30.9	ND	ND	ND	ND
Eyeball	192	133	43.1	ND	ND	ND	276	129	65.7	ND	ND	ND
Harderian gland	801	112	27.0	ND	ND	ND	612	91.4	ND	ND	ND	ND
Heart	793	185	35.1	ND	ND	ND	684	96.8	ND	ND	ND	ND
Kidneys	5940	669	72.8	ND	ND	ND	3370	1070	181	28.3	ND	ND
Large intestine	578	152	51.8	ND	ND	ND	597	160	24.0	ND	ND	ND
Liver	9250	1440	345	124	46.2	ND	6890	1690	589	121	55.6	21.6
Lung	2040	388	51.2	ND	ND	ND	1190	137	52.0	ND	ND	ND
Mandibular gland	564	146	ND	ND	ND	ND	595	77.4	26.7	ND	ND	ND
Medulla oblongata	84.5	ND	ND	ND	ND	ND	116	46.4	ND	ND	ND	ND
Pancreas	721	210	ND	ND	ND	ND	762	121	42.7	ND	ND	ND
Pituitary gland	545	160	ND	ND	ND	ND	578	134	37.4	ND	ND	ND
Prostate gland	436	82.9	ND	ND	ND	ND	542	87.7	ND	ND	ND	ND
Seminal vesicle	431	24.9	199	ND	ND	ND	1540	227	ND	ND	ND	ND
Skeletal muscle	376	49.5	ND	ND	ND	ND	310	48.4	ND	ND	ND	ND
Skin (pigmented area)	776	138	ND	ND	ND	ND	584	194	153	51.5	ND	ND
Skin (white area)	704	41.7	ND	ND	ND	ND	594	134	95.9	ND	ND	ND
Small intestine	3230	253	ND	ND	ND	ND	991	12 300	ND	ND	ND	ND
Spinal cord	73.6	ND	ND	ND	ND	ND	80.2	ND	ND	ND	ND	ND
Spleen	534	77.4	ND	ND	ND	ND	419	96.8	ND	ND	ND	ND
Stomach	3810	195	ND	ND	ND	ND	1500	161	35.6	ND	ND	ND
Testis	209	44.2	ND	ND	ND	ND	207	53.8	ND	ND	ND	ND
Thymus	373	75.5	23.3	ND	ND	ND	364	56.5	ND	ND	ND	ND
Thyroid gland	646	337	ND	ND	ND	ND	973	201	82.7	ND	ND	ND
White fat	490	52.1	ND	ND	ND	ND	310	126	ND	ND	ND	ND

Abbreviation: ND, not detected or BLQ.

3.3. In vitro metabolite profiling

Metabolite profiling was conducted for two types of [¹⁴C]vornorexant with different ¹⁴C‐labeling positions ([pyrazole ring‐¹⁴C]vornorexant and [carbonyl‐¹⁴C]vornorexant) to trace the cleaved metabolites around the oxazinane ring. The metabolites of [¹⁴C]vornorexant in cryopreserved hepatocytes from rats, dogs, and humans were investigated by RI‐HPLC (Figure 3). After 4‐h incubation, the metabolic rates of vornorexant in the rat, dog, and human hepatocytes were 39.4%, 10.2%, and 16.6%, respectively. Vornorexant was more rapidly eliminated from rat hepatocytes compared to dog and human hepatocytes. While in the rat hepatocytes, M10 or M12 were the major metabolites, followed by M1, M3, and M5. In the dog and human hepatocytes, vornorexant was the main component, followed by M10, M12, M1, M3, and M5. No human specific metabolite was observed.

FIGURE 3.

Representative HPLC‐radiochromatograms of each of the [¹⁴C]vornorexant metabolites found in rat (A), dog (B), and human (C) hepatocytes. Rat, dog, or human cryopreserved hepatocytes at 0.5 million cells/mL were incubated at 37°C for 4 h with [pyrazole ring‐¹⁴C]vornorexant or [carbonyl‐¹⁴C]vornorexant.

3.4. In vivo metabolite profiling in rats and dogs

The metabolites in plasma, urine, and feces of rats and dogs after a single oral administration of [pyrazole ring‐¹⁴C]vornorexant or [carbonyl‐¹⁴C]vornorexant at a dose of 3 mg/kg were analyzed by RI‐HPLC (Figure 4 and Table 3).

FIGURE 4.

Representative HPLC‐radiochromatograms of plasma extracts of rats at 0.5 h post‐dose after a single oral administration of (A) [pyrazole ring‐¹⁴C]vornorexant, (B) [carbonyl‐¹⁴C]vornorexant, and (C) of the plasma extract of dogs at 1 h post‐dose after a single oral administration of [carbonyl‐¹⁴C]vornorexant. The drug was administered at a dose of 3 mg/kg in each case.

TABLE 3.

Composition of the metabolites of the drug in the urine and feces of rats and dogs after a single oral administration of [¹⁴C]vornorexant (3 mg/kg).

Metabolite	% of dose
	Rat				Dog
	Urine (0–8 h)		Feces (0–24 h)		Urine (0–24 h)	Feces (0–48 h)
	[P‐14C]vornorexant	[C‐14C]vornorexant	[P‐14C]vornorexant	[C‐14C]vornorexant	[C‐14C]vornorexant	[C‐14C]vornorexant
Vornorexant	–	–	–	–	–	–
M1	–	–	–	–	–	2.6
M2, M22	–	–	–	–	–	1.0
M4	–	–	5.7	4.6	–	5.1
M5	–	–	13.5	11.7	–	3.5
M6	–	–	6.7	5.9	–	–
M8	–	–	–	3.3	1.0	–
M9	–	–	–	–	–	–
M10	47.6	–	10.6	–	–	–
M12	–	13.7	–	9.3	27.4	3.2
M15	–	–	–	–	–	5.7
M16, M17	–	5.0 a	–	8.8 a	5.3	4.6
M18	3.8	–	–	–	–	–
M21	–	–	–	4.1	7.7	2.6
M23	–	–	–	–	–	5.3
M24	–	–	–	–	–	1.2
M31	–	–	–	–	1.0	–
M33	–	–	–	–	–	1.6
Total excretion	52.6	30.6	39.4	54.9	47.5	43.4

Note: [P‐¹⁴C]vornorexant: [pyrazole ring‐¹⁴C]vornorexant, [C‐¹⁴C]vornorexant: [carbonyl‐¹⁴C]vornorexant.

Abbreviation: –, not detected.

^a M17 was only detected.

The main component in rat plasma after the oral administration of [pyrazole ring‐¹⁴C]vornorexant at 0.5 h post‐dose was M10 (56.4% of the plasma radioactivity) (Figure 4). Vornorexant, M1, M3, M9, and M20 accounted for 6.4%, 6.1%, 4.0%, 13.7%, and 7.5% of the radioactivity, respectively. The main component in the rat plasma after the oral administration of [carbonyl‐¹⁴C]vornorexant at 0.5 h post‐dose was M12 (29.7% of the plasma radioactivity). Vornorexant, M1, M3, M16, M17, and M19 accounted for 13.8%, 18.4%, 6.9%, 4.8%, 7.4%, and 8.7% of the radioactivity, respectively.

The main component in dog plasma at 1 h after administration of [carbonyl‐¹⁴C]vornorexant was vornorexant, which accounted for 55.1% of the plasma radioactivity. M1, M3, a mixture of M15 and M32, and M12 accounted for 15.0%, 2.3%, 13.4%, and 4.9% of the radioactivity, respectively. Since M10 and M12 were simultaneously formed by oxidative ring opening and subsequent cleavage, M10 was considered to be formed as a non‐labeled compound. A similar metabolite profile was observed at 4 h after administration of [carbonyl‐¹⁴C]vornorexant.

The major components in rat urine up to 8 h after the oral administration of [pyrazole ring‐¹⁴C]vornorexant or [carbonyl‐¹⁴C]vornorexant were M10 and M12, accounting for 47.6% and 13.7% of dose, respectively (Table 3). The major components in rat feces up to 24 h after the oral administration of [pyrazole ring‐¹⁴C]vornorexant were M5 and M10, accounting for 13.5% and 10.6% of dose, respectively. The major components in rat feces up to 24 h after the oral administration of [carbonyl‐¹⁴C]vornorexant were M5 and M12, accounting for 11.7% and 9.3% of dose, respectively.

The major metabolite in dog urine up to 24 h after the oral administration of [carbonyl‐¹⁴C]vornorexant was M12, accounting for 27.4% of dose, followed by M21 (7.7%) and a mixture of M16 and M17 (5.3%). The major metabolites in dog feces up to 48 h after the oral administration of [carbonyl‐¹⁴C]vornorexant were M15, M23, and M4, accounting for 5.7%, 5.3%, and 5.1% of dose, respectively.

Vornorexant was not detected in the urine and feces of rats and dogs after the oral administration of [¹⁴C]vornorexant. The predominant components in excreta were metabolites in rats and dogs, indicating that the main clearance mechanism of vornorexant is metabolism.

3.5. Identification of the metabolites in vitro and in vivo

The metabolites found in the in vitro and in vivo samples were characterized based on their mass spectral data, summarized in Table S5. A total of 28 metabolites were identified or estimated with a mass accuracy of <3 ppm. LC–MS/MS mass spectra showed several characteristic fragmentation patterns that allowed clear structural estimation of the metabolites. In addition, vornorexant, M1, M2, M3, M10, M11, M12, and M15 were identified by comparison of their retention times and mass spectral data with those of the authentic standards.

3.6. PK of vornorexant and its metabolites in rats and dogs

Among the identified metabolites, M1 and M3, which retain the oxazinane ring, are known to show weaker pharmacological activity than that of vornorexant (Table S6). The plasma concentration–time profiles of vornorexant and its active metabolites, M1 and M3, after a single intravenous or the oral administration of vornorexant to rats and dogs are shown in Figure 5, and the calculated PK parameters are summarized in Table 4.

FIGURE 5.

Plasma concentration–time profiles of vornorexant, M1, and M3 in male rats (left) and dogs (right) after a single intravenous (iv) or oral (po) administration. The oral dose was 3 mg/kg, and the intravenous dose was 1 mg/kg in rats and 0.5 mg/kg in dogs. Data are represented as the means + SD (n = 3). The lower limit of quantification of vornorexant, M1, and M3 was 0.1 ng/mL.

After intravenous administration (1 mg/kg) to rats, the CL_total and V _dss were estimated to be 2150 mL/h/kg and 726 mL/kg, respectively. After the oral administration (3 mg/kg) to rats, plasma vornorexant reached a C _max of 55.4 ng/mL at 0.67 h post‐dose, and rapidly declined with a t _1/2 of 0.96 h. The oral BA was 7.6% in rats. The C _max and AUC_0–∞ of M1 were approximately 1.5‐fold higher than those of vornorexant, while the C _max and AUC_0–∞ of M3 were 0.56‐ and 0.80‐fold those of vornorexant, respectively. Plasma M1 and M3 reached C _max within 1.0 h post‐dose, and rapidly declined with a t _1/2 of approximately 1.0 h.

After intravenous administration (0.5 mg/kg) to dogs, the CL_total and V _dss were estimated to be 163 mL/h/kg and 316 mL/kg. After the oral administration (3 mg/kg) to dogs, plasma vornorexant reached C _max of 2360 ng/mL at 1.3 h post‐dose, and rapidly declined with a t _1/2 of 2.7 h. The oral BA was 58.0% in dogs. The C _max and AUC_0–∞ of M1 were less than 0.33‐fold those of vornorexant, and the C _max and AUC_0–∞ of M3 were less than 0.12‐fold those of vornorexant, respectively. Plasma M1 and M3 reached C _max at 2.0 h post‐dose, and rapidly declined with t _1/2 of 4.6 and 5.2 h, respectively.

3.7. Transfer into the brain and CSF in rats

The brain and CSF penetration of vornorexant, M1, and M3 after a single oral administration of vornorexant was evaluated in rats after the oral administration at an effective dose of 3 mg/kg (Figure 6), and the calculated PK parameters are summarized in Table 5. The concentration in CSF was used as a surrogate for the unbound concentration in brain. Concentration of vornorexant in brain and CSF reached C _max of 7.44 ng/g and 2.14 ng/mL, respectively, at 1.0 h post‐dose, and then decreased as same time‐profile as the plasma concentration. The C _max of M1 and M3 were 1.17 and 5.57 ng/g in brain, and 1.21 and 0.755 ng/mL in CSF, respectively, indicating that in the CNS, the C _max values of M1 and M3 were lower than the C _max of vornorexant in rats. The concentrations of M1 and M3 in brain and CSF changed with the same time‐profile as the corresponding plasma concentrations.

FIGURE 6.

Concentration–time profiles of vornorexant, M1, and M3 in the plasma, brain, and CSF after a single oral administration of vornorexant (3 mg/kg) in rats. Data are represented as the means ± SD (n = 3). The lower limits of quantification of vornorexant, M1, and M3 were 0.1 ng/mL for plasma, 0.5 ng/g for brain, and 0.2 ng/mL for CSF, respectively.

TABLE 5.

Pharmacokinetic parameters of vornorexant, M1, and M3 after a single oral administration of vornorexant to male rats.

Monitored compound	Unchanged form			M1			M3
Tissues	Plasma	Brain	CSF	Plasma	Brain	CSF	Plasma	Brain	CSF
t max (h)	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0
C max (ng/mL or ng/g)	48.4	7.44	2.14	68.4	1.17	1.21	25.3	5.57	0.755
t 1/2 (h)	0.71	0.71	–	0.89	–	–	0.83	0.74	–
AUC0–t (h·ng/mL or h·ng/g)	87.7	13.9	3.55	140	1.63	2.25	48.6	10.9	1.07
fu,p	0.088	–	–	0.323	–	–	0.029	–	–
Unbound C max a (ng/mL)	4.26	–	2.14	22.1	–	1.21	0.734	–	0.755
Unbound AUC0–t a (h·ng/mL)	7.72	–	3.55	45.2	–	2.25	1.41	–	1.07
Unbound AUC ratio to plasma	–	–	0.46	–	–	0.05	–	–	0.76

Note: Oral dose: 3 mg/kg in rats. Data are presented as the mean (n = 3).

Abbreviation: –, not applicable.

^a Unbound AUC_0–t and C _max for plasma were calculated using the free plasma fraction (f_u,p). The CSF drug concentrations were assumed to be the unbound concentrations, because of low protein levels in the CSF.

The unbound CSF/plasma AUC ratios of the unchanged form and M3 were 0.46 and 0.76, respectively, indicating that the CSF concentrations of the unchanged form and M3 were similar to their respective free plasma concentrations. On the other hand, the unbound CSF/plasma AUC ratio of M1 was 0.05, and the CSF concentration of M1 was very low compared to the free plasma concentration (Table 5).

3.8. Evaluation of the substrate for P‐gp

The potential of vornorexant, M1, and M3 as a substrate for P‐gp was evaluated in human P‐gp‐expressing LLC‐PK1 cells. The efflux ratio of vornorexant, M1, M3, and quinidine (positive control) was 2.43, 16.3, 1.43, and 14.8, respectively, indicating that M1 was a strong substrate for P‐gp.

4. DISCUSSION

We investigated the metabolism and disposition of vornorexant and its metabolites in rats and dogs, and the metabolite profiles resulting from in vitro metabolism in rats, dogs, and humans. Furthermore, we also investigated the PK of the active metabolites in rats and dogs and their distribution into CNS in rats, to elucidate the contribution of the metabolites to the drug efficacy.

After the oral administration, [¹⁴C]vornorexant was rapidly (t _max: <1.0 h) and mostly absorbed (74.9%–83.7% of the administered dose) in rats and dogs. The apparent membrane permeability of vornorexant in PAMPA at pH 6.2 was 83.0 (10⁻⁶ cm/s), suggesting that vornorexant has high permeability in intestine. In drug discovery stage, vornorexant was designed to reduce lipophilicity and volume of distribution to achieve a short half‐life. ²⁹ The lipophilicity of compound was reduced, with no compromise of its gastrointestinal absorption. These observations suggest that vornorexant also exhibits good absorbability in humans, just as in animals.

The distribution of vornorexant and its metabolites was investigated as the drug‐derived radioactivity after the oral administration of each [¹⁴C]vornorexant in rats. The drug‐derived radioactivity was rapidly distributed to all tissues. The major distribution was observed in the liver, kidneys, and gastrointestinal tract, and the radioactivity levels in most other tissues were similar to or lower than the level in the blood. Furthermore, the radioactivity in almost all tissues was rapidly and almost completely eliminated within 24 h post‐dose and not remained. Metabolite profiling in rat and human hepatocytes revealed that the human metabolites were also detected in rats. These data suggest that the metabolites detected in human hepatocytes could be rapidly excreted in humans in vivo.

Complete mass balance of [¹⁴C]vornorexant was confirmed in rats and dogs, with the 94.1%–99.0% recovery of the total radioactivity from the excreta up to 168 h after oral administration. The excretion route of drug‐derived radioactivity was both urine and feces in rats and dogs after oral administration. The unchanged form was not excreted to these excreta in the animals, and the excreted radioactivity was derived from metabolites. In human PK study, almost no unchanged form was excreted to urine (<0.1% of dose). ³⁰ These observations indicate that the main clearance mechanism of vornorexant is assumed to be metabolism in human and animals. Further investigation of the main clearance mechanism of vornorexant will be reported in a human mass balance study.

The plasma concentrations of vornorexant after intravenous or the oral administration of vornorexant to rats and dogs were determined by a validated LC–MS/MS method. The PK profile of vornorexant was similar to that in previous reports. ²⁹ The V _dss of vornorexant was small in rats (0.73 L/kg) and dogs (0.32 L/kg), and was close to the total amount of body water in rats (0.67 L/kg) and dogs (0.60 L/kg). ³³ The CL_total of vornorexant was moderate in rats (2150 mL/h/kg) and low in dogs (163 mL/h/kg). In human PK of vornorexant at 10 mg, the Vd/F and CL/F were reported to be 28.9 L and 10.5 L/h, respectively. ³⁰ The small Vd of vornorexant, but not CL, accounts for the short t _1/2 in animals and humans. In addition, the BA was higher in dogs (58.0%) than rats (7.6%). Since [¹⁴C]vornorexant was mostly absorbed in both animals, the difference in the BA between rats and dogs is speculated to be mainly attributable to first‐pass metabolism. Evaluation of the metabolic stability in hepatocytes showed that vornorexant was more stable in dogs and humans than in rats. Considering the superior metabolic stability in human and dog hepatocytes, a high BA of vornorexant in humans is expected.

To clarify the major metabolic pathways, in vitro metabolite profiling of vornorexant was carried out using rat, dog, and human hepatocytes. In addition, in vivo metabolite profiling was conducted in rats and dogs. A total of 28 metabolites were identified or estimated. The results suggested that vornorexant is metabolized via multiple metabolic pathways, as shown in Figure 7. The main metabolic pathways are (1) oxidation of the methyl group in the methylphenyl moiety, (2) oxidative ring opening of the oxazinane ring, and subsequent cleavage at the N‐α‐position of the amide moiety, (3) oxidative ring opening of the oxazinane ring and subsequent dealkylation in the oxazinane moiety, and (4) dehydrogenation of the oxazinane ring. The major circulating metabolites in rats were the cleaved metabolites (M10 and M12), and the unchanged form was minor. In dogs, the unchanged form was predominant component, followed by M1, and then M3. Although the major circulating components differed, the metabolites and metabolic pathways were similar in all the tested species.

FIGURE 7.

Proposed metabolic pathways of vornorexant. The structures of the metabolites were identified or estimated. The characters R, D, and H refer to rat, dog, and human, respectively.

The metabolite profiles in the hepatocytes from each species were considerably similar to that in the corresponding species in vivo. Incubation with human hepatocytes resulted in the formation of several metabolites including M1, M3, M5, M10, and M12. All human metabolites were also detected in rats or dogs, used in the safety assessments of vornorexant. These hepatocytes metabolites were generated in human liver microsomes in a NADPH‐dependent manner (data not shown), suggesting that CYP might be involved.

Among the metabolites of vornorexant, M1 and M3 are active metabolites with weaker potency against OX₁ and OX₂ receptors (Table S6). We investigated the PK profiles of vornorexant and the active metabolites and their distribution into CNS in rats to elucidate the contribution of the active metabolites to the drug efficacy. The drug concentration in CSF was used as a surrogate for the concentration of unbound fraction in brain. In rats, M1 was the predominant component in plasma, followed by the unchanged form, and then M3. However, M1 was hardly distributed to CNS (unbound CSF/plasma AUC ratio: 0.46 for the unchanged form, 0.05 for M1, and 0.76 for M3). Considering that M1 was a substrate of human P‐gp, M1 might be hardly distributed to CSF in rats because rat P‐gp restricted the distribution of M1. Thus, the unchanged form was the predominant component in the rat CSF. The concentration of vornorexant in CSF exceeded the in vitro effective concentration (rOX₁R Kb: 0.20 ng/mL, rOX₂R Kb: 0.36 ng/mL) ²⁹ immediately after administration, while the active metabolites had lower exposure in CSF and weaker pharmacological activity than the unchanged form (Table S6). In dogs and humans, ³⁰ the unchanged form was the predominant component in plasma. Assuming that there are no species differences in the distribution into CNS, the unchanged form may be considered as being the main component in CSF and contribute to the drug efficacy in dogs and humans. Furthermore, the brain and CSF concentrations of vornorexant and its active metabolites changed with the same time‐profiles as the plasma concentration in rats. Thus, the plasma unchanged drug levels are expected to be a possible indicator for the drug efficacy in animals and humans. Active metabolites in plasma were eliminated with a t _1/2 of 1.1–1.2 h in rats and 4.6–5.2 h in dogs. Although the t _1/2 of the active metabolites in dogs was about 1.7–1.9‐fold longer than that of the unchanged form, the plasma concentrations of the active metabolites at 8 h post‐dose were lower than half of the plasma concentration of the unchanged form. These data suggest that vornorexant and its active metabolites are rapidly eliminated from the target tissues, with no residual activity, in rats and dogs, consistent with the short‐acting pharmacological effects with minimal next‐day residual effects in humans.

The overall metabolism and disposition of vornorexant were investigated in rats and dogs, as also the in vitro metabolite profile in humans. [¹⁴C]vornorexant was rapidly and mostly absorbed after the oral administration in rats and dogs. The drug‐derived radioactivity, including metabolites, was rapidly distributed to major organs such as the liver and kidneys in rats, and almost completely eliminated to urine and feces within 24 h post‐dose. In vitro and in vivo metabolite profiling revealed that the main clearance mechanism of vornorexant was metabolism via multiple pathways by oxidation. The metabolic pathways were similar in all tested species, although the main circulating metabolites were different. The CSF concentration–time profiles of vornorexant and its active metabolites, M1 and M3, in rats indicated that the unchanged form mainly contributed to the drug efficacy in rats. Considering the human plasma concentrations of vornorexant and its active metabolites, ³⁰ the unchanged form, but not the active metabolites, was suggested to be main contributor to the drug efficacy in humans. Furthermore, our findings suggest that vornorexant might be absorbed and distributed into CNS immediately after administration to exert drug effect as the unchanged form, and vornorexant and even its metabolites might be rapidly and completely eliminated in humans. Such an ideal pharmacokinetic profile of vornorexant as a hypnotic drug might explain the results of clinical trials, ³¹ which have shown that vornorexant has a rapid onset of action and minimal next‐day residual effects.

AUTHOR CONTRIBUTIONS

Participated in the research design: Konno, Kamigaso, Toki, Terasaka, Hikichi, Endo, Yamaguchi, and Mizuno‐Yasuhira. Conducted the experiments: Kamigaso, Toki, and Terasaka. Performed data analysis: Konno, Kamigaso, Toki, and Terasaka. Wrote or contributed to the writing of the manuscript: Konno, Kamigaso, Terasaka, Yamaguchi, and Mizuno‐Yasuhira.

DISCLOSURES

The authors declare that there are no conflicts of interest.

ETHICS STATEMENT

All animal studies were conducted after the experimental protocols were approved by the Institutional Animal Care and Use Committee.

Supporting information

Table S1.

Konno Y, Kamigaso S, Toki H, et al. Preclinical metabolism and the disposition of vornorexant/TS‐142, a novel dual orexin 1/2 receptor antagonist for the treatment of insomnia. Pharmacol Res Perspect. 2024;12:e1183. doi: 10.1002/prp2.1183

DATA AVAILABILITY STATEMENT

Research data are not shared.