pharmacokinetics, metabolism and excretion of [ 14 c]-lanicemine (azd6765), a novel low-trapping n-...

http://informahealthcare.com/xenISSN: 0049-8254 (print), 1366-5928 (electronic)

Xenobiotica, Early Online: 1–12! 2014 Informa UK Ltd. DOI: 10.3109/00498254.2014.966175

RESEARCH ARTICLE

Pharmacokinetics, metabolism and excretion of [14C]-lanicemine(AZD6765), a novel low-trapping N-methyl-D-aspartic acid receptorchannel blocker, in healthy subjects

Jian Guo1, Diansong Zhou2, Scott W. Grimm1y, and Khanh H. Bui2

1DMPK of Infection Innovative Medicine, AstraZeneca Pharmaceuticals, Waltham, MA, USA and 2Early Clinical Development, AstraZeneca

Pharmaceuticals, Wilmington, DE, USA

Abstract

1. (1S)-1-phenyl-2-(pyridin-2-yl)ethanamine (lanicemine; AZD6765) is a low-trapping N-methyl-D-aspartate (NMDA) channel blocker that has been studied as an adjunctive treatment inmajor depressive disorder. The metabolism and disposition of lanicemine was determinedin six healthy male subjects after a single intravenous infusion dose of 150 mg[14C]-lanicemine.

2. Blood, urine and feces were collected from all subjects. The ratios of Cmax and AUC(0–1) oflanicemine to plasma total radioactivity were 84 and 66%, respectively, indicating thatlanicemine was the major circulating component with T1/2 at 16 h. The plasma clearance oflanicemine was 8.3 L/h, revealing that lanicemine is a low-clearance compound. The meanrecovery of radioactivity from urine was 93.8% of radioactive dose.

3. In urine samples, 10 metabolites of lanicemine were identified. Among which, anO-glucuronide conjugate (M1) was the most abundant metabolite (�11% of the dose inexcreta). In plasma, the circulatory metabolites were identified as a para-hydroxylatedmetabolite (M1), an O-glucuronide (M2), an N-carbamoyl glucuronide (M3) and anN-acetylated metabolite (M6). The average amount of each of metabolite was less than4% of total radioactivity detected in plasma or urine.

4. In conclusion, lanicemine is a low-clearance compound. The unchanged drug andmetabolites are predominantly eliminated via urinary excretion.

Keywords

Antidepressant, major depressive disorder,(1S)-1-phenyl-2-(pyridin-2-yl)ethanamine

History

Received 20 August 2014Revised 4 September 2014Accepted 12 September 2014Published online 26 September 2014

Introduction

Major depressive disorder (MDD) is a serious psychiatric

disorder that affects millions of individuals worldwide. The

most commonly prescribed medications used to treat MDD

include selective serotonin reuptake inhibitors (SSRIs), nor-

epinephrine reuptake inhibitors (NRIs) and dopamine reup-

take inhibitors, working to increase the extracellular levels of

corresponding neurotransmitters in the synaptic cleft by

preventing reuptake into pre-synaptic terminals (Burrows

et al., 1998; Hajos et al., 2004; Jutkiewicz, 2006; Papakostas

et al., 2008). However, clinical studies have shown that up to

45% of patients do not respond to antidepressant treatment

(Rush et al., 2006). Even among those responding to

treatment, remission rates only reached up to 70% (Rush

et al., 2006). In addition, the onset of therapeutic action for

these standard antidepressant treatments is so slow that

several weeks of continuous administration is required to

achieve clinically meaningful changes in symptoms (Katz

et al., 2004; Thompson, 2002). This latency is problematic in

that it prolongs the impairment associated with depression,

leaving patients vulnerable to an increased risk of self-

injurious behavior and suicides (Stahl et al., 2001). As the

existing therapeutic treatments for MDD are often insufficient

for many patients, there is an urgent need to develop better

tolerated, more effective and faster acting antidepressants

for MDD.

In recent years, growing evidences from pre-clinical and

clinical studies suggest that the glutamatergic system is

uniquely central to the treatment of MDD. Glutamate is the

most common abundant excitatory neurotransmitters in

human nervous system, playing an important role in cellular

plasticity and cellular resilience which are involved in

memory, learning and cognition (Sanacora et al., 2008).

The preliminary clinical studies have shown that plasma

yDeceased

Address for correspondence: Jian Guo, DMPK of Infection InnovativeMedicine, AstraZeneca Pharmaceuticals, 35 Gatehouse Drive, Waltham,MA 02421, USA. Tel: +1-781-472-5985. E-mail: [email protected], [email protected]

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levels of glutamate in patients with major depression disease

were significantly higher than those of healthy controls

(Altamura et al., 1995; Mauri et al., 1998; Mitani et al., 2006).

Other studies have provided further evidence that treatment

with standard monoaminergic antidepressants for several

weeks may decrease the plasma glutamate levels in individ-

uals with treatment-resistant depression (Altamura et al.,

1993; Kucukibrahimoglu et al., 2009; Maes et al., 1998).

These findings indicated that glutamate plays a key role in the

action of antidepressants and that the glutamate receptors

are a potential novel target for the treatment of MDD.

Pharmacologically, glutamate receptors have been classified

as ionotropic and metabotropic receptors and channels.

N-methyl-D-asparate (NMDA) receptor is glutamate ionotro-

pic receptor subtypes, which comprises NR1, NR2 (NR2A–

NR2D) and NR3 (NR3A and 3B) subunits (Mathews et al.,

2012). Recent clinical studies support the hypothesis that

N-methyl-D-aspartate (NMDA) receptor antagonists are

effective in MDD patients resistant to conventional anti-

depressant treatments. Specifically, a single low-dose of

ketamine, a high affinity NMDA receptor antagonist,

produced fast-acting and long-lasting antidepressant effects

in patients suffering from MDD (Machado-Vieira et al., 2009;

Zarate et al., 2013), which is very likely mediated by rapid,

transient synthesis of brain-derived neurotrophic factor

followed by alterations in synaptic plasticity (Autry et al.,

2011). However, acute sedative and psychotomimetic side

effects limit the clinical use of ketamine (Krystal et al., 1994;

Machado-Vieira et al., 2008).

(1S)-1-Phenyl-2-(pyridin-2-yl)ethanamine (lanicemine;

AZD6765; Scheme 1) is a low-trapping NMDA channel

blocker with low potential for psychotomimetic dissociative

adverse effects (Sanacora et al., 2013) that has been studied

as an adjunctive treatment for patients with a history of

inadequate response to antidepressants (Dolgin, 2013;

Sanacora et al., 2014; Zarate et al., 2013). The objectives of

this study were to characterize the pharmacokinetics, elim-

ination and metabolism of [14C]-lanicemine in healthy

subjects following a single intravenous infusion, which was

the intended therapeutic route of administration.

Materials and methods

Chemicals

Lanicemine dihydrochloride, [14C]-lanicemine (>99% radio-

chemical purity), [D5]-lanicemine, authentic

standard of M5 4-(1-amino-2-pyridin-2-ylethyl)phenol and

M6 N-(1-phenyl-2-pyridin-2-ylethyl)acetamide were synthe-

sized at AstraZeneca Pharmaceuticals (Wilmington, DE).

HPLC-grade solvents were purchased from Fisher Scientific

(Waltham, MA). All other chemicals were purchased from

Sigma-Aldrich (St. Louis, MO).

Scheme 1. Proposed biotransformation pathway of [14C]-lanicemine after a single 150 mg i.v. administration in healthy make subjects. Asteriskindicates the position of the C-14 label.

2 J. Guo et al. Xenobiotica, Early Online: 1–12

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Formulated drug

[14C]-lanicemine (purity 99%) solution for infusion was

provided by AstraZeneca with a specific radioactivity of

1 mCi/mg.

Clinical study design

This was a phase I, open-label, single-dose study to assess the

pharmacokinetics, metabolism and excretion of [14C]-lanice-

mine after a single intravenous infusion in healthy volunteers

(NCT01217645). This study was performed in accordance

with Good Clinical Practices and in compliance with the

Declaration of Helsinki. The clinical phase of the study was

conducted at Quintiles Drug Research Unit at Guy’s Hospital

(Quintiles Ltd, London, UK). Six healthy male subjects aged

35–60 years with mean body weight of 71.2 kg (SD¼ 10.4 kg)

and mean body mass index of 25.5 kg/m2 (SD 3.2 kg/m2) were

enrolled in this study. Four subjects were Caucasian and two

subjects were of Asian heritage.

Each subject was fasted for 2 h prior to receiving a single

radiolabeled intravenous dose of 150 mg of [14C]-lanicemine

(150mCi, 5.55 mBq) infused at a constant rate over 60 min.

Subjects remained fasting for 1 h after the end of the infusion.

All subjects remained in the clinical facility for 12 days.

Blood samples were collected pre-dose and at 2, 4, 6, 12, 24

and 48 h post-dose in all subjects for biotransformation

analysis. Blood samples for the pharmacokinetic analysis of

blood and plasma radioactivity were collected pre-dose and at

0.25, 0.5, 1, 1.25, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 36, 48, 72, 96,

120, 144, 168, 192, 216 and 240 h. Whole blood and plasma

samples for the measurement of radioactivity were stored

refrigerated at 2–8 �C prior to, and after analysis. Urine

samples were collected pre-dose and at 0–6, 6–12, 12–14 h

and thereafter at 24-h interval through 240 h, while fecal

samples were collected pre-dose and at 24-h intervals through

240 h. Urine and fecal samples were stored at or below

�20 �C until analysis. Subjects were discharged from the

study center after completion of all study related measure-

ment on Day 11 or until a minimum of 90% of the radioactive

dose administered was recovered or less 1% was excreted in

two consecutive 24-h intervals.

Measurements of radioactivity

The analysis of total radioactivity in urine, feces, blood and

plasma was conducted at Covance Laboratories (Harrogate,

UK). The radioactivity in weighted portion of urine and

aliquots of plasma (200 mL) was directly analyzed using a

Packard Tri-Carb liquid scintillation counter (LSC; Canberra

Packard, Pangbourne, Berks) by mixing Ultima Gold scintil-

lation fluid (PerkinElmer UK Ltd) to appropriate amount of

the samples prior to analysis. Aliquot of whole blood (400 mL)

was added to ashless floc and allowed to dry overnight in an

oven 50 �C prior to combustion analysis. Fecal samples were

homogenized in an appropriated volume of deionised water.

Triplicated weighted aliquots of fecal homogenates (200–

500 mg) were added to ashless floc prior to combustion

analysis using Parkard Sample Oxidiser. The resulting 14CO2

were absorbed in Carbsorb and mixed with Permafluor

scintillation fluid, and analyzed by LSC. The lower limits of

quantitation of radioactivity in blood and plasma under these

conditions were 0.29 and 0.21 mmol-lanicemine Eq/L.

Measurement of lanicemine concentration

The concentrations of lanicemine in plasma and urine were

analyzed by BASi (West Lafayette, IN). Concentration

of lanicemine in plasma was determined using validated

LC-MS/MS method. The calibration curves of lanicemine in

plasma and urine were linear over a range of 5–2000 and

5–5000 mg/mL, respectively, using a weighted (1/x2) linear

least square regression. The lower limit of quantitation for

lanicemine was 5 ng/mL in human plasma and urine. [d5]-

Lanicemine was used as internal standard. A 50 mL aliquot of

plasma was mixed with 300 mL of internal standard working

solution in a 96-well plate and centrifuged at 1000 rpm for

5 min. A 10 mL aliquot of each supernatant was injected into

the LC-MS/MS system for quantitative analysis.

Pharmacokinetic analysis

Pharmacokinetic parameters were determined using non-

compartment analysis of plasma concentration versus time

profiles using WinNonlin (Professional Version 5.2. Pharsight

Corp. Mountain View, CA). Total radioactivity concentrations

in whole blood and plasma were converted to lanicemine

nanogram-equivalents based on the molecular weight

(198.3 g) of lanicemine and radioactive specific activity

(1 mCi/mg) of [14C]-lanicemine. Maximum plasma concen-

tration (Cmax) and the time to reach peak concentration (Tmax)

were estimated directly from the experimental data. Area

under the concentration–time curve (AUC) from zero to the

last measurable concentration [AUC(0–t)] and area under the

concentration–time curve from time to infinity [AUC(0–1)]

were calculated by linear up/log down trapezoid summation

AUC(0–t) and extrapolated to infinity by addition of last

quantifiable concentration divided by the elimination rat

constant Ct/lz, where the terminal elimination rat constant

(lz) was determined from linear regression of the terminal

points of the log-linear concentration–time curve. A min-

imum of three data points was used to determine the terminal

linear phase of the profile. The apparent terminal half-life (t1/2)

was determined as ln 2/lz. Clearance (CL) was calculated as

actual dose administered divided by AUC(0–1), and the volume

of distribution at steady state was calculated as CL * MRT,

where MRT was the mean residence time extrapolated to

infinity. The radioactivity in blood cells was calculated from

hematocrit (Ht) and from whole blood and plasma radioactiv-

ity using the equation:

Ccell ¼ ½Cblood � Cplasma � ð1� HtÞ�=Ht:

Preparation of urine and plasma samples formetabolite profile analysis

Feces samples were not analyzed due to low amount of total

radioactivity (ca. 2% recovery over 240 h post-dose).

Urine from 0 to 72 h for each subject was pooled

proportionally to the original sample volume collected at

each time interval. The radioactivity in sample pool repre-

sented >91% of the total radioactivity excreted in urine for

DOI: 10.3109/00498254.2014.966175 Metabolism and excretion of lanicemine in human 3

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each subject. The pooled urine samples were centrifuged at

10 000� g for 10 min to remove debris and then a 20 mL

aliquot of supernatant was evaporated to dryness under

vacuum at 30 �C for 4 h using an HT-4 Series II centrifugal

vacuum evaporator (Geneac Inc., Gardiner, NY). The dry

residues were reconstituted in 1 mL 90:10 water:acetonitrile.

A 50 mL aliquot of reconstituted plasma sample was injected

onto the LC column for metabolite profile analysis.

Plasma samples (0–24 h) for each subject were pooled

according to the Hamilton method (Hamilton et al., 1981).

A 10 mL aliquot of pooled plasma sample from each subject

was mixed with three volumes of acetonitrile for protein

precipitation. The mean recovery of radioactivity following

sample preparation was �85%. The supernatant was con-

centrated to dryness under vacuum at 30 �C for 4 h using an

HT-4 Series II centrifugal vacuum evaporator (Geneac Inc.,

Gardiner, NY). The dry residues were reconstituted in 0.2 mL

90:10 (v:v) water:acetonitrile. A 50 mL aliquot of reconsti-

tuted plasma sample was injected onto the LC column for

metabolite profile analysis.

Deconjugation of urine sample by b-glucuronidase

A 200 mL aliquot of the urine was evaporated to dryness and

reconstituted in 200 mL ammonium acetate buffer (10 mM,

pH 5.0) with �-glucuronidase (1000 units) at pH 7.0. The

deconjugation reactions were carried out at 37 �C for 4 h and

terminated by the addition of acetonitrile as described above.

After centrifugation, supernatants of each sample were

analyzed using LC-MS/MS. Aliquots of samples not treated

with hydrolytic enzymes were used as controls.

Acetylation of M1 in human liver cytosol

Incubations with pooled human liver cytosol contained

0.4 mg/ml of protein, 40 mM of M1, 1 mM EDTA, 1 mM

dithiothreitol, 1 mg/mL acetyl-D,L-carnitine, 0.22 U/mL carti-

nine acetyltransferase and 0.1 mM acetyl Co-A in 50 mM

triethanolamine buffer at pH 7.5 in a total volume of 0.2 ml.

The reactions were initiated by the addition of M5 after a

5-min pre-incubation and were performed at 37 �C for 20 min.

The incubations were terminated by adding 0.6 mL of an ice-

cold mixture of acetonitrile–ethanol (1:1, v/v) and chilling the

resulting mixture on ice. After centrifugation to remove the

precipitated proteins, the supernatant was evaporated to

dryness in vacuo. Each residue was reconstituted in 200 mL

of HPLC mobile phase immediately before analysis using

LC-MS. Incubations containing 10 mM of 4-amino salicylic

acid and sulfamethazine, the selective substrate for NAT1 and

NAT2, respectively, were used as positive controls.

Incubation of M6 in human liver microsomes

Incubations with pooled human liver microsomes contained

0.5 mg/mL of microsomal protein, 50 mM M6 and 1 mM

NADPH in 50 mM phosphate buffer at pH 7.4 in a total

volume of 0.2 mL. The reactions were initiated by addition of

NADPH after a 5-min pre-incubation and were performed at

37 �C for 40 min. The incubations were terminated by adding

0.6 mL of an ice-cold mixture of acetonitrile–ethanol (1:1,

v/v) and chilling the resulting mixture on ice. After

centrifugation to remove the precipitated proteins, the super-

natant was evaporated to dryness in vacuo. Each residue was

reconstituted in 200 mL of HPLC mobile phase immediately

before analysis using LC-MS.

LC-radioactivity analysis for metabolite profiles

Chromatographic analysis of samples from rat mass balance

studies was performed on an AcquityTM UPLC system

(Waters, Milford, MA). The separation was carried out on

an Aquasil C18 column (4.6� 150 mm, 5 mm, Thermo

Scientific, Waltham, MA) at a flow rate of 1.0 mL/min. The

mobile phase consisted of water with 20 mM ammonium

formate (A; pH 4.0) and acetonitrile (B). The analytes were

eluted using the following gradient: 5% solvent B for 2 min, a

linear increase to 25% solvent B over 20 min and maintained

for 17 min, a linear increase to 60% B over 7 min, followed by

90% B for 7 min, then the mobile phase composition was

returned to the starting solvent mixture over 6 min. The LC

elutant was split post-column at approximately a 1:9 ratio

between the LTQ-Orbitrap mass spectrometer (Thermo

Scientific, San Jose, CA) and a HTS PAL faction collector

(LEAP Technologies, Carrboro, NC). The HPLC effluent

of urine and plasma was collected in 7.3-s interval and

9.1-s interval, respectively, into LumaPlate-96-well solid

scintillator-coated microplates (PerkinElmer, Waltham,

MA). The solvent of collected fractions was evaporated to

dryness at room temperature. The radioactivities of collected

sample fractions were determined using a TopCount� NXTTM

microplate scintillation counter (PerkinElmer, Waltham, MA)

at counting time of 5 min. The HPLC radiochromatograms

were created using Laura 3 data handling software (LabLogic

Systems, Sheffield, UK).

Metabolite identification

Identification of metabolites was performed on Thermo LTQ

Orbitarp (Thermo Fisher Scientific) operated in positive

ionization mode with full scan data acquired at a resolving

power of 15 000 and in parallel with data dependent CID MSn

Figure 1. Mean percentage of cumulative radioactivity recovered fromsix healthy male subjects after a single intravenous administration of150 mg [14C]-lanicemine.


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scan mode. CID product ion spectra of the three most intense

ions were acquired within one acquisition cycle. Helium was

used as the collision gas for CID. Normalized collision

energies of 15 and 25% were set for CID MS2 and MS3,

respectively. All scan events were detected in the FT detector

to give accurate mass data for precursor ions and MSn ions

used for metabolite identification.

Results

Mass balance and excretion in urine and feces

The mean recoveries of radioactivity in urine and feces over

0–240 h collection period are summarized in Figure 1.

Following a single i.v. administration of 150 mg of [14C]-

lanicemine infused over 1 h, the mean overall recoveries of

the total radioactivity in urine and feces was 95%, of which

93.8% was recovered in urine and 1.9% in feces. Unchanged

lanicemine accounted for 37.0% of the dose excreted in urine.

At 72 h post-dose, approximately 90% of dose was recovered

in urine, with less than 1% of the dose, on average, being

excreted after 120 h.

Clinical pharmacokinetic results

Mean concentration–time profiles of lanicemine and blood

and plasma radioactivity are depicted in Figure 2. The

summary of pharmacokinetic parameters for lanicemine,

plasma and whole blood radioactivity are presented in

Table 1. Following a single i.v. administration of 150 mg of

[14C]-lanicemine, the mean maximum total radioactivity in

plasma of 1500 ng equivalents (ng-eq/mL) and the mean

Cmax for lanicemine of 1270 ng/mL were achieved approxi-

mately 1 h postdose on average, with Tmax of 1.1 and 1.0 h,

respectively. After reaching the Cmax, the mean terminal

elimination half-life (t1/2) value for lanicemine and total

radioactivity were comparable, approximately 16 h. The

plasma AUC(0–1) value for lanicemine was 62.6% of the

radioactivity AUC(0–1) value, indicating that unchanged

lanicemine accounted for the majority of circulating radio-

activity. The apparent clearance (CL) value of lanicemine was

8.3 L/h, a slightly greater than that of the total radioacitivity

of 5.2 L/h. Relative to total body water 42 L (Davies &

Morris, 1993), the volume of distribution at steady state (Vss)

Figure 2. Mean concentration versus time profiles of plasma lanicemine,total plasma radioactivity and total whole blood radioactivity on(a) linear and (b) semi-logarithmic scales after a single 150 mg i.v.dose of [14C]-lanicemine (n¼ 6).

Table 1. Summary of pharmacokinetic parameters for lanicemine and radioactivity in plasma, whole blood and urine after a singleintravenous administration of 150 mg [14C]-lanicemine in healthy male subjects (n¼ 6).

Parameter Laniceminea Plasma total radioactivitya,bWhole blood

radioactivitya,b

Systemic parametersAUC (mg h/mL) 17.9 (20.8) 28.6 (21.7) 22.6 (19.8)AUC0–t (mg h/mL) 17.7 (20.9) 26.8 (22.7) 20.8 (18.5)Cmax (mg/mL) 1.3 (20.9) 1.5 (19.5) 1.3 (18.1)Tmax (h) 1.0 (1.0, 1.3) 1.1 (1.0, 1.3) 1.0 (1.0, 1.3)T1/2 (h) 16.1 (20.1) 16.9 (25.2) 15.1 (22.4)CL (L/h) 8.3 (20.9) 5.2 (21.7) 6.6 (19.8)Vss (L) 161.0 (9.0) 117.0 (8.2) 135.0 (8.1)

Renal parametersCLr (L/h) 3.07 (45.0) 4.87 (24.6) NA

aValues are reported as geometric mean (Geometric CV%; n¼ 6), except Tmax values, which are reported as median (min – max).bUnits are ng [14C]-lanicemine equivalent/mL for radioactivity in blood or plasma.NA, Not applicable.


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values of total radioactivity and lanicemine were of 117 and

161 L, respectively, indicating some distribution into tissues.

The whole blood-to-plasma radioactivity ratios ranged

from 0.72 to 0.93 cross time points and the calculated

percentage of radioactivity associated with red blood cells

was low, ranging from 20.6% to 38.2%, indicating that

radioactivity in blood was more concentrated in the extracel-

lular fluid than in the blood cells.

Metabolite profiles of [14C]-lanicemine in urine andplasma

Urinary metabolite profiles after i.v. administration of [14C]-

lanicemine ([14C]-lanicemine) are shown in Figure 3. The

relative amounts of metabolites and parent compound are

shown in Table 2. In the radiochromatograms of pooled urine

samples (0–72 h) from the 6 subjects, 10 drug-related

radiochromatographic peaks were detected and tentatively

characterized. Lanicemine was the most abundant component

in pooled urine and represented a mean of 48.0% of the

administered dose. The five major metabolites included a

monohydroxylated metabolite (M1), an O-glucuronide con-

jugate of monohydroxylated metabolite (M2), an N-carbamyl

glucuronide of hydroxylated metabolite (M3), a sulfate of

dihydroxylated metabolite (M4) and an N-carbamyl glucur-

onide (M5), accounting for a mean of 7.2, 10.9, 6.7, 3.3 and

7.1% of the administered dose, respectively.

Plasma metabolite profiles after administration of [14C]-

lanicemine are shown in Figure 4. The relative amounts

of generated metabolites and parent compound are shown

in Table 3. As evidenced in Table 3, lanicemine was the

predominant species in pooled plasma, accounting for

approximately 90% of the total circulating radioactivity. In

the pooled plasma samples (0–24 h), metabolites M1, M2 and

M3 were detected in all six subjects and represented a mean

Figure 3. Representative radioactivity pro-files of [14C]-lanicemine in pooled urinesamples from healthy male subjects aftera single 150 mg i.v. administration of[14C]-lanicemine.

Figure 4. Representative radioactivity profiles of [14C]-lanicemine inplasma samples from healthy male subjects after a single 150 mg i.v.administration of [14C]-lanicemine.

Table 2. Individual percentages of lanicemine-related components inhuman urine (0–72 h) after a single intravenous administration of 150 mg[14C]-lanicemine in healthy male subjects (n¼ 6).

% Administered dose recovered in each subjecta

Metabolites 1 2 3 4 5 6 Mean ± SD

M1 7.0 5.3 6.5 7.4 6.1 10.8 7.2 ± 1.9M2 16.8 7.2 10.5 13.2 5.9 12.1 10.9 ± 4.0M3 9.5 5.1 8.6 5.8 4.8 6.1 6.7 ± 2.0M4 3.6 1.8 3.0 3.2 3.0 5.5 3.3 ± 1.2M5 9.6 5.8 9.7 6.9 3.8 6.4 7.1 ± 2.3M7 1.8 0.6 0.6 1.2 1.6 1.0 1.1 ± 0.5M8 1.2 1.0 1.9 1.1 1.4 2.2 1.5 ± 0.5M9 0.8 0.2 0.3 0.3 0.6 2.3 0.8 ± 0.8M10 0.6 0.4 0.9 0.4 0.5 0.8 0.6 ± 0.2lanicemine 32.0 57.7 47.2 53.9 58.4 38.8 48.0 ± 10.8Total radioactivity 83.0 85.1 89.2 93.4 86.0 85.9 87.1 ± 3.3

aIndividuals (1–3 and 6) are subjects of Caucasian ethnicity andindividuals (4 and 5) are subjects of Asian ethnicity.


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of 2.3, 3.7 and 3.2% of the circulating radioactivity, respect-

ively. M6 was an N-acetylated metabolite and was only

detected in one of the Asian subjects, accounting for 3.1% of

total peak area.

Identification of metabolites

Based on the radiochromatographic profiles of the metabol-

ites, the structures of prominent metabolites were elucidated

by high-resolution accurate mass spectrometry using parent

mass list, or intensity triggered data-dependent product ion

scans on a LTQ-Orbitrap mass spectrometer. The fragment

ions of lanicemine 5 and its metabolites are shown in Table 4.

The proposed prominent metabolic pathways of lanicemine

are shown in Scheme 1.

Lanicemine

The protonated molecule of lanicemine in a full scan MS was

at m/z 199. The base peak in the product ion spectrum was

observed at m/z 182, associated with the loss of the amino

group. Ions with lower abundance were detected at m/z 94

and 106, corresponding to the cleavage of C1–C2 bond

(Table 4).

Metabolite M1

Metabolite M1, one of the most abundant metabolites

detected in urine, displayed a protonated molecule at m/z

215, 16 mass unit higher than that of parent drug. Accurate

mass of protonated M1 (observed at m/z 215.1177 within

�0.9 ppm mass deviation of a calculated value) indicated

its empirical formula as C13H25N2O+ (equivalent to

lanicemine + O), suggesting that M1 was a hydroxylated

metabolite. The most intense MS2 fragment ion was at m/z

198 generated by the loss of amino group, suggesting that the

oxidation did not occurred on the amino group (Table 4). By

comparison of retention time and MS2 spectrum of M1 with

that of synthesized authentic standard, M1 was identified as

4-(1-amino-2-pyridin-2-ylethyl) phenol. This metabolite was

not active at the NMDA receptor.

Metabolite M2

LC-MS scans of M2 showed a protonated molecule at m/z

391, 192 unit higher than that of parent drug. Accurate


1.3 ppm mass deviation of a calculated value) suggested

its empirical formula as C19H23N2Oþ7 (equivalent to

lanicemine + O + 176), suggesting that M2 was a glucuronide

conjugate. The major MS2 fragments were at m/z 374

generated by the loss of amino moiety, indicated that 1

oxygen atom was introduced on an aromatic ring and

O-glucuronidation occurred (Table 4). In the radiochromato-

gram of urine samples treated with �-glucuronidase, the

relative abundant of M2 reduced significantly and no new

signal associated with hydroxylated product was observed

(data not shown), suggesting that M2 was very likely to be an

O-glucuronide conjugate of M1.

Metabolite M3




�0.9 ppm mass deviation of a calculated value) suggested its

empirical formula as C20H23N2Oþ9 . The most intense MS2

fragment ion was at m/z 259, 176 Da less than that of

protonated molecule, indicating that M3 was a glucuronide

conjugate (Figure 5A). Other MS2 fragment ions at m/z 198

and 215 suggested that an oxygen atom was introduced on the

aromatic rings. The accurate mass analysis of fragment ions

at m/z 259 indicated its empirical formula at C14H15N2Oþ3(equivalent to lanicemine + O + CO2). As diagnostic fragment

ion at m/z 418 generated by the loss of amino group [MH+ –

NH3]+ was not observed in the CID mass spectrum, it is very

likely that the primary amine present on the parent drug

interacted with exogenous CO2 to form carbamic acid that

subsequently conjugated to glucuronic acid resulting in an

N-carbamoyl glucuronide. Furthermore, in the radiochroma-

togram of urine samples treated with b-glucuronidase, the

relative abundance of M3 reduced significantly and no new

signal associated with monooxidated product was observed

(data not shown), suggesting that hydroxylation occurred on

the para position of the benzene ring. Therefore, M3 was

tentatively identified as an N-carbamyl glucuronide of a

monohydroxylated metabolite (Scheme 1).

Metabolite M4


311, 12 units higher than that of lanicemine. Accurate mass

analysis of protonated M4 (observed at m/z 311.0692 with

�1.3 ppm mass deviation of a calculated value) suggested

its empirical formula as C13H15N2O5S+ (equivalent to

lanicemine + 2O + SO3). The most intense MS2 fragment

ion was at m/z 214, 97 Da less than that of protonated

molecule. The accurate mass analysis of fragment ions at m/z

214 indicated its empirical formula at C13H12NOþ2 (equivalent

to MH+ – NH2 – HSO3), also suggesting that M4 was a sulfate

conjugate. The MS3 spectrum (311!214!) showed the

fragment ions at m/z 196 and 186, associated with the loss of

water and CO group, respectively (Table 4). Together with

empirical formula of the ion at m/z 214, the presence of these

typical MS3 ions suggested that two oxygen atoms were

introduced, one on benzyl ring and another one on the alkyl

chain. Although minor, the fragment ion generated by the loss

of primary amine was observed at m/z 294, suggesting

Table 3. Percentage of radioactive lanicemine-related components inpooled human plasma (0–24 h) after a single intravenous administrationof 150 mg [14C]-lanicemine in healthy male subjects (n¼ 6).

Percentage of radioactivity (%)

Subjecta M1 M2 M3 M6 Parent

1 2.8 5.7 3.3 NA 88.12 2.6 3.9 3.9 NA 89.73 1.9 2.4 3.6 NA 91.74 2.5 5.0 1.9 NA 89.35 2.3 3.4 4.1 3.1 87.16 1.8 1.8 2.2 NA 93.8Mean 2.3 3.7 3.2 NA 90.0

aIndividuals (1–4) are subjects of Caucasian ethnicity and individuals(5 and 6) are subjects of Asian ethnicity.


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Table 4. Mass spectra data and proposed structures of metabolites of lanicemine in human.

Compounds [M + H]+a Dm/z (ppm) Major product ions Proposed structure and fragmentationb

Lanicemine 199.1230 0.1 MS2: 182, 106, 94

NNH2

182

106

12

M1 215.1177 �0.9 MS2: 198

NNH2

OH

198

M2 391.1505 1.3 MS2: 374, 198

NNH2

OGluc

198

374

M3 435.1394 �0.9 MS2: 259, 215, 198

NHN

O

O

Gluc

OH

198

215 259

M4 311.0692 �1.3 MS2: 294, 214 MS3 (214): 196, 186

NNH2

OHHOSO3H

M5 419.1449 0.0 MS2: 243, 199, 182

NHN

O

O

Gluc

182

199

243

M6 241.1337 0.7 MS2: 182

NHN

O

182

(continued )


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that M4 was an O-sulfate of hydroxylated metabolite. The

positions of hydroxylation could not be determined from mass

spectral analysis.

Metabolite M5



mass of protonated M5 (observed at m/z 419.1449, mass

deviation of a calculated value 50.01 ppm) suggested its

empirical formula as C20H23N2Oþ8 . The base peak in CID

product ion spectrum of M5 was at m/z 243, 176 units less

than that of protonated molecule, indicating that M5 was a

glucuronide conjugate (Table 4). The accurate mass analysis

of fragment ions at m/z 243 indicated its empirical formula at

C14H15N2Oþ2 (equivalent to lanicemine + CO2). The fragment

ion generated by the loss of primary amine was not observed

in the CID mass spectrum, suggesting that M5 was formed by

the reaction of primary amine with CO2, forming carbamic

acid and subsequent glucuronidation with glucuronic acid.

Based on these data, M5 was tentatively identified as an

N-carbamoyl glucuronide of lanicemine.

Metabolite M6


241, 42 mass unit higher than that of parent drug. The

accurate mass of protonated M6 was observed at m/z

241.1337 (within 0.7 ppm mass deviation of a calculated

value) gave its empirical formula as C15H17N2O+ (equivalent

to lanicemine + C2H2O). The major MS2 fragment ion was at

m/z 182 generated by the loss of amino group connected with

an acetyl group. By comparison of chromatographic retention

time and MS2 spectrum of M6 with synthesized authentic

Table 4. Continued

Compounds [M + H]+a Dm/z (ppm) Major product ions Proposed structure and fragmentationb

M7 257.1284 �0.2 MS2: 198

NHN

O

OH

198

M8 433.1606 1.5 MS2: 374, 257, 198

NHN

OGluc

O

257

374

198

M9 465.1499 �1.0 MS2: 289, 245, 228

NHN

O

O

Gluc

OH

OCH3

289

228

245

M10 299.1386 �1.4 MS2: 281, 199, 182

NHN

O

O

OH

182

199

aObserved accurate mass of protonated molecule.bLanicemine and metabolites have S configuration on C-1.


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standard, M6 was identified to be N-(1-phenyl-2-pyridin-2-

ylethyl)acetamide (Table 4). This metabolite was not active at

the NMDA receptor.

Metabolite M7

M7 showed a protonated molecule at m/z 257, 58 mass

units higher than that of parent drug. Accurate mass

analysis of protonated M7 (observed at m/z 257.1284 within

�0.2 ppm mass deviation of a calculated value) suggested

its empirical formula as C15H17N2Oþ2 (equivalent to

lanicemine + C2H2O2), one oxygen atom more than that

of M6. The most intense MS2 fragment ion was at m/z 198

generated by the loss of substituted amino group, suggesting

that hydroxylation occurred on the aromatic rings and the

primary amine was acetylated to form an amidic metabolite

(Table 4). Although the exact location of hydroxylation could

not be determined from mass spectral analysis, the para-

hydroxylation was confirmed in in vitro assay using standard

M1 incubated with human liver cytosol and using standard

M6 incubated with human liver microsomes (data not shown).

Therefore, M7 was identified as (R)-N-(1-(4-hydroxyphenyl)-

2-(pyridin-2-yl)ethyl)acetamide.

Metabolite M8

M8 showed a protonated molecule at m/z 433, 234 mass units

higher than that of parent drug. The accurate mass of

protonated M8 (observed at m/z 433.1606 within 1.5 ppm

mass deviation of a calculated value) suggested its empirical

formula as C21H25N2Oþ8 . The most abundant MS2 fragment

ion was at m/z 198 generated by the loss of glucuronosyl

group and substituted amino group, suggesting that hydrox-

ylation occurred on the aromatic rings (Table 4). However,

the positions of hydroxylation could not be determined from

mass spectral analysis. In the radiochromatogram of urine

samples treated with glucuronidase, the relative abundant of

M8 reduced significantly (data not shown), revealing that M8

formed via O-glucuronidation rather than N-glucuronidation.

Therefore, M8 was tentatively identified as an O-glucuronide

conjugate of lanicemine with acetylation occurred on the

primary amine.

Metabolite M9

M9 displayed a protonated molecule at m/z 465, 266 mass

units higher than that of parent drug. Accurate mass

measurement of M9 gave a molecular ion at m/z 465.1499

and an empirical formula of as C21H25N2Oþ10 (D¼�0.1 ppm).

The most abundant MS2 fragment ion was at m/z 289, 176 Da

less than that of protonated molecule, revealing that M9 was a

glucuronide conjugate. The accurate mass analysis of frag-

ment ion at m/z 228 indicated its empirical formula as

C14H14NOþ2 , demonstrating that this fragment ion was

generated by the cleavage of C1–N bond, and also indicating

that the M9 was an N-carbamoyl glucuronide (Figure 5b). The

difference of elemental composition between fragment ion at

Figure 5. CID product ion mass spectraof (a) M3 (MH+¼ 435) and (b) M9(MH+¼ 465).


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m/z 228 and the corresponding fragment ion of parent drug at

m/z 182 (C13H12N+) was CH2O2, suggesting that one oxygen

atom and one methoxyl group was introduced into the phenyl

ring (Table 4). The exact location of hydroxylation and

methoxylation could not be determined from mass spectral

analysis.

Metabolite M10

Metabolite M10 showed a protonated molecule at m/z 299,

100 mass unit higher than that of parent compound. Accurate

mass analysis of M20 gave a protonated molecular ion at

m/z 299.1386 and an empirical formula of C17H19N2Oþ3(D¼�1.4 ppm; equivalent to lanicemine + C3H5O3). The

parent drug related fragment ions at m/z 182 and 199

observed in MS2 spectrum of M10 indicated that no metabolic

reactions occurred on the aromatic rings and introduced

groups were connected to the primary amine. Based on these

data, M10 was tentatively identified as a succinic amide

of lanicemine.

Discussion

The objective of this study was to evaluate the excretion,

pharmacokinetics and metabolism of lanicemine in man after

a single i.v. dose (150 mg) of [14C]-lanicemine. The results

of this study showed that, in healthy subjects, drug related

materials were excreted almost completely in urine. On

average, urinary recovery of total radioactivity accounted for

93.8% of the administered dose, whereas the radioactivity

excreted in feces over 240 h was lower than 2%, indicating

that biliary and/or intestinal excretion of lanicemine in human

is negligible.

The ratios of Cmax and AUC(0–1) of lanicemine to plasma

total radioactivity were 84 and 66%, respectively, indicating

that lanicemine represented the predominant circulating

component. The mean plasma clearance of lanicemine at

8.3 L/h revealed that lanicemine was a low-clearance com-

pound relative to a hepatic blood flow of 87 L/h (Davies &

Morris, 1993). Moreover, mean lanicemine plasma concen-

tration–time profile, plasma radioactivity and whole blood

radioactivity showed a mono-exponential decline following

tmax. The slopes for radioactivity and whole blood radioactiv-

ity generally paralleled that of plasma lanicemine (Figure 2),

indicating that parent drug was the primary driver for

overall systemic distribution and elimination of [14C]-radio-

activity. In addition, Vss of plasma lanicemine was higher

than that of plasma radioactivity, suggesting that the meta-

bolic products of lanicemine have a lower volume of

distribution than parent drug. The mean blood cell-to-

plasma radioactivity ratios, ranging from 20.6% to 38.2%,

indicated the low distribution and the absence of accumula-

tion of lanicemine and corresponding metabolites in blood

cells.

The mean renal clearance of unbound lanicemine in human

was 3.07 L/h and is slightly lower than the glomerular

filtration rate (3.9 L/h) in kidney, which was calculated by

multiplying the creatinine clearance with the fraction

unbound of lanicemine (fu¼ 52%) in human plasma. This

result indicated that active tubular secretion probably does not

contribute to the renal elimination of lanicemine.

As CLR of lanicemine was just 37% of the systemic

clearance (CL) of lanicemine in plasma, hepatic metabolism

played an important role in the elimination of lanicemine in

man. In this study, a total of 10 excretory metabolites of

lanicemine were detected in urine, which accounted for

approximately 39% of dosed radioactivity. The proposed

biotransformation pathways of lanicemine in man are

illustrated in Scheme 1. The major route of metabolism is

the para-hydroxylation of phenyl ring, leading to the forma-

tion of major metabolite M1 and its glucuronide conjugate

M2. The other major metabolic pathways involved in

N-acytelation (M6, M7 and M8) and N-carbamoyl glucur-

onidation (M2, M4 and M9). Among them, the M1 and M6

were identified by comparison of retention time and MS2

spectrum with that of authentic standards. The structure and

metabolic pathway of M7 was confirmed by the incubation of

M1 in human liver cytosol in the presence of acetyl Co-A to

determine the position of hydroxyl group on the phenyl ring.

The structures of M2 and M8 were assigned by selectively

hydrolysis of M2 and M8 in urine using bacterial �-glucur-

onidase, revealing that the formation of M2 and M8 are

primarily via O-glucuronidation instead of N-glucuronidation

(Zenser et al., 1999). The chemical structures of other

metabolites were tentatively assigned based on the mass

spectral data.

The detected circulatory metabolites included a para-

hydroxylated metabolite (M1), O-glucuronide (M2), an

N-carbamoyl glucuronide (M3) and an N-acetylated metab-

olite (M6). The average amount of each of circulatory

metabolites was less than 4% of total detected peak area

(less than 5% of parent drug) in radiochromatograms. M6 was

only observed in plasma from one Asian subject with 3.1% of

the total radioactivity. As it is a low abundant circulatory

metabolite, M6 formation in Asian subjects is unlikely to lead

to the significant differences in lanicemine related activities

between ethnic groups.

In conclusion, the drug-related radioactivity was mainly

excreted in urine after a single intravenous administration of

[14C]-lanicemine in healthy subjects. The metabolite profile of

lanicemine in urine showed that the primary biotransform-

ation pathways of lanicemine in man include oxidation,

N-acetylation and N-carbamoyl glucuronidation. Given

lanicemine and its metabolites are excreted via multiple

elimination pathways, including metabolism and renal excre-

tion, it can be anticipated that the impaired hepatic or renal

function will exert moderate influence on the elimination

of lanicemine.

Acknowledgements

The authors acknowledge Quintiles Drug Research Unit at

Guy’s Hospital (London, UK) for conduct of the clinical mass

balance study; Covance Laboratories (Harrogate, UK) for

determination of [14C] radioactivity in whole blood, plasma,

urine and feces; BASi (US) for determination of concentra-

tion of lanicemine in human plasma and urine; and other

colleagues for their contributions to this study.

Declaration of interest

This study was sponsored by AstraZeneca Pharmaceuticals.


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