Comparison of the ex vivo receptor occupancy profile of ketamine to several NMDA receptor antagonists in mouse hippocampus

European Journal of Pharmacology 715 (2013) 21–25

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia

Comparison of the ex vivo receptor occupancy profile of ketamine to several NMDA receptor antagonists in mouse hippocampus Brian Lord a,n, Cindy Wintmolders b, Xavier Langlois b, Leslie Nguyen a, Tim Lovenberg a, Pascal Bonaventure a a b

Janssen Research and Development, LLC, 3210 Merryfield Row, San Diego, CA 92121-1126, United States Janssen Research and Development, LLC, Turnhoutseweg 30, 2340 Beerse, Belgium

art ic l e i nf o

a b s t r a c t

Article history: Received 19 March 2013 Received in revised form 4 June 2013 Accepted 21 June 2013 Available online 16 July 2013

NMDA receptor antagonists, particularly these targeting the GluN2B subunit are of therapeutic interest for the treatment of severe mood disorders. The receptor occupancy profiles of several NMDA receptor antagonists (30 mg/kg, s.c.) were compared in mouse hippocampus by ex vivo autoradiography using [3H]MK-801, a non-selective NMDA channel blocker, and [3H]ifenprodil a selective GluN2B antagonist. Subcutaneous administration of ketamine ((RS)-2-(2-Chlorophenyl)-2-(methylamino)cyclohexanone) and memantine (3,5-dimethyladamantan-1-amine) inhibited [3H]MK-801 but not [3H]ifenprodil binding in mouse hippocampus. Ketamine reached maximal occupancy of [3H]MK-801 binding sites after 15 min and rapidly cleared from the brain with no significant level of occupancy measured at the 1 h time point. Memantine significantly occupied [3H]MK-801 binding sites throughout the 6 h time course. The selective GluN2B antagonist CP101,606 ((1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol) and Ro 25-6981 ((αR,βS)-α-(4-Hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidinepropanol maleate) inhibited [3H]ifenprodil but not [3H]MK-801 binding and significant levels of occupancy (above 50%) were measured throughout the 6 h time course. These data highlight the unique quick pulse target engagement profile of ketamine compared to other NMDA receptor antagonists. & 2013 Elsevier B.V. All rights reserved.

Keywords: Ketamine GluN2B NMDA Autoradiography Receptor occupancy

1. Introduction NMDA receptors are critical for glutamate-mediated excitatory signaling, participating in synaptic transmission and triggering synaptic plasticity. The NMDA receptor forms a heterotetramer between two GluNR1 and two GluNR2 subunits. Four distinct isoforms of the GluNR2 subunit are expressed in vertebrates and are referred to GluN2A through D (Ozawa et al., 1998; Yamakura and Shimoji, 1998; Chazot and Hawkins, 1999; Dingledine et al., 1998). Related gene families of GluNR3 A and B subunits have an inhibitory effect on receptor activity (Henson et al., 2012). NMDARs are sites of action of important neuroactive agents including ketamine. Ketamine, despite its psychotomimetic potential, is gaining recognition as a rapidly acting antidepressant for individuals with severe and refractory mood disorders (Zarate et al., 2006; Machado-Vieira et al., 2009). Low doses of ketamine infused over an hour result in an acute antidepressant effect that can persist for days (Zarate et al., 2006). Ketamine, a phenycyclidine (PCP) derivative, is a noncompetitive, high affinity NMDA

n

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0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.06.028

antagonist. Ketamine blocks the NMDA receptor at the PCP site within the ionotropic channel. Non-competitive NMDA receptor antagonists like ketamine produce psychomimetic effects when used acutely. To try to eliminate these side-effects, subtype selective antagonists, particularly these targeting the GluN2B subunit have been developed (Loftis and Janowsky, 2003; Gogas, 2006). In the present study, we compared the ex vivo receptor occupancy profile of ketamine to several NMDA receptor antagonists, traditional NMDA channel blockers MK-801 ([5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine) and memantine alongside selective GluN2B antagonists ifenprodil ((1R*,2S*)-erythro-2(4-Benzylpiperidino)-1-(4-hydroxyphenyl)-1-propanol hemitartrate), CP101,606 ((1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol), Ro 25-6981 ((αR,βS)-α-(4-Hydroxyphenyl)-βmethyl-4-(phenylmethyl)-1-piperidinepropanol maleate) and eliprodil (α-(4-Chlorophenyl)-4-[(4-fluorophenyl)methyl]-1-piperidineethanol) (Whittemore et al., 1997; Ogden and Traynelis, 2011). Noteworthy, ifenprodil and eliprodil display some affinity for sigma receptors (Hashimoto and London 1993; Hashimoto and London, 1995; Ishima and Hashimoto, 2012). In addition ifenprodil has a known interaction with polyamines (Reynolds and Miller, 1989; Schoemaker et al., 1990), G protein-activated inwardly rectifying K+ channels (Kobayashi et al.,

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2006) and adrenergic one receptor (Chenard et al., 1991). Receptor occupancy was measured by ex vivo autoradiography using [3H]MK801 and [3H]ifenprodil, two commercially available tracers. A short incubation protocol was used to minimize dissociation. To mask [3H] ifenprodil binding to non-NMDA receptor sites, sigma and polyamine blockers were added (Guscott et al., 2003). Under these experimental conditions our [3H]ifenprodil protocol measures occupancy of the GluN2B site whereas our [3H]MK801 protocol measures occupancy within the ion channel.

analysis by liquid chromatography tandem mass spectrometry. The compounds were extracted using protein precipitation method and analyzed on a SCIEX API 4000 Q-Trap, using a Shimadzu SCL-10ADvp LC. Column: Zorbax, SB-C18, 2.1  50 mm, 5u. “A” Buffer: 0.1% formic acid in water. “B” Buffer 0.1% formic acid in acetonitrile. Gradient: 5% B–95% B in about 2 min. Compounds were detected with mulitple reaction monitoring with an injection volume of 10 μL and a flow rate of 0.7 ml/min. 2.4. Drugs and materials

2. Materials and methods 2.1. Drug treatment All animal work described in this paper was done in accordance with the Guide Care for and Use of Laboratory Animals adopted by the US National Institutes of Health. Animals were allowed to acclimate for 7 days after receipt. They were group housed in accordance with institutional standards, received food and water ad libitum and were maintained on a 12 h light/dark cycle. Male C57/bl6 mice (Jackson Laboratories Bar Harbor, Maine, USA) approximately 25–35 g in body weight were used. Drugs were formulated at 3 mg/ml in 20% Hydroxpropyl-beta-Cyclodextrin and delivered in a volume of 10 ml/kg for a final dose of 30 mg/ kg. Animals were treated subcutaneously with drugs (three animals per time point). Brains were rapidly frozen on powdered dry ice and stored at
80 1C before sectioning. 2.2. Ex vivo radioligand binding Hippocampal tissue sections of 20-micron thickness were prepared for autoradiography as previously described by Langlois et al. (2001). [3H]ifenprodil and [3H]MK-801 binding were modified from Guscott et al. (2003). For [3H]ifenprodil binding, tissue sections were incubated with 3 nM [3H]ifenprodil in 50 mM Tris HCl buffer (pH 7.4) containing 100 μM (+)3-PPP(R(+)-3-(3-Hydroxyphenyl)-N-propylpiperidine hydrochloride), 1 mM GBR-12909 (1-[2-[Bis-(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl) piperazine dihydrochloride), and 1 mM GBR-12935 (1-(2-Diphenylmethoxyethyl)-4-(3-phenylpropyl)piperazine dihydrochloride) for 1 min at 4 1C. Sections were rinsed in 50 mM Tris HCl on ice four times for 20 min per rinse followed by two dips in ice cold deionized water. Non-specific binding was characterized with 10 μM Ro-25-6981. For [3H]MK-801 tissue sections were incubated with 20 nM [3H]MK-801 in 30 mM HEPES buffer (pH 7.4) containing 1 mM EDTA (ethylenediaminetetraacetic acid), 100 mM Glutamate and 100 mM Glycine for 1 min at room temperature. Sections were rinsed on ice two times for 20 min in incubation buffer followed by two dips ice cold deionized water. Non-specific binding was characterized with 10 μM MK-801. Sections were dried under a stream of cold air following rinses. Digitized images were acquired with β-Imager (Biospace, Paris, France) following a three hour acquisition. Labeling the receptor on tissue section rather than homogenates and using a short incubation with the radioligand minimizes dissociation of the drug-receptor complex formed in vivo (Schotte et al., 1993). 2.3. Pharmacokinetics and Bioanalysis Following s.c. administration animals were euthanized with carbon dioxide then blood and brains removed. Blood was placed in tubes containing EDTA, centrifuged for 5 min at 11,000g, and plasma removed and stored at
80 1C. Remaining brain tissue following sectioning was homogenized in three volumes of sterile water. Plasma and brain homogenate was stored at
20 1C until

[3H]ifenprodil (1.48 TBq/mmol or 40.0 Ci/mmol) and [3H]MK801 (0.832 TBq/mmol or 22.5 Ci/mmol) were purchased from Perkin-Elmer (Waltham, Massachusetts, USA). Eliprodil, ifenprodil hemitartrate, memantine hydrochloride, (+)MK-801 maleate, Ro 25-6981 maleate were purchased from Tocris Bioscience (Bristol, United Kingdom). CP101,606, (+)ketamine hydrochloride, (+)3PPP, GBR 12909, GBR 12935, Tris HCl, HEPES, EDTA, Glycine, Glutamate and Hydroxypropyl-beta-Cyclodextrin were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). 2.5. Data analysis All data are presented as mean 7S.E.M. unless otherwise stated. Quantitative analysis of images was performed as described by Langlois et al. (2001). The percentage of receptor occupancy was determined as described by Barbier et al. (2007). Pharmacokinetic data was obtained by applying a non-compartmental model with the mean plasma or brain concentration with WinNonlin version 5.2 (Pharsight, Sunnyvale, CA, USA).

3. Results Mice were treated subcutaneously with 30 mg/kg of CP101,606, Ro 25-6981, ifenprodil, eliprodil, MK-801, memantine, ketamine (structures shown in Fig. 1) and [3H]ifenprodil or [3H]MK801 binding occupancy was measured in hippocampus at several time points. Representative digital images after treatment with CP101,606 or memantine are shown in Figs. 2 and 3, respectively. Full time courses are presented in Fig. 4. CP101,606, Ro 25-6981, ifenprodil and eliprodil exhibited significant level of occupancy at the [3H]ifenprodil binding site (maximal occupancy 460%). CP101,606 and Ro 25-6981 almost fully occupied the [3H]ifenprodil binding site and all four compounds maintained occupancy for this site at 445% throughout the 6 h time course. Later these compounds failed to significantly occupy the [3H]MK-801 binding site (maximal occupancy o25%). Conversely, ketamine, MK-801 and memantine had 4 50% maximal [3H]MK-801 occupancy and negligible level of occupancy at the [3H]ifenprodil binding site. The kinetics of ketamine differed from the other channel blockers with maximal level of occupancy observed after 15 min and a rapid clearance resulting in no significant level of occupancy at 1 h. The blood-brain barrier did not impair the ability of any of the drugs to cross into the brain (see Table 1) or limit the partitioning of all the drugs from the plasma into the brain noted by the high brain to plasma ratios. There was high exposure in both plasma and brain compartments which were maintained throughout the entire 6 h time course leading to terminal half-life of approximately 1 h or greater of all drugs tested with the exception of ketamine. Ketamine exhibited high initial concentration followed by rapid clearance in both plasma and brain marked by a half-life of roughly 0.5 h in both compartments. Although the kinetics of ketamine differs from the rest of the drugs tested, overall there was good correlation between receptor occupancy level and

B. Lord et al. / European Journal of Pharmacology 715 (2013) 21–25

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Fig. 3. Occupancy of [3H]ifenprodil and [3H]MK801 binding sites by memantine 30 min after subcutaneous administration of 30 mg/kg in the mouse hippocampus. Digital images obtained after with the β-imager. Note the 50% inhibition of [3H]MK801 binding with memantine. In comparison, memantine could not prevent [3H] ifenprodil binding.

Fig. 1. Structure of CP101,606, Ro 25-6981, ifenprodil, eliprodil, MK-801, memantine and ketamine.

Fig. 2. Occupancy of [3H]ifenprodil and [3H]MK801 binding sites by CP101,606 30 min after subcutaneous administration of 30 mg/kg in the mouse hippocampus. Digital images obtained after with the β-imager. Note the almost complete inhibition of [3H]ifenprodil binding with CP101,606. In comparison, CP101,606 could not prevent [3H]MK801 binding.

plasma/brain concentration, i.e. receptor occupancy levels drop when plasma/brain levels drop.

4. Discussion The receptor occupancy profiles of several NMDA receptor antagonists were compared in mouse hippocampus by ex vivo autoradiography using [3H]MK-801, a non-selective channel blocker and [3H]ifenprodil a selective GluN2B antagonist. The ex vivo [3H] MK-801 and [3H]ifenprodil binding protocols have been used in the literature but on brain membranes (Guscott et al., 2003). The protocol described in this paper used autoradiography and has several advantages to in vitro homogenate binding assays. With ex vivo receptor binding experiments, the unlabeled drug is administered

peripherally to the animal; thereafter, the animal is sacrificed and the brain processed for ex vivo receptor labeling. The advantage of labeling the receptor on tissue sections rather than in tissue homogenates has been demonstrated in the literature (Schotte et al., 1989, 1993). The dissociation of the drug-receptor complex formed in vivo can be kept minimal by immediate freezing of the brains, omitting pre-incubations of the sections, and by using short incubations with the radioligand. The short incubation time is a critical step of ex vivo autoradiographic protocol. The high sensitivity of the β-imager allows us to further decrease the incubation time to 1 min compared to the standard incubation time that we are generally using in our laboratory for ex vivo autoradiography experiments of 10 min (Schotte et al., 1996). The results from the present study demonstrate that reliable measure of ex vivo receptor occupancy and thus proof of target engagement can be measured for several NMDA receptor antagonists. With short incubation and imaging time this method is suitable for rapid screening of compounds. Our data are in agreement with the data reported by Guscott et al. (2003) showing that after intraperitoneal administration ifenprodil and CP101,606 significantly occupied [3H]ifenprodil binding sites but not [3H]MK801 in rat brain membranes. However, in contrast to the data reported by Murray et al. (2000) we did not observed any significant level of occupancy of [3H]MK-801 binding sites after subcutaneous administration of Ro-25-6981 or CP101,606. [3H] MK-801 binding assay protocols can be biased towards the investigation of a particularly modulatory site (Murray et al., 2000). Under our experimental conditions (no pre washes and high glutamate/glycine concentration) our [3H]MK-801 binding protocol does not measure occupancy of the ifenprodil binding site. We did not use [3H]Ro 25-6981 to measure GluN2B occupancy, [3H]Ro 25-6981 is more selective than [3H]ifenprodil (Mutel et al., 1998; Cyr et al., 2001), but to the best of our knowledge is not commercially available. All the compounds investigated in this study occupied mouse brain NMDA receptors in a time-dependent manner that reflected the plasma and brain kinetics of the drugs. Our dose selection of 30 mg/kg was much higher than what was used in the clinic and this dose was chosen to address the occupancy profiles of these drugs and not mimic the low doses that were used to demonstrate efficacy. Ketamine exhibited rapid occupancy of the NMDA channel with rapid clearance off the receptor. This unique kinetic profile might explain the rapid antidepressant effect observed with ketamine. Although the occupancy of ketamine is transient the antidepressant effects can persist for days (Zarate et al., 2006). This pathophysiology of ketamine in depression is unclear. It has

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CP 101,606

Ketamine

100

% Receptor Occupancy

% Receptor Occupancy

100 80 60 40 20

80 60 40 20

0

0 0

1

2

3

4

5

6

7

0

1

2

3

Time (h)

Ro 25-6981

80 60 40 20

7

5

6

7

5

6

7

60 40 20 0

0

1

2

3

4

5

6

7

0

1

2

3

Time (h)

4

Time (h)

Ifenprodil

Memantine

100

% Receptor Occupancy

100

% Receptor Occupancy

6

80

0

80 60 40 20

80 60 40 20 0

0 0

1

2

3

4

5

6

7

0

1

2

Time (h)

3

4

Time (h)

Eliprodil

100

% Receptor Occupancy

5

MK-801

100

% Receptor Occupancy

% Receptor Occupancy

100

4

Time (h)

80 60 40 20 0 0

1

2

3

4

5

6

7

Time (h) Fig. 4. Occupancy of [3H]ifenprodil and [3H]MK801 binding sites by CP101,606, Ro 25-6981, ifenprodil, eliprodil, ketamine, MK-801 and memantine in function of time after subcutaneous administration of 30 mg/kg in the mouse hippocampus. Occupancy vs. time curves for [3H]ifenprodil receptor occupancy in the mouse hippocampus depicted by dotted line. Occupancy vs. time curves for [3H]MK-801 receptor occupancy in the mouse hippocampus depicted by full line. Results are expressed as average percent receptor occupancy vs. vehicle treated mice 7 standard error of the mean (n ¼3).

Table 1 Mouse pharmacokinetic parameters after 30 mg/kg s.c. administration of the compounds (n¼ 15, 3 mice per time point). Plasma

CP101,606 Ro 25-6981 Ifenprodil Eliprodil MK-801 Memantine Ketamine

Brain

Cmax (ng/ml)

Tmax (h)

AUCinf (h ng/ml)

T1/2 (h)

Cmax (ng/ml)

Tmax (h)

AUCinf (h ng/ml)

T1/2 (h)

B/P ratio

2315.80 1902.70 1136.60 1611.90 2634.60 3099.30 2565.60

0.50 0.25 0.25 2.00 1.00 0.50 0.25

3496.14 4724.34 6849.15 10004.80 8241.38 13408.19 3144.75

0.77 0.97 3.12 3.15 1.57 2.14 0.43

1988.67 6300.40 3707.47 5336.80 13268.80 18512.67 5981.73

1.00 0.25 0.25 1.00 1.00 2.00 0.25

4581.59 18983.97 21698.31 47021.29 39679.83 99349.53 5239.40

0.88 0.85 3.11 4.97 1.44 2.89 0.47

0.86 3.31 3.26 3.31 5.04 5.97 2.33

B. Lord et al. / European Journal of Pharmacology 715 (2013) 21–25

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