A Novel M1 PAM VU0486846 Exerts Efficacy in Cognition Models without Displaying Agonist Activity or Cholinergic Toxicity

Article abstract of DOI:10.1021/acschemneuro.8b00131

Selective activation of the M₁ subtype of muscarinic acetylcholine receptor, via positive allosteric modulation (PAM), is an exciting strategy to improve cognition in schizophrenia and Alzheimer's disease patients. However, highly potent M₁ ago-PAMs, such as MK-7622, PF-06764427, and PF-06827443, can engender excessive activation of M₁, leading to agonist actions in the prefrontal cortex (PFC) that impair cognitive function, induce behavioral convulsions, and result in other classic cholinergic adverse events (AEs). Here, we report a fundamentally new and highly selective M₁ PAM, VU0486846. VU0486846 possesses only weak agonist activity in M₁-expressing cell lines with high receptor reserve and is devoid of agonist actions in the PFC, unlike previously reported ago-PAMs MK-7622, PF-06764427, and PF-06827443. Moreover, VU0486846 shows no interaction with antagonist binding at the orthosteric acetylcholine (ACh) site (e.g., neither bitopic nor displaying negative cooperativity with [³H]-NMS binding at the orthosteric site), no seizure liability at high brain exposures, and no cholinergic AEs. However, as opposed to ago-PAMs, VU0486846 produces robust efficacy in the novel object recognition model of cognitive function. Importantly, we show for the first time that an M₁ PAM can reverse the cognitive deficits induced by atypical antipsychotics, such as risperidone. These findings further strengthen the argument that compounds with modest in vitro M₁ PAM activity (EC₅₀ > 100 nM) and pure-PAM activity in native tissues display robust procognitive efficacy without AEs mediated by excessive activation of M₁. Overall, the combination of compound assessment with recombinant in vitro assays (mindful of receptor reserve), native tissue systems (PFC), and phenotypic screens (behavioral convulsions) is essential to fully understand and evaluate lead compounds and enhance success in clinical development.

Full text of DOI:10.1021/acschemneuro.8b00131

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Article
A novel M1 PAM VU0486846 exerts efficacy in cognition models
without displaying agonist activity or cholinergic toxicity
Jerri M. Rook, Jeanette L Bertron, Hyekyung P. Cho, Pedro M Garcia-Barrantes, Sean P Moran, James T
Maksymetz, Kellie D Nance, Jonathan W Dickerson, Daniel H Remke, Sichen Chang, Joel Harp, Anna L.
Blobaum, Colleen M Niswender, Carrie K. Jones, Shaun R. Stauffer, P. Jeffrey Conn, and Craig W Lindsley
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00131 • Publication Date (Web): 27 Apr 2018
Downloaded from http://pubs.acs.org on April 28, 2018
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A novel M1 PAM VU0486846 exerts efficacy in cognition models without
displaying agonist activity or cholinergic toxicity
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Jerri M. Rook,1,4† Jeanette L. Bertron,2† Hyekyung P. Cho,1,4† Pedro M. GarciaꢀBarrantes,4† Sean P.
Moran,1,4 James T. Maksymetz,1,4 Kellie D. Nance,1,4 Jonathan W. Dickerson,1,4 Daniel H. Remke,1,4
Sichen Chang,4 Joel M. Harp,3 Anna L. Blobaum,1,4 Colleen M. Niswender,1,4,5 Carrie K. Jones,1,4 Shaun
R. Stauffer,1 P. Jeffrey Conn1,4,5 and Craig W. Lindsley1,2,3,4*
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1Department of Pharmacology, Department of Chemistry, Department of Biochemistry, Vanderbilt Center for
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Neuroscience Drug Discovery, Vanderbilt Kennedy Center, Vanderbilt University, Nashville, TN 37232ꢀ6600,
USA
†These authors contributed equally to this work.
Keywords: M1, muscarinic acetylcholine receptor, agonist, positive allosteric modulator (PAM), agoꢀPAM,
cognition, schizophrenia
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Abstract: Selective activation of the M1 subtype of muscarinic acetylcholine receptor, via positive
allosteric modulation (PAM), is an exciting strategy to improve cognition in schizophrenia and
Alzheimer’s disease patients. However, highly potent M1 agoꢀPAMs, such as MKꢀ7622, PFꢀ06764427,
and PFꢀ06827443, can engender excessive activation of M1, leading to agonist actions in the prefrontal
cortex (PFC) that impairs cognitive function, induce behavioral convulsions, and results in other classic
cholinergic adverse events (AEs). Here, we report a fundamentally new and highly selective M1 PAM,
VU0486846. VU0486846 possesses only weak agonist activity in M1ꢀexpressing cell lines with high
receptor reserve and is devoid of agonist actions in the PFC, unlike previously reported agoꢀPAMs MKꢀ
7622, PFꢀ06764427 and PFꢀ06827443. Moreover, VU0486846 shows no interaction with antagonist
binding at the orthosteric acetylcholine (ACh) site (e.g., neither bitopic nor displaying negative
cooperativity with [3H]ꢀNMS binding at the orthosteric site), no seizure liability at high brain exposures,
and no cholinergic AEs. However, as opposed to agoꢀPAMs, VU0486846 produces robust efficacy in the
novel object recognition model of cognitive function. Importantly, we show for the first time that an M1
PAM can reverse the cognitive deficits induced by atypical antipsychotics, such as risperidone. These
findings further strengthen the argument that compounds with modest in vitro M1 PAM activity (EC50s >
100 nM) and pureꢀPAM activity in native tissues display robust proꢀcognitive efficacy without AEs
mediated by excessive activation of M1. Overall, the combination of compound assessment with
recombinant in vitro assays (mindful of receptor reserve), native tissue systems (PFC), and phenotypic
screens (behavioral convulsions) is essential to fully understand and evaluate lead compounds and
enhance success in clinical development.
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INTRODUCTION
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Of the five muscarinic acetylcholine receptor subtypes (M1ꢀM5), extensive biochemical, genetic
and patient data have implicated selective activation of M1 as an attractive approach for the treatment of
the cognitive deficits associated with both schizophrenia and Alzheimer’s disease (AD).1ꢀ15 Early efforts
to selectively activate M1 with panꢀmuscarinic agonists ultimately failed due to a lack of subtype
selectivity, resulting in severe gastrointestinal (GI) disturbances and SLUD (salivation, lacrimation,
urination, defecation) due to activation of peripheral M2 and M3.16 However, Phase II and Phase III
clinical trials with these agonists demonstrated proꢀcognitive efficacy.17,18 Thus, the field moved to
targeting less conserved allosteric binding sites on the M1 receptor and achieved unprecedented levels of
muscarinic subtype selectivity with positive allosteric modulators (PAMs).1,2,12ꢀ14 BQCA was the first of
these compounds disclosed (Figure 1).19ꢀ21 However, BQCA and related analogs, such as the subsequent
clinical compound MKꢀ7622,22 are robust agoꢀPAMs demonstrating direct agonist activity in addition to
PAM activity in both in vitro recombinant systems and native tissues).19ꢀ23 In addition, they appear to
interact with the orthosteric site, which may contribute to their severe seizure liability and other
cholinergic adverse effects (AEs), as well as limit their proꢀcognitive efficacy.19ꢀ23 An early compound
derived from our internal highꢀthroughput screening (HTS) campaign,24 VU0453595 (VU595), proved to
be a pureꢀPAM, devoid of direct agonist activity, in both recombinant and native systems. Additionally,
VU595 was devoid of AEs and possessed proꢀcognitive activity. However, VU0453595 is a weak M1
PAM with a potency at M1 in the low ꢀM range.25 Derived from another HTS hit, VU6004256, is a more
potent M1 PAM, with agonist activity in high receptor reserve recombinant cell lines, yet is devoid of
behavioral convulsions and cholinergic AEs in rodents.26,27 Unlike the structurally related M1 agoꢀPAM
PFꢀ0676442728 and the reported M1 PAM PFꢀ06767832,29 VU6004256 displays proꢀcognitive efficacy in
rodents.26,27 Furthermore, despite very low agonist activity in a recombinant in vitro cell line, PFꢀ
06827443 still elicited robust cholinergic AEs across species and induced seizures in mice.29 Recently, we
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demonstrated that similar to MKꢀ7622,22 PFꢀ06827443 is a potent agonist in native systems (prefrontal
cortex (PFC), and that agonist activity in native tissues accurately predicts the magnitude of in vivo
efficacy/toxicity.30 Thus, the field does not have a robust in vivo M1 PAM with the ideal properties to
critically evaluate the role of M1 in CNS biology. Therefore, we are reporting VU0486846, a highly
selective, potent M1 PAM devoid of agonist activity in native tissues and cholinergic AEs, with robust
proꢀcognitive efficacy, for the benefit of the research community to study selective M1 activation in vitro
and in vivo.
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HO
O
N
OH
O
N
N
O
N
O
N
F
N
OMe
N
N
BQCA (1)
MK-7622 (2)
VU0453595 (3)
O
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N
OH
OH
HO
HN
F
O
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HN
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N
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F
N
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VU6004256 (4)
PF-06764427 (5)
PF-06827443 (6)
Figure 1. Structures of reported M1 PAMs with varying degrees of M1 PAM potency, varying degrees of
M1 agoꢀPAM activity in recombinant cell lines and native tissues, and a diverse range of cholinergic AEs.
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RESULTS DISCUSSION
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Ligand Design Toward the Next Generation of M1 PAMs. Having exhausted our indoleꢀbased series
of M1 PAMs (e.g., VU6004256),26,27 we revisited where the M1 PAM field began – with BQCA.19ꢀ22 The
discovery of the (1S,2S)ꢀ2ꢀaminocyclohexanꢀ1ꢀol amide moiety, as replacement for the carboxylic acid
functionality of BQCA, as in 7, was a waterꢀshed event.31 Even more salient was the recognition that the
secondary NꢀH of (1S,2S)ꢀ2ꢀaminocyclohexanꢀ1ꢀol amide moiety formed an intramolecular hydrogen
bond (IMHB) with the carbonyl oxygen of the quinolone core.29ꢀ31 It was this recognition that led to the
tricyclic core of MKꢀ7622 and the azaindole core of PFꢀ06764427. Utilizing the 6,6ꢀfused ring system of
BQCA and 7, we simultaneously increased sp3 character, while bringing the Lewis basic oxygen into the
ring by scaffold hopping to a benzomorpholine core 8 (Figure 2). By virtue of this juxtaposition, we also
created a new chiral center, and the opportunity for enantioselective M1 PAM activity while still
maintaining the key intramolecular hydrogen bonding (IMHB) between the secondary amide NꢀH and the
benzomorpholine oxygen atom.
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Figure 2. Ligand design concept to scaffold hop from the classical M1 PAM quinolone core of 1 and 7 to
a novel, benzomorphoine core 8, bearing a new chiral center and increased sp3 character.
Chemistry and Limited Structure-Activity Relationships (SAR).
The chemistry to access
benzomorpholineꢀbased M1 PAMs was straightforward (Scheme 1). Following a standard route to the
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benzomorpholine core, commercial ethyl 2,3ꢀdibromopropanoate 9 was condensed with 2ꢀaminophenol to
provide the key racemic heterocyclic core 10 in 65% yield. Benzylation with 4ꢀbromobenzyl bromide
proceeded smoothly delivering 11, suitable for diversification, in 74% yield. A quantitative hydrolysis of
the ester gave 12, which then underwent a HATUꢀmediated amide coupling with (1S,2S)ꢀ2ꢀ
aminocyclohexanꢀ1ꢀol to provide 13 as a mixture of diastereomers 13. Finally, a copperꢀmediated
coupling with pyrazole produced the putative M1 PAM 14, in 65% yield.
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Scheme 1. Synthesis of benzomorpholine-based M1 PAM 14.
o
aReagents and conditions: (a) 2ꢀaminophenol, CH3CN, K2CO3, 60 C, 65%; (b) 4ꢀbromobenzyl bromide,
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K2CO3, CH3CN, 80 C, 74%; (c) KOH, THF/H2O (2:1), rt, 98%; (d) (1S,2S)ꢀ2ꢀaminocyclohexanꢀ1ꢀol,
HATU, DIEA, DMF, rt, 80%; (e) pyrazole, (1S,2S)ꢀN1,N2ꢀdimethylcyclohexaneꢀ1,2ꢀdiamine, CuI, K3PO4,
dioxane, rt, 65%.
Upon testing, 14 proved to be a human M1 PAM (EC50 = 0.92 ꢀM, pEC50 = 6.1±0.13, 77±7%
acetylcholine (ACh) Max) with weak M1 agonist activity (EC50 >10 ꢀM, 22% ACh Max). The
diastereomers at the benzomorpholine chiral center were readily separable by column chromatography,
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affording isomerꢀ1, 15 (VU0486834) and isomerꢀ2, 16 (VU0486846). A single crystal Xꢀray structure
determined the absolute stereochemistry of 16 (Figure 3) as the (R)ꢀenantiomer and highlighted the
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IMHB.32 Moreover, enantioselective M1 PAM activity was noted, with 15 (EC50 = 0.63 ꢀM, pEC50
=
9
6.21±0.05 89±2% ACh Max and weak agonist activity EC50 >10 ꢀM, 16% ACh Max) being less potent
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than VU0486846 (EC50 = 0.31 ꢀM, pEC50 = 6.53±0.05, 85±2% ACh Max and weak agonist activity EC50
= 4.0 ꢀM, pEC50 = 5.39±0.07, 39±3% ACh in our high expression line). These positive data necessitated
a more in depth pharmacological evaluation of novel M1 PAM VU0486846 (VU0486846), which also
possessed a favorable central nervous system multiparameter optimization (CNS MPO) score (>5).33
Figure 3. Structures and human M1 PAM activities of 14ꢀ16, as well as a single Xꢀray crystal structure of
the more active (R)ꢀenantiomer 16 (VU0486846).
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Molecular pharmacology studies and adverse effect liability. As shown previously,26,27 in our high
expression human M1 cell line, VU0486846 was a moderately potent M1 PAM (EC50 = 0.31 ꢀM, pEC50 =
6.53±0.05, 85±2% ACh Max) with weak, partial agonist activity (EC50 = 4.5 ꢀM, pEC50 = 5.37±0.07,
29±6% ACh). In our rat M1 cell line (also a high expression system), VU0486846 was an equipotent
PAM (EC50 = 0.25 ꢀM, pEC50 = 6.63±0.06, 83±1% ACh Max) with weak, partial agonist activity (EC50 =
5.6 ꢀM, pEC50 = 5.27±0.05, 26±6% ACh). In contrast, in our mouse M1 cell line with low expression,
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VU0486846 showed little agonism (9±1% ACh Max), and good M1 PAM potency (EC50 = 0.6 ꢀM, pEC50
= 6.25±0.10, 77±4% ACh Max). Similarly, in our low expression M1 dog line, VU0486846 was a PAM
(EC50 = 0.38 ꢀM, pEC50 = 6.41±0.03, 78±1% ACh Max) with minimal agonist activity (EC50 > 10 ꢀM,
18±0.2 % ACh Max). Agonism (full/partial) is a receptor reserve/expressionꢀdependent driven
pharmacology, whereas PAM activity is conserved across varying degrees of expression/reserve,
potentiating the EC20 orthosteric agonist effect.23 Therefore, the lower expression cell lines are more
indicative of the native system. Demonstrating a lack of agonist activity in these in vitro systems or native
tissues, such as the PFC, more accurately predicts a lack of CNS AEs.23,27,30 The lack of agonist activity in
the lower expressing cell line by VU0486846, suggests that this compound will be devoid of seizure
liability in vivo. A point worth mentioning is the potential for overꢀstimulation of M1 with either a highly
potent M1 PAM or a potent M1 agoꢀPAM exists, and would thereby lose the mechanistic advantage of
potentiation by these allosteric modulators. As the in vitro cellular functional assays employ a subꢀ
threshold concentration of ACh (typically an EC20 concentration), in vivo PAM potency is arguably
underꢀestimated, as cholinergic tone in many brain regions may approach an EC100 levels and leftward
shifts in PAM potency are observed with increasing ACh concentration. For example (Supplemental
Figure 1),32 with a very low level of ACh (an ~EC9), VU0486846 exhibited an M1 PAM potency of
430±120 nM (84±2% ACh Max), but as ACh concentration was increased to an EC70, M1 PAM potency
improved to 68±11nM (90±1% ACh Max).32 Similarly, PFꢀ06827443 also showed an increase in M1
PAM potency from 40±6 nM to 7±1 nM at the same ACh concentration range.32 Thus, a very potent
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PAM or agoꢀPAM in vitro may lead to overꢀactivation of M1 in vivo leading to M1ꢀmediated seizure
liability. After in vitro and in vivo evaluation of M1 PAMs across multiple chemotypes, M1 functional
potencies in the 100ꢀ400 nM potency range in vitro in our cellꢀbased systems have proven to be a first
step towards avoiding seizure liability in vivo. As shown in Figure 4, M1 PAM VU0486846 was highly
selective for M1 against both human M2ꢀM5 (Figure 4A) and rat M2ꢀM5 (Figure 4B). While M1
VU0486846 had no interaction with the orthosteric site (Figure 4C), i.e., no displacement of [3H]ꢀNMS
binding, other M1 PAMs, such as 1,2,5 and 6, with reported adverse effect liability, show weak to
significant displacement of [3H]ꢀNMS, and in some cases found to possess negative
cooperativity.19,20,23,25,27ꢀ30 With regard to this parameter, we feel M1 PAMs should not interact with the
orthosteric site, and that PAMs with negative cooperativity can present as proꢀcognitive efficacy (reversal
of scopolamineꢀinduced deficits) when, in reality, they are inducing a doseꢀdependent reversal of
scopolamine binding. This is further supported by ligands such as 2, which failed to show efficacy in a
rat novel object recognition (NOR) model, yet the same structural class of compounds were efficacious in
scopolamine challenge models.23,34,35
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Figure 4. Molecular pharmacology profile of 16. A) Human M1 CRC (EC50 = 308 nM) versus M2ꢀM5
(inactive); B) Rat M1 CRC (EC50 = 253 nM) versus M2ꢀM5 (inactive); C) Inhibition of orthosteric
radioligand binding with [3H]ꢀNMS by VU0486846 and atropine control. VU0486846 does not
substantially inhibit binding of [3H]ꢀNMS up to 30 ꢀM.
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Finally, prior to any further characterization, a high dose (100 mg/kg intraperitoneal (i.p.))
phenotypic mouse screen was performed to assess seizure liability, with a clean profile allowing a PAM
to advance in development. Whereas seizure activity in mouse indicates overꢀstimulation of M1 (either
too potent of an M1 PAM or robust agoꢀPAM), the latter are compounds not suitable for further
advancement down the lead optimization work flow.22,26 VU0486846 was devoid of seizure liability in
mice up to 3 hours at a 100 mg/kg dose; by comparison, both 222 and 526 displayed Racine scale 4/5
behavioral convulsions that develop rapidly and lasted for the 3 hour duration of the study.
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Electrophysiology studies in layer V medial prefrontal cortex (mPFC) pyramidal neurons reveal
that VU0486846, in contrast to other ago-PAMs, maintains the activity-dependence of muscarinic
agonists. A critical feature of an M1 PAM that appears to differentiate proꢀcognitive efficacy from
cognitive disruption is agonist effects in a native system such as the rodent PFC.20,23,25,27ꢀ30 As agonist
activity in cell lines can be accentuated or disguised by the level of receptor expression, assessment in
native systems is the ultimate arbiter.30 As we recently reported, 2 (MKꢀ7622), 5 (PFꢀ06764427) and 6
(PFꢀ06827443) have agonist activity in the mouse PFC, which overꢀactivates M1 in the absence of
endogenous ACh release and disrupts PFC function potentially leading to the observed lack of proꢀ
cognitive efficacy in rodent models. In contrast, pure M1 PAMs do not.23,30 Evaluation of VU0486846 in
this same assay was shown to induce no significant change in field excitatory post synaptic potentials
(fEPSPs) electrically evoked in layer II/III and recorded in layer V, and therefore maintain activity
dependence of PFC function (Figure 5A). A submaximal concentration of the prototypical acetylcholine
receptor (AChR) agonist carbachol (10 µM) does not induce a significant longꢀterm depression (LTD) of
layer V fEPSPs (Figure 5B) as previously shown,23,25,30 but can be potentiated to a robust LTD by the M1
PAM VU0486846 (Figure 5C), similar to other previously reported M1 pureꢀPAMs such as
VU045359525 and VU055164.23 Furthermore, at the ventral hippocampalꢀmPFC synapse, a pathway
thought to be dysregulated in schizophrenia,36,37 a submaximal concentration (3ꢁM) of the muscarinic
acetylcholine receptor (mAChR) agonist oxotremorineꢀM (OxoM), does not induce a significant LTD of
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layer V fEPSPs evoked by optical stimulation of ventral hippocampal afferents (Figure 5D). However,
VU0486846 can robustly potentiate the effects of 3 µM OxoM to induce a substantial LTD at the ventral
hippocampalꢀPFC synapse. A significant increase in the magnitude of LTD was observed with coꢀ
application of 3 µM VU0486846 + 10 µM CCh compared to 10 µM CCh alone (p<0.05) (Figure 5F). In
addition, the magnitude of LTD observed with coꢀapplication of 10 µM VU0486846 + 3 µM OxoM
compared to 3 µM OxoM alone (p<0.05) was also significantly enhanced (Figure 5G). Therefore, in this
native system, VU0486846 displays no agonist activity on its own but can potentiate the effects of
cholinergic agonists and thus holds promise for utility as an in vivo tool to assess the proꢀcognitive
efficacy of M1 PAMs in rodent models.
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Figure 5. The M1 PAM VU0486846 can robustly potentiate a submaximal cholinergic agonistꢀinduced
longꢀterm depression (LTD) in layer V of the prelimbic mPFC evoked by either electrical or optical
stimulation (A) Time course graph showing that bath application of 3 µM VU0486846 for 20 min led to
no significant change in fEPSP slope. (B) Time course graph showing that bath application of 10 µM
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carbachol (CCh), a cholinergic agonist, induces a negligible LTD of fEPSPs in layer V electricallyꢀ
evoked by stimulation of layer II/III in the mPFC. (C) 10 min pretreatment of the M1 PAM VU0486846
(3 ꢁM) followed by a 10 min coꢀapplication of PAM and 10µM CCh led to a robust LTD of electrically
evoked fEPSP slope. Inset shows representative fEPSP traces for each condition for baseline (black trace)
and 50 min after CCh washout (gray trace) (D) Time course graph showing that bath application of 3 µM
OxoM, a selective muscarinic agonist, for 10 min led to an acute depression followed by a minimal LTD
of optically evoked fEPSPs (ofEPSP) measured 46ꢀ50 min following drug washout. (E) Under similar
conditions bath application 10 min pretreatment of the M1 PAM VU0486846 (10 ꢁM) followed by a 10
min coꢀapplication of PAM with 3 µM OxoM led to a robust LTD of ofEPSP slope. Inset shows
representative ofEPSP traces for each condition for baseline (black trace) and 50 min after OxoM
washout (blue trace). (F) Quantification of fEPSP slope 46ꢀ50 min following drug washout (shaded area)
indicates a significant depression of fEPSP slope with 3 µM VU0486846 + 10 µM CCh compared to 10
µM CCh alone (n = 9 brain slice experiments per group). (G) Quantification of ofEPSP slope 46ꢀ50 min
following drug washout (shaded area) indicates a significant depression of ofEPSP with 10 µM
VU0486846 + 3 µM OxoM compared to 3 µM OxoM alone (n = 6ꢀ8 brain slice experiments per group).
Scale bars denote 0.5 mV and 5ms. Data are expressed as mean ± SEM *denotes p<0.05, student’s tꢀtest.
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Drug Metabolism Studies. Due to the differentiated pharmacological profile of VU0486846 (16) from
the other M1 PAMs (Figure 1),19,20,23,25,27ꢀ30 we evaluated VU0486846 in a battery of in vitro and in vivo
drug metabolism and pharmacokinetic (DMPK) assays. VU0486846 was predicted to be a low to
moderately cleared compound in hepatic microsomes (human CLhep = 11.1 mL/min/kg; rat CLhep = 23
mL/min/kg; cyno CLhep = 43 mL/min/kg) with attractive fraction unbound in plasma (human fu = 0.12, rat
fu = 0.11, cyno fu = 0.016) and rat brain homogenate (fu = 0.03). The CYP450 profile was acceptable for a
tool compound (3A4 IC50 = 5.0 ꢀM; 1A2 IC50 > 30 ꢀM; 2C9 IC50 = 18.8 ꢀM; 2D6 IC50 = 29.8 ꢀM), but
precluded further advancement as a putative clinical candidate due to the inhibition of 3A4. Like other
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M1 PAMs harboring the (1S,2S)ꢀ2ꢀaminocyclohexanꢀ1ꢀol amide moiety, CNS exposure varied across
rodent species (rat Kp = 0.18, Kp,uu = 0.05; mouse Kp = 0.67; Kp,uu = 0.17), but the compound was not a
human Pꢀgp substrate (MDCK ER = 0.9, Papp = 30 x 10ꢀ6 cm/s). In vivo PK for VU0486846 was
evaluated in both rats and nonꢀhuman primates. From a standard rat PK IV/PO crossꢀover study,
VU0486846 showed high clearance (CLp = 89 mL/min/kg) and a lack of an in vitro:in vivo correlation
(IVIVC) from predicted in vitro parameters. 16 also displayed a 1.2 hour halfꢀlife, a reasonable volume
(Vss = 1.8 L/kg), and excellent oral bioavailability (%F = 95.9) in rat. A similarly designed study in male
cynomolgus monkey was more favorable, with a 4.2 hour halfꢀlife, moderate clearance (CLp = 18
mL/min/kg), good volume (Vss = 1 L/kg) and good oral bioavailability (%F= 37) from the amorphous
solid. No adverse events or cholinergic side effects were noted in these PK studies. While VU0486846
was highly selective versus M2ꢀM5, we wanted a broader assessment of ancillary pharmacology prior to
initiating extensive in vivo studies. Evaluation of VU0486846 in a Eurofins Lead Profiling panel of 68
GPCRs, ion channels, and transporters revealed no significant activity at any target in the panel (no
inhibition >50%@10 ꢀM).32 Thus, we were ready to assess the behavioral pharmacology of a structurally
novel M1 PAM with good CNS penetration, an attractive DMPK profile, devoid of agonist activity in the
PFC, and free from seizure/cholinergic liability in mouse, the most sensitive species.
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Behavioral Pharmacology Assessment. Prior to behavioral pharmacology assessment for efficacy of
the novel M1 PAM VU0486846, we evaluated the potential of VU0486846 to produce cholinergic or CNS
adverse events in both mice and rats. Thus, we performed a Modified Irwin Toxicology Battery
(Supplemental Figure 2)32 test in mice dosed at 100 mg/kg i.p., which mirrored the mouse seizure study.
Over the 3 hour evaluation period, VU0486846 did not produce any cholinergic side effects or
pronounced adverse effects in mice, as opposed to nonꢀselective muscarinic agonists and M1 agoꢀ
PAMs.23,27 Moreover, exposure from this study was more than sufficient to elicit adverse pharmacology.
Here, total plasma levels reached 18.2 ꢀM (30x above the mouse EC50), free plasma levels were 2 ꢀM,
with correlating high brain levels (total brain reached 12.2 ꢀM, free brain levels were 342 nM). A similar
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Modified Irwin Toxicology Battery (Supplemental Figure 3)32 test in rats was also performed at 56.6
mg/kg i.p, and, as with mouse, VU0486846 did not produce any cholinergic side effects or pronounced
adverse effects in rats, as opposed to nonꢀselective muscarinic agonists/M1 agoꢀPAMs, over the 3 hour
evaluation period. Here, total terminal plasma exposure reached 4.4 ꢀM (489 nM free) with acceptable
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brain levels (1.15 ꢀM total, 32 nM free). We attempted a related nonhuman primate (NHP) assessment,
but the physiochemical properties and exposure limitations of VU0486846 precluded definitive data;
however, we achieved total plasma levels in NHP of ~ 2 ꢀM, without any adverse effects. The
physiochemical properties of VU0486846 did not allow for higher dose formulations, and is another
limitation, beyond 3A4 inhibition, that precluded further development towards a clinical candidate.
Previously, we demonstrated that M1 PAMs with no agonist activity in native systems (PFC),
afforded robust efficacy in the novel object recognition (NOR) paradigm, whereas M1 PAMs and agoꢀ
PAMs with agonist activity in the PFC showed little efficacy in NOR.23,27,30 As shown in Figure 6A, PAM
VU0486846 doseꢀdependently enhanced recognition memory in rats, with a minimum effective dose of 3
mg/kg i.p (n = 12; p = 0.0133; F = 4; R2 = 0.2143). Thus, suggesting that VU0486846 could serve as an
M1 PAM to investigate efficacy in other models of cognition. The cognitive and negative symptom
clusters of schizophrenia are a major focus of our lab. However, a major unmet need in the schizophrenia
patient population is the added cognitive dysfunction induced by standard atypical antipsychotics.38ꢀ40,
Indeed, first line agents, such as risperidone, are known to engender cognitive deficits in humans (and in
rodent models), where cognitive dysfunction is already a major symptom of the disease.38ꢀ41 In Figure
6B, we show for the first time that an M1 PAM can reverse the cognitive deficits induced by risperidone
(n = 11ꢀ13; p < 0.0001; F = 6.682; R2 = 0.3430), and suggest coꢀadministration of an M1 PAM with an
atypical antipsychotic might attenuate cognitive impairments in schizophrenia patients. In these studies,
VU0486846 doseꢀdependently reversed risperidoneꢀinduced deficits in acquisition of contextual fear
conditioning. When coꢀadministered with 3 mg/kg risperidone, 10 mg/kg VU0486846 restored
conditioned freezing to vehicleꢀtreated control levels, with a minimum effective dose (MED) of 1 mg/kg.
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Future efforts will evaluate the cognitive dysfunction induced by other typical/atypical antipsychotics and
the ability of VU0486846 to reverse these deficits.
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Figure 6. Novel object recognition and contextual fear conditioning. A) VU0486846 doseꢀ
dependently enhanced recognition memory in rats. Pretreatment with 3 and 10 mg/kg VU0486846 (i.p.,
10% Tween 80 in water, 30 min) prior to exposure to identical objects significantly enhanced recognition
memory assessed 24 hr later. B) VU0486846 doseꢀdependently reversed risperidoneꢀinduced deficits in
acquisition of contextual fear conditioning. When coꢀadministered with 3 mg/kg risperidone, 10 mg/kg
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VU0486846 restored conditioned freezing to vehicleꢀtreated control levels. Data are expressed as mean ±
S.E.M (n = 11ꢀ13). Statistical analysis was conducted using a oneꢀway analysis of variance. If significant
(p < 0.05), comparison of group effects relative to the vehicle group was completed using a Dunnett’s
test, * p < 0.05, ** p < 0.01, *** p < 0.001.
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CONCLUSIONS
Here we report the discovery of a novel M1 PAM with enhanced potency, physiochemical properties, and
DMPK profile to assess M1 function in CNS disorders. VU0486846 is a highly selective, potent M1
PAM, devoid of agonist activity in the PFC, as well as cholinergic or other adverse effects in mice, rats
and NHP, which results in robust proꢀcognitive activity in rodent models. These data afford the
community a new in vivo tool compound to study M1 PAM activity in mouse and rat models, as well as
supporting the pursuit of M1 PAMs as either a monotherapy to enhance cognitive function or has an
adjunct therapy with current standard of care therapies for individuals suffering from cognitive deficits,
such as schizophrenia patients. Further studies with VU0486846 (16) are underway and will be reported
in due course.
METHODS
Chemical Synthesis and Purification.
All 1H & 13C NMR spectra were recorded on Bruker AVꢀ400 (400 MHz) or Bruker AVꢀNMR (600 MHz)
instrument. Chemical shifts are reported in ppm relative to residual solvent peaks as an internal standard
set to δH 7.26 or δC 77.0 (CDCl3) and δH 3.31 or δC 49.0 (CD3OD). Data are reported as follows:
chemical shift, multiplicity (s =singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet),
integration, coupling constant (Hz). IR spectra were recorded as thin films and are reported in
wavenumbers (cmꢀ1). Low resolution mass spectra were obtained on an Agilent 1200 LCMS with
electrospray ionization. High resolution mass spectra were recorded on a Waters QtofꢀAPIꢀUS plus
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Acquity system. The value ꢀ is the error in the measurement (in ppm) given by the equation ꢀ = [(ME –
MT)/ MT] × 106, where ME is the experimental mass and MT is the theoretical mass. The HRMS results
were obtained with ES as the ion source and leucine enkephalin as the reference. Optical rotations were
measured on a Perkin Elmerꢀ341 polarimeter. Analytical thin layer chromatography was performed on
250 ꢁM silica gel 60 F254 plates. Visualization was accomplished with UV light, and/or the use of
ninhydrin, anisaldehyde and ceric ammonium molybdate solutions followed by charring on a hotꢀplate.
Chromatography on silica gel was performed using Silica Gel 60 (230ꢀ400 mesh) from Sorbent
Technologies. Analytical HPLC was performed on an Agilent 1200 analytical LCMS with UV detection
at 214 nm and 254 nm along with ELSD detection. Solvents for extraction, washing and chromatography
were HPLC grade. All reagents were purchased from Aldrich Chemical Co. and were used without
purification. All polymerꢀsupported reagents were purchased from Biotage, Inc. Flameꢀdried (under
vacuum) glassware was used for all reactions. All reagents and solvents were commercial grade and
purified prior to use when necessary. Highꢀresolution mass spectrometry (HRMS) data were obtained
using a Micromass QꢀTof APIꢀUS mass spectrometer.
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Ethyl 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylate (10): A solution of 2ꢀaminophenol (15740 mg,
144.28 mmol), potassium carbonate (33717 mg, 240.46 mmol), ethyl 2,3ꢀdibromopropionate (25000 mg,
96.18 mmol) and acetonitrile (300 mL) was heated to 80 °C and was allowed to stir for 16 hours. The
reaction was diluted with water and extracted with EtOAc (3x). The layers were separated and the
organic phases were combined, washed with brine (1x), dried with MgSO4, filtered, and concentrated in
vacuo. Crude product was purified by flash chromatography (Teledyne ISCO CombiꢀFlash system, 0ꢀ
1
30% EtOAc in hexanes) to provide a brown oil. (14595mg, 70.43mmol, 73% yield). H NMR (400MHz,
CDCl3) δ 6.93 (dd, J = 7.92 Hz, 1.4 Hz, 1H), 6.79 (td, J = 7.5 Hz, 1.4 Hz, 1H), 6.72 (td, J = 7.6 Hz, 1.6
Hz, 1H), 6.61 (dd, J = 7.7 Hz, 1.6 Hz, 1H), 4.81ꢀ4.79 (m, 1H), 4.26 (m, 2H), 3.63ꢀ3.55 (m, 2H), 1.28 (t, J
= 7.2, 3H). 13C NMR (100MHz, CDCl3) δ 169.4, 142.9, 132.6, 121.6, 119.5, 116.9, 115.7, 72.7, 61.5,
42.6, 14.1 ppm. HRMS (TOF, ES+) calc’d for C11H13NO3, 207.0895; found, 207.0892.
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Ethyl 4-[(4-bromophenyl)methyl]-2,3-dihydro-1,4-benzoxazine-2-carboxylate (11). A solution of
ethyl 3,4ꢀdihydroꢀ2Hꢀbenzo[b][1,4]oxazineꢀ2ꢀcarboxylate (2165 mg, 10.45 mmol), potassium carbonate
(1831 mg, 13.06 mmol), 4ꢀbromobenzyl bromide (3263.88 mg, 13.06 mmol), and acetonitrile (18 mL)
was heated to 80 °C and allowed to stir for 16 hours. The reaction was diluted with water and extracted
with EtOAc (3x). The layers were separated and the organic phases were combined, washed with brine
(1x), dried over MgSO4, filtered, and concentrated in vacuo. Crude product was purified using flash
crhomatographys (Teledyne ISCO CombiꢀFlash system 0ꢀ40% EtOAc in hexanes) to provide an off white
solid. (2935mg, 7.80mmol, 74% yield). 1H NMR (400MHz, CDCl3) δ 7.45 (d, J = 8.4 Hz, 2H), 7.14 (d, J
= 8.3 HZ, 2H), 6.97 (dd, J = 7.9 Hz, 1.5 Hz, 1H), 6.80 (td, J = 7.7 Hz, 1.5 Hz, 1H), 6.72 (td, J = 7.6 Hz,
1.4 Hz, 1H), 6.61 (dd, J = 8.0 Hz, 1.3 Hz, 1H), 4.83 (t, J = 4.0 Hz, 1H), 4.45ꢀ4.17 (m, 4H), 3.51 (d, J =
4.1 Hz, 2H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (100MHz, CDCl3) δ 169.2, 142.9, 136.7, 134.4, 131.7,
128.7, 121.8, 121.0, 119.0, 116.6, 112.8, 72.5, 61.6, 54.4, 48.6, 14.1 ppm. HRMS (TOF, ES+) calc’d for
C18H18BrNO3, 375.0470; found, 375.0467.
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4-[(4-Bromophenyl)methyl]-2,3-dihydro-1,4-benzoxazine-2-carboxylic acid (12). A solution of ethyl
4ꢀ[(4ꢀbromophenyl)methyl]ꢀ2,3ꢀdihydroꢀ1,4ꢀbenzoxazineꢀ2ꢀcarboxylate (2935 mg, 7.8 mmol), potassium
hydroxide (437.7 mg, 7.8 mmol), THF (16 mL), and Water (8 mL) was heated to 60 °C and allowed to
stir for 3 hours. The reaction was diluted in water, acidified to pH=3, extracted with EtOAc (3x), and
concentrated in vacuo to afford the desired material, which was carried forward to the next step without
1
any further purification (2715 mg, quantitative yield). H NMR (400MHz, CDCl3) δ 7.40 (d, J = 8.3 Hz,
2H), 7.13 (d, J = 8.2 Hz, 2H), 6.96 (dd, J = 7.8 Hz, 1.4 Hz, 1H), 6.82 (td, J = 8.0 Hz, 1.4 Hz, 1H), 6.73
(td, J = 7.4 Hz, 1.4 Hz, 1H), 6.63 (dd, J = 8.0 Hz, 1.1 Hz, 1H), 4.90 (t, J = 3.8, 1H), 4.44ꢀ4.28 (m, 2H),
3.52 (d, J = 3.80 Hz, 2H). 13C NMR (100MHz, CDCl3) δ 174.4, 142.7, 136.5, 134.5, 131.9, 129.0, 122.3,
121.3, 119.4, 116.8, 113.2, 72.4, 54.6, 48.4 ppm. HRMS (TOF, ES+) calc’d for C16H14BrNO3, 347.0157;
found, 347.0150.
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4-[(4-bromophenyl)methyl]-N-[(1S,2S)-2-hydroxycyclohexyl]-2,3-dihydro-1,4-benzoxazine-2
carboxamide (13): A solution of 4ꢀ[(4ꢀbromophenyl)methyl]ꢀ2,3ꢀdihydroꢀ1,4ꢀbenzoxazineꢀ2ꢀcarboxylic
acid (2700 mg, 7.75 mmol), N,Nꢀdiisopropylethylamine (6.08 mL, 34.8 9mmol), HATU (3243.3 mg, 8.53
mmol), and DMF (18 mL) was preꢀstirred for 10 min, to which was then added (1S,2S)ꢀ2ꢀ
aminocyclohexanol (1071.69 mg, 9.31 mmol) and reaction was allowed to stir at room temperature for 12
hours. Reaction was diluted with water and extracted with EtOAc (3x). The layers were separated and the
combined organics were washed with brine (1x), dried over MgSO4, filtered, and concentrated in vacuo.
Crude product was purified using flash chromatography (Teledyne ISCO CombiꢀFlash system, 0ꢀ5%
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MeOH in DCM) to provide an off white solid (2785 mg, 6.2535 mmol, 80% yield, 8:1 d.r.). H NMR
(400MHz, CDCl3) δ 7.44 (d, J = 8.3 Hz, 2H), 7.16 (d, J = 8.3 Hz, 2H), 6.92 (dd, J = 7.9 Hz, 1.3 Hz, 1H),
6.83 (td, J = 7.7 Hz, 1.3 Hz, 1H), 6.70 (td, J = 7.7 Hz, 1.2 Hz, 1H), 6.63 (dd, J = 8.1 Hz, 1.1 Hz, 1H), 6.50
(d, J = 7.4 Hz, 1H), 4.72 (dd, J = 6.7 Hz, 2.9 Hz, 1H), 4.43ꢀ4.42 (m, 2H), 3.72ꢀ3.63 (m, 1H), 3.59 (dd, J =
11.9 Hz, 3.0 Hz, 1H), 3.51ꢀ3.46 (m, 1H), 3.28ꢀ3.23 (m, 1H), 2.06ꢀ1.97 (m, 2H), 1.77ꢀ1.69 (m, 2H), 1.38ꢀ
1.17 (m, 5H). 13C NMR (100MHz, CDCl3) δ 170.2, 141.9, 136.5, 134.8, 131.8, 128.8, 122.6, 121.0,
118.6, 116.3, 113.2, 74.9, 73.8, 55.5, 54.7, 49.0, 34.1, 31.2, 24.4, 23.8 ppm. HRMS (TOF, ES+) calc’d for
C22H25BrN2O3, 444.1049; found, 444.1048.
(2R)-N-[(1S,2S)-2-hydroxycyclohexyl]-4-[(4-pyrazol-1-ylphenyl)methyl]-2,3-dihydro-1,4-
benzoxazine-2-carboxamide (VU0486846, 16). A degassed solution of 4ꢀ[(4ꢀbromophenyl)methyl]ꢀNꢀ
[(1S,2S)ꢀ2ꢀhydroxycyclohexyl]ꢀ2,3ꢀdihydroꢀ1,4ꢀbenzoxazineꢀ2ꢀcarboxamide (900 mg, 2.02 mmol),
pyrazole (192.61 mg, 2.83 mmol), potassium phosphate (943.74 mg, 4.45 mmol), copper (I) iodide
(115.46 mg, 0.61 mmol), transꢀN,N'ꢀdimethylcyclohexaneꢀ2ꢀdiamine (0.09 mL, 0.61mmol) and 1,4ꢀ
Dioxane (6.7 mL) was heated to 100 °C and was allowed to stir for 24 hours. The reaction was diluted in
water and extracted with EtOAc (3x). The layers were separated and the organic phases were combined,
washed with saturated NH4Cl (1x) and saturated brine (1x), dried over MgSO4, filtered, and concentrated
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in vacuo. Crude product was purified using flash chromatography (Teledyne ISCO CombiꢀFlash system,
0ꢀ60% EtOAc in hexanes) to provide a white solid (219 mg, 0.506 mmol, 25% yield). 1H NMR (400MHz,
CDCl3) δ 7.89 (d, J = 2.3 Hz, 1H), 7.70 (d, J = 1.2 Hz, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 8.4 Hz,
2H), 6.91 (dd, J = 8.2 Hz, 1.0 Hz, 1H), 6.84 (td, J = 8.2 Hz, 1.2 Hz, 1H), 6.73ꢀ6.68 (m, 2H), 6.52 (d, J =
7.5 Hz, 1H), 6.44 (t, J = 2.0 Hz, 1H), 4.73 (dd, J = 6.7 Hz, 2.9 Hz, 1H), 4.51ꢀ4.40 (m, 2H), 3.73ꢀ3.61 (m,
2H), 3.53ꢀ3.39 (m, 1H), 3.30ꢀ3.24 (m, 1H), 2.01 (d, J = 10.9 Hz, 2H), 1.76ꢀ1.68 (m, 2H), 1.38ꢀ1.20 (m,
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5H). C NMR (100MHz, CDCl3) δ 170.3, 142.0, 140.9, 139.4, 135.7, 134.9, 128.1, 126.7, 122.6, 119.5,
118.5, 116.2, 113.3, 107.5, 74.8, 73.8, 55.5, 54.8, 49.0, 34.1, 31.2, 24.4, 23.8 ppm. HRMS (TOF, ES+)
calc’d for C25H28N4O3, 432.2161; found, 432.2161; [α]D21 +58.571 (c = 0.91, MeOH).
Cell lines.
Chinese Hamster Ovary (CHO) cells stably expressing muscarinic receptor isoforms were maintained in
Ham’s Fꢀ12 growth medium containing 10% FBS, 20 mM HEPES, antibiotic/antimycotic, 500 µg/mL
G418 in the presence of 5% CO2 at 37 ºC. For Giꢀcoupled M2 and M4 receptors, chimeric Gqi5 was stably
coꢀexpressed to elicit Ca response. To determine the functional activity at dog M1, the dog M1 fullꢀlength
open reading frame (ORF) was amplified from the dog hippocampus cDNAs (Zyagen, San Diego, CA) .
The ORF was then subcloned into the EcoR I and Xho I sites of pcDNA3.1 (+) vector (Life Technologies,
Carlsbad, CA). Sequencing of the plasmid confirmed the presence of dog M1 ORF (XM_540897). CHO
cells were transfected with dog M1 expression plasmid using Fugene 6 (Promega, Madison, WI), the
transfected cells were incubated with the selection medium containing 1 mg/ml G418 for 2 weeks, and the
resulting polyclones were used for the calcium mobilization assay described below.
Calcium mobilization assay.
To determine the potency and efficacy of M1 agoꢀPAMs, calcium flux was measured using the Functional
Drug Screening System (FDSS7000, Hamamatsu, Japan) as previously described (Rook et al 2016).
Briefly, All muscarinic receptorꢀCHO cells including multi species M1 and M2ꢀM5 cells were plated in
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blackꢀwalled, clearꢀbottomed 384 well plates (Greiner BioꢀOne, Monroe, NC) at 20,000 cells/well in 20
ꢁL of growth medium without G418 the day before assay. The following day, cells were washed with
assay buffer (Hank’s balanced salt solution, 20 mM HEPES, and 2.5 mM probenecid) and immediately
incubated with 20 µL of 1.15 µM Fluoꢀ4ꢀacetomethoxyester (Fluoꢀ4 AM) dye solution prepared in assay
buffer for 45 min at 37 °C. During the incubation time, all compounds were serial diluted (1:3) in DMSO
for 10 point concentrationꢀresponse curves (CRC), and further diluted in assay buffer at starting final
concentration 30 ꢀM using Echo liquid handler (Labcyte, Sunnyvale CA). Dye was removed and
replaced with assay buffer. Immediately, calcium flux was measured using the FDSS7000. The CRC of
compounds or vehicle was added to cells for 2.5 min and then an EC20 concentration of acetylcholine
(ACh) was added and incubated for 1 min. ECmax concentration was also added to cells that were
incubated with DMSO vehicle to calculate the EC20 calcium response. Using a four point logistical
equation in GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA), the concentration response
curves were generated for determination of the potency and efficacy of the agonist and PAM.
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Radioligand binding assay
Competition binding assays were performed using [3H]ꢀNꢀmethylscopolamine ([3Hꢀ]NMS, Perkin Elmer.
Boston, MA) as previously described.48 Briefly, compounds were serial diluted 1:3 in DMSO for an 11
point CRC, then further diluted for a final top concentration of 30 ꢀM in binding buffer (20 mM HEPES,
10 mM MgCl2, and 100 mM NaCl, pH 7.4). Membranes from rat M1ꢀCHO cells (10 µg) were incubated
with the serial diluted compounds in the presence of a Kd concentration of [3Hꢀ]NMS, 0.088 nM, at room
temperature for 1 hr with constant shaking. Nonspecific binding was determined in the presence of 10 µM
atropine. Binding was terminated by rapid filtration through GF/B Unifilter plates (PerkinElmer) using a
Brandel 96ꢀwell plate Harvester (Brandel Inc., Gaithersburg, MD), followed by three washes with iceꢀ
cold harvesting buffer (25 mM TrisꢀHCl, pH 7.4, 150 mM NaCl). Plates were airꢀdried overnight, 50 ꢀl
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of Microscint20 added to the plate, and radioactivity was counted using a TopCount Scintillation Counter
(PerkinElmer Life and Analytical Sciences).
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Drug Metabolism Methods
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In vitro: Protein binding of M1 PAMs were determined in plasma via equilibrium dialysis employing
SingleꢀUse RED Plates with inserts (ThermoFisher Scientific, Rochester, NY). Briefly plasma (220 µL)
was added to the 96 well plate containing test article (5 µL) and mixed thoroughly. Subsequently, 200 µL
of the plasmaꢀtest article mixture was transferred to the cis chamber (red) of the RED plate, with an
accompanying 350 µL of phosphate buffer (25 mM, pH 7.4) in the trans chamber. The RED plate was
sealed and incubated 4 h at 37 °C with shaking. At completion, 50 µL aliquots from each chamber were
diluted 1:1 (50 µL) with either plasma (cis) or buffer (trans) and transferred to a new 96 well plate, at
which time iceꢀcold acetonitrile (2 volumes) was added to extract the matrices. The plate was centrifuged
(3000 rpm, 10 min) and supernatants transferred to a new 96 well plate. The sealed plate was stored at ꢀ
20°C until LC/MS/MS analysis.
A cocktail of substrates for cytochrome P450 enzymes (1A2: Phenacetin, 10 µM; 2C9: Diclofenac, 5
µM; 2D6: Dextromethorphan, 5 µM; 3A4: Midazolam, 2 µM) were mixed to assess the ability of
compounds to inhibit the major cytochrome P450 enzymes. A reaction mixture of 100 mM Kpi, pH 7.4,
0.1 mg/mL human liver microsomes (HLM) and Substrate Mix was prepared and aliquoted into a 96ꢀ
deepwell block. Test compound and positive control (in duplicate) were then added such that the final
concentration of test compound ranged from 0.1 – 30 µM. The plate was vortexed briefly and then preꢀ
incubated at 37 ºC while shaking for 15 minutes. The reaction was initiated with the addition of NADPH
(1 mM final concentration). The incubation continued for 8 min and the reaction quenched by 2x volume
of cold acetonitrile containing internal standard (50 nM carbamazepine). The plate was centrifuged for 10
minutes (4000 rcf, 4 ºC) and the resulting supernatant diluted 1:1 with water for LC/MS/MS analysis. A
12 point standard curve of substrate metabolites over the range of 0.98 nM to 2000 nM. The IC50 values
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for each compound were obtained for the individual CYP enzymes by quantitating the inhibition of
metabolite formation for each probe substrate. A 0 µM compound condition (or control) was set to 100%
enzymatic activity and the effect of increasing test compound concentrations on enzymatic activity could
then be calculated from the % of control activity. Curves were fitted using XLfit 5.2.2 (fourꢀparameter
logistic model, equation 201) to determine the concentration that produces halfꢀmaximal inhibition (IC50).
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The in vitro biotransformation of compounds was investigated using hepatic microsomes incubated
with or without NADPH (1 mM). Reactions were terminated by adding 1 volume of acetonitrile, and
proteins were removed by centrifugation. The supernatants were saved for HPLC/UV/MS analysis.
Samples were analyzed on a Waters Acquity UPLC system with PDA detector with a flow rate of 0.5
mL/min (A: Water/Acetonitrile 95/5 with 0.1% formic acid; B: Acetonitrile) and a Waters Xevo QTOF
mass spectrometer with positive ESI source.
The metabolic stability of M1 PAMs was investigated in multiꢀspecies hepatic microsomes (BD
Biosciences, Billerica, MA) using substrate depletion methodology (% test article remaining).
A
potassium phosphateꢀbuffered reaction mixture (0.1 M, pH 7.4) of test article (1 µM) and microsomes
(0.5 mg/mL) was preꢀincubated (5 min) at 37 °C prior to the addition of NADPH (1 mM). The
incubations, performed in 96ꢀwell plates, were continued at 37 °C under ambient oxygenation and
aliquots (80 µL) were removed at selected time intervals (0, 3, 7, 15, 25 and 45 min). Protein was
precipitated by the addition of chilled acetonitrile (160 µL), containing glyburide as an internal standard
(50 ng/mL), and centrifuged at 3000 rpm (4°C) for 10 min. Resulting supernatants were transferred to
new 96ꢀwell plates in preparation for LC/MS/MS analysis. The in vitro halfꢀlife (t1/2, min, Eq. 1),
intrinsic clearance (CLint, mL/min/kg, Eq. 2) and subsequent predicted hepatic clearance (CLhep,
mL/min/kg, Eq. 3) was determined employing the following equations:
(1) t1/2 = Ln(2) / k ; where k represents the slope from linear regression analysis (% test article
remaining)
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(2) CLint = (0.693 / t1/2) (rxn volume / mg of microsomes) (45 mg microsomes / gram of liver) (20a
a
gm of liver / kg body weight); scaleꢀup factors of 20 (human) and 45 (rat); Q = heptatic blood
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Q⋅CLint
Q +CLint
CLhep =
(3)
In vivo: All animal studies were approved by the Vanderbilt University Medical Center Institutional
Animal Care and Use Committee. The animal care and use program is fully accredited by the Association
for Assessment and Accreditation of Laboratory Animal Care, International.
Male SpragueꢀDawley rats (n=2) weighing around 300g were purchased from Harlon laboratories
(Indianapolis, IN) and implanted with catheters in the carotid artery and jugular vein. The cannulated
animals were acclimated to their surroundings for approximately one week before dosing and provided
food and water ad libitum. IV cassette PK experiments in rats were carried out according to methods
described previously (Bridges et al. Pharmacol. Res. Perspect. 2014; reference 49). Briefly, A cassette of
compounds (n = 4–5/cassette) were formulated from 10 mM solutions of compounds in DMSO. In order
to reduce the absolute volume of DMSO that was administered, the compounds were combined and
diluted with ethanol and PEG 400 to achieve a final concentration of 0.4–0.5 mg/mL for each compound
(2 mg/mL total) administered in each cassette. The final dosing solutions consisted of approximately 10%
ethanol, 40% PEG400, and 50% DMSO (v/v). Each cassette dose was administered IV via the jugular
vein to two dualꢀcannulated (carotid artery and jugular vein) adult male Sprague–Dawley rats, each
weighing between 250 and 350 g (Harlan, Indianapolis, IN) for a final dose of 0.2–0.25 mg/kg per
compound. Whole blood collections via the carotid artery were performed at 0.033, 0.117, 0.25, 0.5, 1, 2,
4, 7, and 24 hours post dose and plasma samples prepared for bioanalysis.For tissue distribution studies in
cassette format, brain dissection and blood collections via the carotid artery were performed at 0.25 hr
post dose. Blood samples were collected into chilled, EDTAꢀfortified tubes, centrifuged for 10 minutes at
3000 rpm (4 °C), and resulting plasma aliquoted into 96ꢀwell plates for LC/MS/MS analysis. The brain
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samples were rinsed in PBS, snap frozen and stored at ꢀ80 °C. Prior to LC/MS/MS analysis, brain
samples were thawed to room temperature and subjected to mechanical homogenation employing a Miniꢀ
Beadbeater™ and 1.0 mm Zirconia/Silica Beads (BioSpec Products). Discrete IV PK experiments in rats
(n=2) were carried out analogously at a dose of 1.0 mg/kg in 10% EtOH, 50% PEG 400, 40% saline,
while discrete PO PK experiments in rats (n=2) were carried out using a 3 mg/kg dose of compounds in a
fine microsuspension in 30% Captisol in H2O via oral gavage to fasted animals. Whole blood collections
via the carotid artery were performed at 0.117, 0.25, 0.5, 1, 2, 4, 7, and 24 hours post dose. Plasma
samples were prepared for bioanalysis as described above.
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Determination of the pharmacokinetic profile of VU0486846 following a single IV bolus dose of 1
mg/kg (Ethanol: Peg400: Saline, 10:60:30, v/v/v) and a single PO dose of 3 mg/kg (30% aqueous
Captisol) to male Cynomolgus monkeys (n=3) was carried out at Frontage Laboratories (Exton,
Pennsylvania). Blood was collected at standard PK time points and plasma prepared for bioanalysis as
per internal protocols at Frontage. All bioanalysis and calculation of PK parameters was carried out by
Frontage and a final report issued upon study completion.
Liquid Chromatography/Mass Spectrometry Analysis.: M1 PAMs were analyzed via electrospray
ionization (ESI) on an AB Sciex APIꢀ4000 (Foster City, CA) tripleꢀquadrupole instrument that was
coupled with Shimadzu LCꢀ10AD pumps (Columbia, MD) and a Leap Technologies CTC PAL autoꢀ
sampler (Carrboro, NC). Analytes were separated by gradient elution using a Fortis C18 2.1 x 50 mm,
3.5 µm column (Fortis Technologies Ltd, Cheshire, UK) thermostated at 40°C. HPLC mobile phase A
was 0.1% formic acid in water (pH unadjusted), mobile phase B was 0.1% formic acid in acetonitrile (pH
unadjusted). The gradient started at 30% B after a 0.2 min hold and was linearly increased to 90% B over
0.8 min; held at 90% B for 0.5 min and returned to 30% B in 0.1 min followed by a reꢀequilibration (0.9
min). The total run time was 2.5 min and the HPLC flow rate was 0.5 mL/min. The source temperature
was set at 500 °C and mass spectral analyses were performed using multiple reaction monitoring (MRM)
utilizing a TurboꢀIonspray® source in positive ionization mode (5.0 kV spray voltage). All data were
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analyzed using AB Sciex Analyst 1.4.2 software. For in vivo studies, the final PK parameters were
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calculated by noncompartmental analysis using Phoenix (version 6.2) (Pharsight Inc., Mountain View,
CA).
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Mouse Plasma-Brain exposure
M1 PAMs were dissolved in 10% Tween 80 at the concentration of 1ꢀ10 mg/mL (base form) and
administered intraperitoneally to male C57BL/6J (Jackson Laboratory, Sacramento, CA) mice aged 7ꢀ9
weeks at a volume of 10 ml/kg. The blood and brain were collected at 0.25 h. Animals were euthanized
and decapitated, and the brains were removed, thoroughly washed with cold saline and immediately
frozen on dry ice. Trunk blood was collected in EDTA coated Eppendorf tubes, and plasma was separated
by centrifugation and stored at ꢀ80°C until processed for LC/MS/MS analysis as previously described.
Three animals were used for each time point.
Electrophysiology Methods
Animals: All animal studies were approved by the Vanderbilt University Medical Center Institutional
Animal Care and Use Committee and were conducted in accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals. Male C57BL6/J mice (Jackson laboratories) were
used in electrophysiology and behavioral studies (6ꢀ10 weeks old). Animals were group housed 4−5 per
cage, maintained on a 12 h light/dark cycle, and provided food and water ad libitum.
Extracellular field electrophysiology: Briefly, 6ꢀ10 week old male C57BL6/J mice were anesthetized
using a mixture of ketamine and xylazine (100mg/kg and 10mg/kg, respectively, intraperitoneal injection)
then transcardially perfused with iceꢀcold cutting solution (in mM: 230 sucrose, 2.5 KCl, 8 MgSO4, 0.5
CaCl2, 1.25 NaH2PO4, 10 Dꢀglucose, 26 NaHCO3), and then the brains were removed and submerged in
iceꢀcold cutting solution. Coronal slices containing the prelimbic prefrontal cortex were cut at 400 µm
using Leica VT1200 vibratome and were transferred to a holding chamber containing NMDGꢀHEPES
recovery solution (in mM: 93 NMDG, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 Dꢀglucose, 5
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sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 10 MgSO4, 0.5 CaCl2, 12 NꢀacetylꢀLꢀcysteine, pH 7.3,
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<310 mOsm) for 8ꢀ10 minutes at 32 ºC. Slices were then transferred to a room temperature holding
chamber for at least 1.5 hours containing ACSF (in mM: 126 NaCl, 1.25 NaH2PO4, 2.5 KCl, 10
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glucose, 26 NaHCO3, 2 CaCl2, 1 MgSO4) supplemented with 600ꢀµM sodium ascorbate for slice
viability. All buffers were continuously bubbled with 95% O2/5% CO2. Subsequently, slices were
transferred to a 30ꢀ32 °C submersion recording chamber (Warner Instruments) where they were perfused
with ACSF at a rate of 2 mL/min. Field excitatory postsynaptic potentials (fEPSPs) were recorded from
layer V of the prelimbic cortex and evoked electrically by a concentric bipolar stimulating electrode (200
µs duration, 0.05 Hz; interꢀpulse interval of 50 ms) in the superficial layers IIꢀIII as described previously
(Ghoshal et al., 2017). VU0486846 was diluted to the appropriate concentrations in DMSO (<0.1% final)
in ACSF and applied to the bath for 20 minutes using a peristaltic pump perfusion system. Carbachol and
OxoM (Tocris Bioscience, Bristol, UK) were diluted in H2O. See supplemental methods for additional
information.
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Stereotaxic Viral Injections: Mice underwent stereotaxic injections for viralꢀmediated gene transfer of
channelrhodopsinꢀ2 (ChR2) at 4−5 weeks of age. Mice were anaesthetized with 1ꢀ2% isoflurane for the
duration of the surgery and were administered carprofen (10mg/kg, s.c.) before the surgery commenced.
Following a craniotomy, one injection per hemisphere (800 nL at a rate of 100 nL/min) of AAV5ꢀ
CaMKIIꢀChR2ꢀ EYFP (UNC Viral Vector Core) was delivered into the target regions through a 28G
needle attached to a 10ꢁL Hamilton syringe. Injection site coordinates were as follows (relative to
bregma): vHipp [ML: ± 3.4, AP: −3.4, DV: −4.0]. Carprofen (10 mg/kg, s.c.) was administered for at
least 72 hours post procedure. Recordings were made 4−5 weeks following surgery to allow sufficient
expression of ChR2 in axon terminals within the PFC.
Optical extracellular field electrophysiology: Brain slices from viral injected mice were prepared in a
similar manner described above. For studies involving optical stimulation, blue light (470 nm) was
delivered using a HighꢀPower LED (Thorlabs Inc. Newton, NJ), which was mounted to the epiꢀ
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illumination port of an Olympus BX50WI upright microscope (Olympus). Blue light was shined onto
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slices through the 40x objective lens at 0.05 Hz (Maximum light intensity at the site of illumination is 2.6
mW).
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Behavioral Pharmacology Studies
Animals: In vivo studies utilized either 7 – 8 week old male C57Bl/6 mice (Jackson Laboratory,
Sacramento, CA) or male Sprague Dawley rats weighing 275 – 300 g (Envigo, Indianapolis, IN). The
animals were cared for in accordance with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals. All experimental procedures were approved by the Vanderbilt University Animal
Care and Use Committee.
Novel Object Recognition Task: Novel object recognition memory was assessed as previously
described26. Briefly, rats were habituated to an empty novel object recognition (NOR) arena. On test day,
rats were administered vehicle (10% tween 80) or VU0486846 (1 ꢀ 10 mg/kg, intraperitoneally (i.p.), 1
ml/kg, n = 12) and returned to their home cage for 30 min. Rats were then placed in the NOR arena
containing 2 identical objects for 10ꢂmin. Following the exposure period, rats were placed back into their
home cages for 24ꢂhrs. The rats were then returned to the arena in which one of the previously exposed
(familiar) objects was replaced by a novel object and exploration behavior was assessed for 3ꢂmin. Time
spent exploring each object was scored by an observer blinded to the experimental conditions and the
recognition index was calculated as [(time spent exploring novel object) − (time spent exploring familiar
object)]/total time exploring objects.
Contextual Fear Conditioning: The effects of VU0486846 on acquisition of contextual fear
conditioning were evaluated in rats as previously described.42 Rats were given a 30 min pretreatment of
vehicle (10% tween 80), risperidone alone (3 mg/kg, i.p., 1 ml/kg) or risperidone coꢀadministered with
VU0486846 (1–10 mg/kg) and placed in a soundꢀattenuating conditioning chamber (Med Associates).
Following a 2 min habituation period, rats received three shockꢀtone pairing trials (30 s 3000 Hz 80 dB
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