Qualification of LSP1-2111 as a brain penetrant group III metabotropic glutamate receptor orthosteric agonist

Article abstract of DOI:10.1021/ml400338f

LSP1-2111 is a group III metabotropic glutamate receptor agonist with preference toward the mGlu4 receptor subtype. This compound has been extensively used as a tool to explore the pharmacology of mGlu4 receptor activation in preclinical animal behavioral

Full text of DOI:10.1021/ml400338f

Letter
pubs.acs.org/acsmedchemlett
Qualification of LSP1-2111 as a Brain Penetrant Group III
Metabotropic Glutamate Receptor Orthosteric Agonist
Manuel Cajina,† Megan Nattini,† Dekun Song,† Gennady Smagin,† Erling B. Jørgensen,‡
Gamini Chandrasena,† Christoffer Bundgaard,‡ Dorthe Bach Toft,‡ Xinyan Huang,† Francine Acher,§
and Dario Doller†,*
†Lundbeck Research USA, 215 College Road, Paramus, New Jersey 07652, United States
‡H. Lundbeck A/S, Ottiliavej 9, 2500 Copenhagen-Valby, Denmark
§Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite
Saints-Peres, 75270 Paris Cedex 06, France
́
Paris Descartes, 45 rue des
̀
S
* Supporting Information
ABSTRACT: LSP1-2111 is a group III metabotropic
glutamate receptor agonist with preference toward the
mGlu4 receptor subtype. This compound has been extensively
used as a tool to explore the pharmacology of mGlu4 receptor
activation in preclinical animal behavioral models. However,
the blood−brain barrier penetration of this amino acid
derivative has never been studied. We report studies on the
central nervous system (CNS) disposition of LSP1-2111 using
quantitative microdialysis in rat. Significant unbound concen-
trations of the drug relative to its in vitro binding affinity and
functional potency were established in extracellular fluid
(ECF). These findings support the use of LSP1-2111 to
study the CNS pharmacology of mGlu4 receptor activation through orthosteric agonist mechanisms.
KEYWORDS: Metabotropic glutamate receptors, G protein-coupled receptors, mGlu4 receptor, orthosteric agonist
he investigation of potential therapeutic indications of
(4, also obtained as a 1:1 mixture of diastereoisomers). This
Tmetabotropic glutamate 4 (mGlu4) receptor activation is shows an improved selectivity profile of 40-, 100-, and 300-fold
versus the mGlu6, 7, and 8 receptors, respectively.6
an active area of drug discovery research.1 The selective
activation of the mGlu4 receptor can be achieved using two
different molecular mechanisms: orthosteric agonists (compet-
ing with L-glutamate, 1; see Chart 1) or noncompetitive
positive allosteric modulators (PAMs).2
The interest of a number of drug discovery organizations,
including our group, has focused in the PAM mechanism.1,2,7
However, recent reports prompted us to explore the orthosteric
agonist mechanism. Specifically, preclinical pharmacology
studies with LSP1-2111 reported efficacy using in vivo rodent
models of Parkinson’s disease,8 anxiety,9 and psychosis.10,11
LSP1-2111 was also efficacious in acquisition of fear learning
and memory models.12 These results in multiple animal models
are both compelling and provocative. To our best knowledge,
there are no reports of the concentrations of LSP1-2111 in
tissues relevant to central pharmacological actions (plasma,
brain, cerebrospinal fluid (CSF), or brain extracellular fluid
(ECF)) in any species, in particular rat. This led to an
investigation of central drug disposition and pharmacokinetics
of LSP1-2111 following systemic administration in rat, to
examine whether the compound is able to cross the blood−
brain barrier (BBB) and achieve relevant exposures at the
receptor location.13,14
A number of mGlu receptor agonists with selectivity for
Group III (mGlu4, 6, 7, and 8 receptors) versus Group I
(mGlu1 and 5 receptors) and Group II (mGlu2 and 3
receptors) have been reported. From a drug design perspective,
L-AP4 (2) may be considered as a starting lead compound
toward the discovery of mGlu4 receptor agonists with
improved subtype selectivity. LSP1-2111 [3; (2S)-2-amino-4-
(hydroxy(hydroxy(4-hydroxy-3-methoxy-5-nitrophenyl)-
methyl)phosphoryl)butanoic acid, used as a 1:1 mixture of
equipotent diastereoisomers at the benzylic position] is a
preferential agonist of the mGlu4 and mGlu6 receptors, where
the terminal phosphonic acid fragment in 2 is replaced by a
hydroxymethyl-phosphinic acid pharmacophore, with an extra
substituted aryl group.3,4 While highly selective (>100-fold)
against Group I and II mGlu receptors, LSP1-2111 (3) has 1-,
25-, and 30-fold preference over mGlu6, mGlu7, and mGlu8
receptors, respectively.5 Recently, the discovery of a highly
selective mGlu4 agonist was reported, known as LSP4−2022
Received: September 7, 2013
Accepted: December 12, 2013
Published: December 12, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Chart 1. Chemical Structures of Selected mGlu4 Receptor
Orthosteric Agonists (1−4), Marketed CNS Drugs (5−10),
and mGlu2/3 Receptor Agonists which Reached Clinical
Trials (11−14) Containing Amino Acid Functionality
Table 1. In Vitro and in Silico Attributes of LSP1-2111 (3)
h.mGlu4 EC50 (FLIPR)
h.mGlu4 Emax (FLIPR)
h.mGlu4 IC50 ([3H]-L-AP4 binding)
molecular weight
1.5 μM
85%
8.6 μM
364 amu
LogD7.4
−0.7
cLogP
−2.6
polar surface area
196 Å2
aqueous solubility pH 7.4
pKa
>1.8 mg/mL
11.7; 6.6; 2.0
<0.1 × 10−6 cm/s
A → B = 0.2 × 10−6 cm/s
B → A = 0.5 × 10−6 cm/s
0.73
passive permeability PAPP (PAMPA)
MDCK/MDR1 permeability PAPP
human plasma unbound fraction, fu
rat or mouse plasma unbound fraction, fu
>0.99
rat or mouse brain homogenate unbound
fraction, UBBR
>0.99
a
rat microsomal CLint
3.1 mL/min (low)
0.1 L/min (low)
14% inhibition at 30 μM
score 1 (nontoxic)
score 1 (nontoxic)
nontoxic
b
human microsomal CLint
c
human hERG Ionworks
d
cytotoxicity assay
d
reactive oxygen species assay
d
mitochondrial membrane potential assay
e
broad selectivity screen
no cross-reactivity observed
at 10 μM
a
b
Rat hepatic blood flow Qr = 20 mL/min. Human hepatic blood flow
c
d
e
Qh = 1.5 L/min. Conducted at Millipore. See ref 21. Binding and
functional activities.20,21
Strategies to establish BBB permeability for typical (e.g.,
lipophilic) CNS drugs have changed over the past decade. Prior
focus on total brain-to-plasma ratios has shifted to using
parameters such as unbound drug concentration or unbound
brain-to-plasma ratios during the lead optimization of CNS
drugs.15 The compounds are often of lipophilic nature, show
high levels of nonspecific binding to matrix components, and
cross membranes through transcellular mechanisms. In general,
an aim of the CNS drug design process is to avoid significant
interactions with efflux transporters. However, the physico-
chemical characteristics of amino acids such as LSP1-2111
(Table 1) are markedly different from those for a typical CNS
drug, which impacts their systemic disposition attributes. These
compounds show low cell membrane passive permeability
through the transcellular mechanism, which may lead to
unbound brain-to-plasma ratios deviating from unity, as the free
drug hypothesis would predict.16 They remain largely in the
vascular compartment upon peripheral dosing, and in the brain
they are mainly distributed in the extracellular space. They are
mostly distributed as unbound in plasma and brain
parenchyma. Therefore, we felt a different strategy was required
to establish BBB penetration than that typically used for
lipophilic drugs.
It is worth noting that a number of amino acid derivatives
believed to act directly at brain receptors are used in the clinic
for a number of CNS indications; for example, levodopa (5),
baclofen (6), gabapentin (7), pregabalin (8), vigabatrin (9),
and α-methyltyrosine (10). In addition, related amino acid
mGlu2/3 agonists LY354740 (11) and LY404039 (12), as their
corresponding prodrugs LY544344 (13) and LY2140023 (14),
have reached the clinical stage. Our plan was to establish the
concentration−time profile of LSP1-2111 in brain ECF using
quantitative microdialysis techniques. This would measure the
ability of LSP1-2111 to cross the BBB and access the
orthosteric binding site on the extracellular space of the cell
membrane-bound mGlu4 receptor.1,2
Prior to performing microanalysis studies, we characterized
LSP1-2111 in terms of its physicochemical and in vitro ADMET
profile (Table 1). Consistent with its hydrophilic nature, the
experimental LogD7.4 (−0.7) and the in silico cLogP (−2.6) are
low.17 The polar surface area (196 Å2) is well above the typical
values considered optimum for lipophilic CNS drugs.18 The
kinetic aqueous solubility measured in an assay developed and
optimized for evaluating lipophilic compounds exceeded the
detection limit (>800 μM). The thermodynamic solubility was
high as well (>1.8 mg/mL). Consistent with its highly polar
nature, the transcellular permeability was determined to be low
by two different measures: a PAMPA assay and the MDCK/
MDR1 assay (PAPP < 0.5 × 10−6 cm/s in both cases). The PAPP
ratio B → A/A → B = 2.5 might suggest LSP1-2111 is a
moderate P-glycoprotein (P-gp) substrate; albeit, these
permeability values are very low and within experimental
error. Human, rat, and mouse plasma unbound fractions are
high (fu = 0.73, >0.99, and >0.99, respectively). Rat and mouse
brain homogenate unbound fraction (>0.99 in both cases)
indicate this compound has very low nonspecific binding to
brain tissue. Both rat and human microsomal intrinsic clearance
(CLint) are low, suggesting LSP1-2111 is poorly metabolized by
hepatic cytochrome P450 enzymes. Standard in vitro toxicology
assays indicated a rather safe profile, including low potential for
hERG channel inhibition.19 Lastly, a broad binding screen
against 70 GPCRs, ion channels, and enzymes as well as a
functional screen toward 56 GPCRs demonstrated a highly
selective cross-reactivity profile for LSP1-2111.20,21
We then determined the distribution characteristics of LSP1-
2111 within CNS, using in vivo microdialysis to generate a time
course profile of extracellular space fluid concentrations using a
MetaQuant dialysis probe (Figure 1). The method follows the
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ACS Medicinal Chemistry Letters
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Figure 2. Plasma, brain, and CSF concentration time profile of LSP1-
2111 upon subcutaneous (SC) administration at 10 mg/kg (N = 4).
Only standard error bars larger than marker size are shown.
were 30 μg × h/mL, 0.7 μg × h/g, and 3.5 μg × h/mL,
respectively.
The average CSF concentrations for LSP1-2111 also
Figure 1. Rat plasma and ECF dialysate concentration time profile of
LSP1-2111 following subcutaneous (SC) administration at 10 and 30
mg/kg. The ECF concentration values have been corrected for
recovery loss based on the 96% in vivo probe recovery under the same
flow rate.23
declined from 1.7 0.5 μg/mL (4.6 μM) to 0.3
0.1 μg/
mL (0.7 μM) over the 6 h period following SC dose
administration. Since protein content in CSF is well below
that of plasma, these concentrations essentially represent the
free drug. Thus, the maximum CSF concentration of LSP1-
2111 (4.6 μM) is ca. 2-fold below its binding affinity, ca. 2-fold
above its functional EC50 (Table 1), and ca. 2.5-fold the
corresponding ECF concentration (1.8 μM) at 1 h for the 10
mg/kg dose. The CSF AUC0−6 is approximately 12% of the
principle that microdialysis extraction becomes quantitative
(i.e., concentration recovery is close to 100%) when the dialysis
flow rate is decreased from the usual rates of 1.5−2 μL/min
down to 0.1 μL/min. In this study, in vitro recovery of the
probes was 96% under similar low flow rate conditions.22
In the behavioral studies reported in the literature, LSP1-
2111 was dosed via intraperitoneal (IP) administration.8−12 In
our hands, preliminary work comparing brain and plasma
exposures in rat upon IP or subcutaneous (SC) dosing
demonstrated very similar average concentration values of
LSP1-2111, but with a significantly higher inter-animal
variability in the IP route (Supporting Information, Figure
S1). Thus, we chose the SC route of administration for all our
in vivo brain penetration studies with LSP1-2111.
Plasma concentrations in the microdialysis samples were
dose-proportional and consistent with those found in the
preliminary exposure studies. They reached maximum values of
11 1 μg/mL (31 μM) and 41 7 μg/mL (114 μM) at 10
and 30 mg/kg, respectively, 60 min after subcutaneous dose.
ECF concentrations were dose-proportional, reaching values of
up to 0.7 0.4 μg/mL (1.8 μM) and 3.5 μg/mL (9.8 μM) at
10 and 30 mg/kg, respectively. These concentrations are
commensurate with the in vitro binding affinity and functional
potency of LSP1-2111 (Table 1 and those reported in the
literature).3,7
Further insight into the CNS disposition of LSP1-2111 was
gained upon studying its distribution into CSF and brain tissue
after systemic administration. Tissue concentrations were
measured in rats after dosing LSP1-2111 (10 mg/kg, SC)
formulated in saline at pH 7.4. Results from a 6-h time course
study are shown in Figure 2.
Plasma concentrations decreased with a t1/2 of 20 min. The
average plasma concentrations of LSP1-2111 during the 0.5−6
h time interval decreased from 20.1 3.7 μg/mL to 0.15
0.05 μg/mL, with a maximum unbound plasma concentration
of 55.2 μM at 0.5 h. Plasma, brain, and CSF AUC0−6 values
corresponding plasma AUC0−6
.
The average brain homogenate concentrations of LSP1-2111
range between 0.36 0.07 μg/g (1 μM) at 30 min and 0.03
0.01 μg/g (0.08 μM) at 4 h. This essentially follows the rate of
change in plasma concentrations over the same period. The 6 h
drug concentration is 0.14 0.05 μg/g (0.4 μM). Based on
AUC0−6, the brain-to-plasma ratio for LSP1-2111 is 2.4%. This
low value is in agreement with previous reports for related
compounds such as 11 and 13.24 As a general practice in our
experimental protocols, brain tissue was not perfused to
eliminate capillary blood prior to homogenization in the
measurement of LSP1-2111 concentration. Thus, the concen-
trations measured in the brain tissue are mostly arising from
LSP1-2111 in blood contained in brain capillaries, outside of
the CNS structure (1.9% wet brain weight).25 This precludes a
clear determination of BBB penetration based on brain
concentrations of LSP1-2111, especially when taken into
consideration experimental variability.
It is noteworthy that despite their low passive permeability
characteristics, sporadic reports have disclosed that similar
amino acid compounds are orally absorbed in preclinical
species, presumably facilitated by active transport processes.26
Thus, we decided to explore whether intestinal absorption of
LSP1-2111 could be facilitated by putative transporters. Results
from an in vivo rat pharmacokinetic study in Sprague−Dawley
rats are shown in Figure 3, and key parameters are listed in
Table 2. Plasma clearance is low (6.2 mL/min × kg) compared
with the characteristic hepatic blood flow of 75 mL/min × kg.
The volume of distribution at steady state is very low (Vss = 0.1
L/kg), indicating the majority of the compound dosed remains
in the vascular compartment. Unfortunately, oral bioavailability
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ACS Medicinal Chemistry Letters
Letter
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental protocols and characterization data for the
preparation of LSP1-2111, time course for brain and plasma
exposures of LSP1-2111 using IP dosing, experimental
parameters for the bioanalysis of LSP1-2111 in plasma, brain,
ECF, and CSF, and efficacious doses of LSP1-2111 in all
reported preclinical models. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (201)-350-0341. E-mail: dado@lundbeck.com.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Figure 3. Plasma concentration time profile of LSP1-2111 after oral
administration in SD rats (N = 2). Only standard error bars larger than
marker size are shown.
Notes
The authors declare no competing financial interest.
Table 2. Summary of Rat Pharmacokinetic Parameters for
LSP1-2111 Generated Using Noncompartmental Analysis in
WinNonlin 5.2
ACKNOWLEDGMENTS
■
a
We are grateful to Dr. Stevin Zorn and Dr. Klaus Bæk
Simonsen for support of this work. We thank the reviewers of
this manuscript for their valuable criticism.
b
b
parameter
units
mg/kg
intravenous (IV)
oral (PO)
10
dose
t1/2
1
h
0.3
ABBREVIATIONS
Cmax
tmax
Clp
ng/mL
92
■
h
1.4
ADMET, absorption-distribution-metabolism-elimination-tox-
icity; PAM, positive allosteric modulator; CSF, cerebrospinal
fluid; ECF, extracellular fluid; CNS, central nervous system; SC,
subcutaneous; IP, intraperitoneal; P450, cytochrome P450;
GPCR, G protein-coupled receptor; BBB, blood−brain barrier;
SD, standard deviation
mL/min × kg
L/kg
6.2
0.1
2.8
Vss
AUCinf
F
μg × h/mL
%
0.2
0.8
a
LSP1-2111 (3) was dosed as a solution in saline buffered to pH 7.4,
at a dose volume of 5 mL/kg. Mean values (N = 2).
b
REFERENCES
■
(1) Cel
́
anire, S.; Campo, B. Recent advances in the drug discovery of
metabotropic glutamate receptor 4 (mGluR4) activators for the
treatment of CNS and non-CNS disorders. Expert Opin. Drug
Discovery 2012, 7, 261−280.
was poor (0.8%), and Cmax was 92 ng/mL (253 nM unbound
drug concentration), showing that very low systemic exposure
was obtained following the oral route of administration. Plasma
t1/2 was short (0.3 h), as a reflection of a very low Vss.
In summary, we report the use of quantitative microdialysis
for the determination of brain ECF concentrations for LSP1-
2111, an amino acid tool compound extensively used to study
behavioral pharmacology of mGlu4 receptor activation. The
ECF concentrations achieved at the doses tested, which partly
overlap with those showing efficacy in rat models, are
commensurate with the compound’s mGlu4 receptor binding
affinity and functional EC50 values. Due to the particular
physicochemical and ADMET attributes of highly polar amino
acids, the total brain-to-plasma ratio does not necessarily
provide an appropriate measure of brain penetration. The oral
bioavailability of LSP1-2111 in rat is low, suggesting the
absence of intestinal transporters to facilitate its absorption.
LSP1-2111 is a useful tool compound to explore the potential
of mGlu4 agonists as therapies for CNS and peripheral
disorders. Thus, selective orthosteric agonists should provide
valuable options to complement the ongoing efforts in
potentiating the mGlu4 receptor via positive allosteric
modulation.
(2) Lindsley, C. W.; Hopkins, C. R. Metabotropic glutamate receptor
4 (mGlu4)-positive allosteric modulators for the treatment of
Parkinson’s disease: historical perspective and review of the patent
literature. Expert Opin Ther. Pat. 2012, 22, 461−481.
́
(3) Beurrier, C.; Lopez, S.; Revy, D.; Selvam, C.; Goudet, C.;
Lherondel, M.; Gubellini, P.; Kerkerian-LeGoff, L.; Acher, F.; Pin, J. P.;
́
Amalric, M. Electrophysiological and behavioral evidence that
modulation of metabotropic glutamate receptor 4 with a new agonist
reverses experimental parkinsonism. FASEB J. 2009, 23, 3619−3628.
(4) Acher F.; Selvam C.; Pin J.-P. Diastereoisomers of Hypophos-
phorous acid derivatives. World patent application WO 2010/106526.
March 19, 2010.
(5) Flor, P. J.; Acher, F. C. Orthosteric versus allosteric GPCR
activation: the great challenge of group-III mGluRs. Biochem.
Pharmacol. 2012, 84, 414−424.
(6) Goudet, A. C.; Vilar, B.; Courtiol, T.; Deltheil, T.; Bessiron, T.;
Brabet, I.; Oueslati, N.; Rigault, D.; Bertrand, H.-O.; McLean, H.;
Daniel, H.; Amalric, M.; Acher, F.; Pin, J.-P. A novel selective
metabotropic glutamate receptor 4 agonist reveals new possibilities for
developing subtype selective ligands with therapeutic potential. FASEB
J. 2012, 26, 1682−1693.
(7) Bennouar, K. E.; Uberti, M. A.; Melon, C.; Bacolod, M. D.;
Jimenez, H. N.; Cajina, M.; Kerkerian-Le Goff, L.; Doller, D.;
Gubellini, P. Synergy between L-DOPA and a novel positive allosteric
modulator of metabotropic glutamate receptor 4: implications for
122
dx.doi.org/10.1021/ml400338f | ACS Med. Chem. Lett. 2014, 5, 119−123

ACS Medicinal Chemistry Letters
Letter
Parkinson’s disease treatment and dyskinesia. Neuropharmacology
2013, 66, 158−169.
Identification of potent, selective, and orally bioavailable agonists for
mGlu2/3 receptors. J. Med. Chem. 2007, 50, 233−40.
(8) Lopez, S.; Bonito-Oliva, A.; Pallottino, S.; Acher, F.; Fisone, G.
Activation of metabotropic glutamate 4 receptors decreases L-DOPA-
induced dyskinesia in a mouse model of Parkinson’s disease. J.
Parkinson’s Dis. 2011, 1, 339−346.
(9) Wieron
́
ska, J. M.; Stachowicz, K.; Pałucha-Poniewiera, A.; Acher,
F.; Branski, P.; Pilc, A. Metabotropic glutamate receptor 4 novel
́
agonist LSP1-2111 with anxiolytic, but not antidepressant-like activity,
mediated by serotonergic and GABAergic systems. Neuropharmacology
2010, 59, 627−634.
́
(10) Wieronska, J. M.; Stachowicz, K.; Acher, F.; Lech, T.; Pilc, A.
Opposing efficacy of group III mGlu receptor activators, LSP1-2111
and AMN082, in animal models of positive symptoms of
schizophrenia. Psychopharmacology 2012, 220, 481−494.
́ ́ ́
(11) Wieronska, J. M.; Acher, F. C.; Sławinska, A.; Gruca, P.; Łason-
Tyburkiewicz, M.; Papp, M.; Pilc, A. The antipsychotic-like effects of
the mGlu group III orthosteric agonist, LSP1-2111, involves 5-HT1A
signalling. Psychopharmacology 2013, 227, 711−725.
(12) Davis, M. J.; Iancu, O. D.; Acher, F. C.; Stewart, B. M.; Eiwaz M,
A.; Duvoisin, R. M.; Raber, J. Role of mGluR4 in acquisition of fear
learning and memory. Neuropharmacology 2013, 66, 365−372.
(13) Green, A. R.; Gabrielsson, J.; Fone, K. C. F. Translational
neuropharmacology and the appropriate and effective use of animal
models. Br. J. Pharmacol. 2011, 164, 1041−1043.
(14) Gabrielsson, J.; Green, A. R.; Van der Graaf, P. H. Optimising in
vivo pharmacology studiespractical PKPD considerations. J.
Pharmacol. Toxicol. Methods 2010, 61, 146−156.
(15) Di, L.; Rong, H.; Feng, B. Demystifying brain penetration in
central nervous system drug discovery. J. Med. Chem. 2013, 56, 2−12.
(16) Smith, D.; Di, L.; Kerns, E. H. The effect of plasma protein
binding on in vivo efficacy: misconceptions in drug discovery. Nat. Rev.
Drug Discovery 2010, 9, 929−939.
(17) In silico parameters calculated using MOE (Molecular Operating
system, 2012).
(18) Wager, T. T.; Chandrasekaran, R. Y.; Hou, X.; Troutman, M.
D.; Verhoest, P. R.; Villalobos, A.; Will, Y. Defining desirable central
nervous system drug space through the alignment of molecular
properties, in vitro ADME, and safety attributes. ACS Chem. Neurosci.
2010, 1, 420−434.
(19) Persson, M.; Løye, A. F.; Mow, T.; Hornberg, J. J. A high
content screening assay to predict human drug-induced liver injury
during drug discovery. J. Pharmacol. Toxicol. Methods 2013, 68, 302−
313.
(20) Broad binding counterscreen conducted at Cerep (www.cerep.
com).
(21) Broad functional counterscreens conducted at Euroscreen
(www.euroscreen.com).
(22) Cremers, T. I.; de Vries, M. G.; Huinink, K. D.; van Loon, J. P.;
v d Hart, M.; Ebert, B.; Westerink, B. H.; De Lange, E. C. Quantitative
microdialysis using modified ultraslow microdialysis: direct rapid and
reliable determination of free brain concentrations with the
MetaQuant technique. J. Neurosci. Methods 2009, 178, 249−254.
(23) For experimental conditions and bioanalytical methods for
LSP1-2111 see Supporting Information.
(24) Rorick-Kehn, L. M.; Perkins, E. J.; Knitowski, K. M.; Hart, J. C.;
Johnson, B. G.; Schoepp, D. D.; McKinzie, D. L. Improved
bioavailability of the mGlu2/3 receptor agonist LY354740 using a
prodrug strategy: in vivo pharmacology of LY544344. J. Pharmacol.
Exp Ther. 2006, 316, 905−13.
(25) Triplett, J. W.; Hayden, T. L.; McWhorter, L. K.; Gautam, S. R.;
Kim, E.; Bourne, E. D. W. A. Determination of gallium concentration
in “blood-free” tissues using a radiolabeled blood marker. J. Pharm. Sci.
1985, 74, 1007−1009.
(26) Monn, J. A.; Massey, S. M.; Valli, M. J.; Henry, S. S.;
́
Stephenson, G. A.; Bures, M.; Herin, M.; Catlow, J.; Giera, D.; Wright,
R. A.; Johnson, B. G.; Andis, S. L.; Kingston, A.; Schoepp, D. D.
Synthesis and metabotropic glutamate receptor activity of S-oxidized
variants of (−)-4-amino-2-thiabicyclo-[3.1.0]hexane-4,6-dicarboxylate:
123
dx.doi.org/10.1021/ml400338f | ACS Med. Chem. Lett. 2014, 5, 119−123