Research Article

Discovery of a Highly Selective Sigma-2 Receptor Ligand,

1-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2

(1H)-yl)butyl)-3-methyl-1H-benzo[d]imidazol-2(3H)-one (CM398),

with Drug-Like Properties and Antinociceptive Effects In Vivo

Sebastiano Intagliata,

Abhisheak Sharma,

Tamara I. King,

Christophe Mesangeau,

Michael Seminerio,

Frederick T. Chin,

Lisa L. Wilson,

Rae R. Matsumoto,

4,7

Jay P. McLaughlin,

Bonnie A. Avery,

and Christopher R. McCurdy

1,3,8

Received 1 May 2020; accepted 16 June 2020

Abstract.

The sigma-2 receptor has been cloned and identiﬁed as Tmem97, which is a

transmembrane protein involved in intracellular Ca

regulation and cholesterol homeostasis.

Since its discovery, the sigma-2 receptor has been an extremely controversial target, and

many efforts have been made to elucidate the functional role of this receptor during

physiological and pathological conditions. Recently, this receptor has been proposed as a

potential target to treat neuropathic pain due to the ability of sigma-2 receptor agonists to

relieve mechanical hyperalgesia in mice model of chronic pain. In the present work, we

developed a highly selective sigma-2 receptor ligand (sigma-1/sigma-2 selectivity ratio >

1000), 1-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-3-methyl-1H-

benzo[d]imidazol-2(3H)-one (CM398), with an encouraging in vitro and in vivo pharmaco-

logical proﬁle in rodents. In particular, radioligand binding studies demonstrated that CM398

had preferential afﬁnity for sigma-2 receptor compared with sigma-1 receptor and at least

four other neurotransmitter receptors sites, including the norepinephrine transporter.

Following oral administration, CM398 showed rapid absorption and peak plasma concentra-

tion (Cmax) occurred within 10 min of dosing. Moreover, the compound showed adequate,

absolute oral bioavailability of 29.0%. Finally, CM398 showed promising anti-inﬂammatory

analgesic effects in the formalin model of inﬂammatory pain in mice. The results collected in

this study provide more evidence that selective sigma-2 receptor ligands can be useful tools in

the development of novel pain therapeutics and altogether, these data suggest that CM398 is

a suitable lead candidate for further evaluation.

KEY WORDS: sigma receptors; sigma-2 receptor; neuropathic pain; formalin assay; pharmacokinetic.

INTRODUCTION

Neuropathic pain is a major clinical problem that results

in a drastic reduction to the quality of life in patients who are

affected with this chronic condition (1,2). It represents one of

the most frequent causes of adult disability and a consider-

able health-care cost which consists of medical expenses and

lost workdays. It has been estimated that approximately 20

million individuals in the USA suffer from some form of

peripheral neuropathy (3). The most common reasons of

developing peripheral neuropathy include physical injury

(trauma), chronic diseases (diabetic neuropathy), and expo-

sure to toxins (3). Despite the fact that neuropathic pain is a

common medical problem, there are only a few effective

treatment options, each with their own limitations, thus the

management of chronic pain is quite complicated and

challenging. First-line treatments include antid epressant

Department of Medicinal Chemistry, College of Pharmacy, Univer-

sity of Florida, Gainesville, Florida 32610, USA.

Department of Pharmaceutics, College of Pharmacy, University of

Florida, Gainesville, Florida 32610, USA.

Department of BioMolecular Sciences, School of Pharmacy, The

University of Mississippi, University, Mississippi 38677, USA.

Department of Basic Pharmaceutical Sciences, West Virginia

University, Morgantown, West Virginia 26506, USA.

Department of Radiology, Stanford University School of Medicine,

Stanford, California 94305, USA.

Department of Pharmacodynamics, College of Pharmacy, University

of Florida, Gainesville, Florida 32610, USA.

Present Address: Dean’sOfﬁce, Touro University California College

of Pharmacy, Vallejo, CA 94592, USA.

To whom correspondence should be addressed. (e–mail:

[email protected]ﬂ.edu)

The AAPS Journal (2020) 22:94

DOI: 10.1208/s12248-020-00472-x

1550-7416/20/0000-0001/0

2020 American Association of Pharmaceutical Scientists

agents such as tr icyclic antidepr essants (TCAs) and

serotonin-norepinephrine reuptake inhibitors (SNRIs) or

anticonvulsant drugs such as calcium channel alpha-2-delta

ligands (gabapentinoids) (4). Nonsteroidal anti-inﬂammatory

drugs (NSAIDs) may be used for mild pain relief, whereas

narcotic agents are generally prescribed for pain that does not

respond to the ﬁrst-line treatments (4). Although most of the

prescribed drugs are effective for alleviating pain symptoms,

they cause several side effects and possess notable liabilities

that include sedation, diplopia, and dizziness in case of

antiepileptic drugs (5), or tolerance, respiratory depression,

dependence, and addiction in case of opioids (6). Therefore,

there is a strong need for new therapeutics with fewer side

effects and increased effectiveness for pain management. To

this end, several new potential targets to treat neuropathic

pain have been proposed, and novel agents have shown

efﬁcacy in preclinical models, and some have currently

reach ed investigation in clinical trials (7). Among these

approaches, sigma receptor ligands emerged as promising

tools for alleviating chronic pain (8). Sigma receptors are

transmembrane proteins present throughout the central

nervous system as well as in peripheral tissues (e.g., spleen,

liver, and kidney) (9). Two different receptor subtypes were

reported and identiﬁed as sigma-1 and sigma-2. The sigma-1

receptor was cloned over two decades ago, and the human

crystal structure has been reported (10,11), whereas puriﬁca-

tion and cloning of the sigma-2 receptor have only recently

been published in 2017 (12). The sigma-2 receptor has been

conﬁrmed as Tmem97 (transmembrane protein 97) (12).

Several literature reports suggest a role for the sigma-1

receptor in pain modulation, and either selective or non-

selective sigma-1 receptor ligands have shown antinociceptive

effects in animal models ( 13–16). In particular, the sigma-1

receptor antagonist S1RA (E-52862) (Fig. 1) has completed

successfully phase I clinical trials studies and is currently

under phase II investigation (17,18). Another example of

sigma-1 receptor antagonist is [

F]FTC-146 (Fig. 1) which

has entered into clinical trials as a positron emission

tomography and magnetic resonance imaging (PET/MRI)

diagnostic agent to pinpoint peripheral nerve injury (19–22).

Despite many years of research efforts focused on the

validation of the sigma-1 subtype as an effective target to

treat neuropathic pain (23), the evaluation of the sigma-2

receptor potential as a therapeutic target for pain target has

Fig. 1. Chemical structure and afﬁnity values of selective sigma-1 antagonists S1RA (E-52862) and [

F]FTC-146 which

are current under clinical studies; selective sigma-2 agonist UKH-1114 and non- selective sigma-1/sigma-2 ligand AZ66

which have showed antineuropathic pain effects in mice; selective sigma-2 benzimidazolone-based analog CM397

94 Page 2 of 11 The AAPS Journal (2020) 22:94

also started to gain momentum. Most recently, the phenyl

methanobenzazocine derivative (UKH-1114, Fig. 1) as sigma-

2/Tmem97 agonist was reported to be able to produce

antinociceptive effects when administered IT to spared nerve

injury (SNI) mice, suggesting antineuropathic pain effects of

sigma-2 ligands (24). Moreover, the non-selective sigma-1/

sigma-2 ligand AZ66 (Fig. 1) demonstrated the ability to

alleviate multiple modalities of chronic pain with reduced

liabilities compared to opioids (16).

Our research group has been working for many years in

the development of selective sigma receptor ligands, including

benzimidazolone-based selective sigma-2 receptor li gands

(25,26). Several of our compounds have been used as

effective chemical probes in the elucidation of the putative

roles of the sigma receptor subtypes in many diseases such as

cancer, drug addiction, and pain (27–31). More recently, our

efforts have been focused on the translational research of

many of our lead molecules into effective diagnostic or

pharmacotherapies through a multi-disciplinary and inte-

grated approach. Herein, we report the synthesis, in vitro/in

vivo pharmacology evaluation, and preclinical pharmacoki-

netic studies of CM398, a highly selective sigma-2 receptor

ligand (sigma-1/sigma-2 selectivity ratio > 1,000), that demon-

strates anti-inﬂammatory analgesic effects in mice as an initial

proof-of-concept.

MATERIALS AND METHODS

Chemistry

Reagents and starting materials were obtained from

commercial suppliers and were used without puriﬁcation.

Precoated silica gel GF Uniplates from A naltech were used

for thin-layer chromatography (TLC). Column chromatog-

raphy was performed on silica gel 60 (Sorbent Technolo-

gies).

Hand

C NMR spectra were obtained on a Bruker

APX400 (400 and 100 MHz, respectively) in CDCl

and

DMSO-d

solution. Chemical shift (δ) values are given in

parts per million ( ppm) using tetramethylsilane (TMS) and

DMSO or CHCl

as the internal standard; coupling

constants (J values) are given in hertz (Hz). For signal

multiplicities, the following abbreviations are used as

follows: s (singlet), d (doublet), dd (doublets of doublet),

ddd (doublet of doublet of doublets), t (triplet), br s (broad

singlet), and m (multiplet). The mass spectra (MS) w ere

recorded on a WATERS ACQUITY Ultra Performance LC

with ZQ detector in ES I or A PCI mode. The h igh

resolution mass spectra (HRMS) were recorded on a Waters

Micromass Q-TOF Micro m ass spectrometer w ith a lock

spray sourc e. Chemical names we re generated using

ChemDraw Ultra (CambridgeSoft, version 10.0).

N-Methyl-2-nitroaniline (2). A solution of 1-ﬂuoro-2-

nitro-benzene (2.00 g, 14.17 mmol) in water (5 mL) was

added 40% aqueous methylamine ( 5 mL) at room temper-

ature, and the reaction mixture stirred at room temperature

under nitrogen. After 2 h, the reaction mixture was poured

into a sat urated aq ueous s olution of sodium chlo ride

(50 mL) and extracted with ethyl acetate (3 × 50 mL). The

organic layer was dried over anhydrous sodium sulfate,

concentrated in va cuo, and the residue was puri ﬁed by ﬂash

column chromatography (hexane/ethyl acetate 9:1) to afford

2 as an orange oil (2.00 g, 93%).

H NMR (400 MHz,

DMSO-d

) δ 8.14 (d, J 0 5.5 Hz, 1H), 8.01 (dd, J 0 8.6,

1.5 Hz, 1H), 7.49 (ddd, J 0 8.3, 6.9, 1.5 Hz, 1H), 6.91 (dd, J 0

8.8, 1.3 Hz, 1H), 6.62 (ddd, J 0 8.3, 7.0, 1.2 Hz, 1H), 2.91 (d,

J 0 5.0 Hz, 3H).

C NMR (101 MHz, DMSO-d

) δ 146.59,

137.19, 131.48, 126.72, 115.49, 114.80, 30.24. MS (ESI

) m/z

153 [M + H]

-Methylbenzene-1,2-d iamine (3). Asolutionof2

(2.00 g, 13.14 mmol) in methanol (20 mL) was added 10%

palladium on carbon (0.10 g) in a portion wise manner. After

addition and stirring for an additional 2 h under hydroge-

nated (H

) atmosphere at room temperature, the reaction

mixture was then ﬁltered through a pad of celite, and the

ﬁltrate was concentrated in vacuo to afford 3 as brown

residue, which was used in the next step without further

puriﬁcation.

H NMR (400 MHz, DMSO-d

) δ 6.55 (t, J 0 7.3

Hz, 2H), 6.48–6.35 (m, 2H), 4.56 (d, J 0 5.9 Hz, 1H), 4.42 (s,

2H), 2.70 (d, J 0 3.6 Hz, 3H).

C NMR (101 MHz, DMSO-d

)

δ 137.85, 135.75, 118.40, 117.37, 114.46, 109.78, 30.91. MS

(ESI

) m/z 123 [M + H]

1-Meth yl-1H-benzo[d]imidazol-2(3H)-one (4). Amix-

ture of 3 (2.00 g, 16.40 mmol), 1,1′-carbonyldiimidazole

(3.18 g, 19.60 mmol) in anhydrous tetrahydrofuran (25 mL)

was stirred at 65°C for 5 h. The mixture was poured onto

water and extracted with ethyl acetate (3 × 50 mL). The

extract was washed with a saturated aqueous solution of

sodium chloride (35 mL) and dried over anhydrous sodium

sulfate. The solvent was removed in vacuo, and the obtained

residue was crystallized from hexane-ethyl acetate (1:1) to

afford 4 as white crystals (1.90 g, 79%).

H NMR (400 MHz,

DMSO-d

) δ 10.82 (s, 1H), 7.06–6.93 (m, 4H), 3.25 (s, 3H).

NMR (101 MHz, DMSO-d

) δ 155.07, 131.58, 128.85, 121.47,

121.13, 109.23, 108.21, 27.03. MS (ESI

) m/z 149 [M + H]

1-(4-Bromobutyl )-3-methyl-1H-benzo[d]imidazol-2( 3H)-

one (5). K

(0.56 g, 4.05 mmol) and 1,4-dibromobutane

(1.12 mL, 9.45 mmol) were added, under mechanical stirring,

to a solution of 4 (0.20 g, 1.35 mmol) in anhydrous DMF

(8 mL). The reaction mixture was heated at 60°C for 3 h.

After cooling, the mixture was poured into 100 mL of water,

extracted with eth yl acetate (3–40 mL), washed with a

saturated aqueous solution of sodium chloride, and dried

over anhydrous sodium sulfate. The solvent was removed in

vacuo, and the residue was puriﬁed by ﬂash column

chromatography (petroleum ether/ethyl acetate 7:3) to afford

5 as a colorless oil (0.27 g, 70%).

H NMR (400 MHz, CDCl

)

d 7.06–7.03 (m, 2H), 6.96–6.91 (m, 2H), 3.88–3.86 (m, 2H),

3.41–3.38 (m, 2H), 3.63 (s, 3H), 1.89–1.85 (m, 4H).

C NMR

(101 MHz, CDCl

) d 154.41, 130.10, 129.17, 121.26, 121.23,

107.53, 107.50, 40.08, 33.13, 29.66, 27.15, 26.98. MS (ESI) m/z

305 [M + Na]

(

Br) and 307 [M + Na]

(

Br).

1-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-

yl)butyl)-3-methyl-1H- benzo[d]imidazol-2(3H)-one Hydro-

chloride (CM398). K

(0.08 g, 0.62 mmol) and 6,7-

dimethoxy-1,2,3,4- tetrahydroisoquinoline hydrochloride

(0.05 g, 0.21 mmol) were added, under mechanical stirring,

to a solution of 5 (0.05 g, 0.18 mmol) in anhydrous DMF

(3 mL). The reaction mixture was heated at 40°C for 1 h.

94 Page 3 of 11

The AAPS Journal (2020) 22:94

After cooling, the mixture was poured into 20 mL of water,

extracted with eth yl aceta te (3–30 mL), washed with a

saturated aqueous solution of sodium chloride, and dried

over anhydrous sodium sulfate. The solvent was removed in

vacuo, and the residue was puriﬁed by ﬂash c olumn

chromatography (methylene chloride/methanol 95:5) to af-

ford 6, which was converted into the hydrochloride salt by

addition of HCl/dioxane and isolated as a white solid (0.026 g,

34%).

H NMR (400 MHz, DMSO-d6) d 11.26 (br s, 1H),

7.24–7.22 (m, 1H), 7.16–7.14 (m, 1H), 7.08–7.06 (m, 2H), 6.79

(s, 1H), 6.77 (s, 1H), 4.35–4.32 (m, 1H), 4.13–4.09 (m, 1H),

3.87 (t, J 0 7.2 Hz, 2H), 3.73–3.56 (m, 7H), 3.33–3.19 (m, 7H),

2.89–2.85 (m, 1H), 1.83–1.71 (m, 4H).

C NMR (101 MHz,

DMSO-d6) d 153.49, 148.16, 147.51, 129.57, 128.65, 123.23,

120.72, 119.77, 111.40, 109.65, 107.64, 107.60, 55.47, 55.41,

54.22, 50.99, 48.51, 39.65, 26.77, 25.11, 24.14, 20.49. HRMS

calcd for C

[M + H]

396.2287, found 396.2268.

Competition Binding Assays

Radioligand binding studies at sigma and non-sigma

receptors were performed using competition binding assays

in homogenates of rat brain tissues and following previously

reported procedures (26,32). Speciﬁc information regarding

tissues, radioligands, and drugs used in the assays are

reported in Table I.

Metabolic Stability in Rat Liver Microsomes

Phase-I metabolism stability of CM398 was studied in rat

liver microsomes. The incubation mixture consisted of liver

microsomal protein (1 mg/mL), CM398 (1 μM), phosphate

buffer (100 mM, pH 7.4), and reduced nicotinamide adenine

dinucleotide phosphate (NADPH, 2 mM). Verapamil (1 μM)

was used as a positive control to assess the metabolizing

capacity of rat liver microsomes. NADPH deﬁcient micro-

somal reaction was performed as a negative control to reveal

the non-NADPH-dependent degradation, non-speciﬁc

binding, and chemical instability of the compound in the

reaction mixture. In a 96-well plate, 178 μL of buffer, 2 μLof

CM398/verapamil stock solution (100 μM), and 10 μL of liver

microsomes (protein 20 mg/mL) were added and equilibrated

for 5 min in a shaking water bath at 37°C. Total organic

content in reaction mixture was 1%. The reaction was started

by adding 10 μL of NADPH (40 mM). For the negative

control reaction, volume of NADPH was replaced by

phosphate buffer. An aliquot (20 μL) from the incubation

mixture was quenched at 0, 5, 10, 15, 30, and 45 min with

80 μL of ice cold acetonitrile containing internal standard (IS)

to terminate the microsomal reaction. The samples were

centrifuged at 4°C for 15 min at 12,000 rpm, and supernatants

were analyzed using U PLC/MS-MS for residual CM398

content. The positive and negative controls were also

processed similarly. The area under the curve (AUC) was

calculated for the analyte and IS in MassLynx, and the ratio

of the analyte to IS AUC was used to plot re lative

concentration vs. time and calculate percent degradation of

CM398 in the reaction mixture. The elimination half-life

1/2

) was calculated as Eq. 1.

1=2

¼ −0:693=k ð1Þ

Where k is the slope of the line obtained by plotting natural

logarithmic of percentage of CM398 remaining in the reaction

mixture vs. incubation time. Intrinsic clearance (CL

int

) and

whole liver clearance (CL

int,H

) were calculated from the

following equations:

int

¼ k  Incubation volumeðÞ= Microsomal proteinðÞð2Þ

int;H

¼ CL

int

 MPPGL  liver scaling factor ð3Þ

where MPPGL represents the microsomal protein per gram of

Sprague Dawley rat liver (45 mg/g) (33), and liver scaling factor

(40 g/Kg) (34) represents the liver weight per body weight of

Sprague Dawley rats. In vivo hepatic clearance was extrapolated

through well-stirred model (Eq. 4) including microsomal protein

binding (f

umic

)(35). The f

umic

was derived using the equation

Table I. Binding afﬁnities of CM398 for sigma and non-sigma receptors and speciﬁc conditions used for the competition binding assays

Target Ki

(nM) Tissue Radioligand Nonspeciﬁc binding

Sigma receptors Sigma-1

Sigma-2

560 ± 8.72

0.43 ± 0.015

Rat brain

5nM[

H](+)-pentazocine

3nM[

H]di-o-tolylguanidine

10 μM

haloperidol

10 μM

haloperidol

Monoamine transporters Dopamine 32.90 ± 1.9 Rat striatum 0.5 nM [

H]WIN 35,428 50 μM cocaine

Serotonin 244.2 ± 2.4 Rat brainstem 0.2 nM [

H]paroxetine 1.5 μM imipramine

Norepinephrine > 1000 Rat cerebral cortex 0.5 nM [

H]nisoxetine 4 μM desipramine

Other neurotransmitter receptors Dopamine (D

) > 1000 Rat brain 5 nM [

H](−)sulpiride 1 μM haloperidol

Serotonin (5-HT

) > 1000 Rat brain 2 nM [

H]ketanserin 1 μM mianserin

NMDA > 10,000 Rat brain 5 nM [

H]TCP 10 μM cyclazocine

Opioid >1000 Rat brain 1 nM [

H]naloxone 1 μM naloxone

Affinities (Ki values in nanomolar) were determined in brain tissue homogenates

The values represent ± S.E.M. from replicate assays. Values of > 10,000 represent less than 30% displacement of the radioligand at that

concentration.

94 Page 4 of 11 The AAPS Journal (2020) 22:94

(Eq. 5) of Hallifex and Houstan (36). Hepatic extraction ratio was

also determined using Eq. 6.

Hepatic Clearance CL

ðÞ

¼ Q  f

 CL

int

umic

ðÞ= Q þ f

 CL

int

umic

ðÞðÞð4Þ

umic

¼ 1=1 þ C  10

0:072 logP=DðÞ

2þ0:067logP=D−1:126

ðÞ

ð5Þ

Hepatic extraction ratio ¼ CL

=Q ð6Þ

where f

(6.2%) is the unbound fraction of CM398 in rat

plasma, logP/D (3.1) is the partition coefﬁcient, C is the

microsomal protein concentration, and Q is the hepatic blood

ﬂow in Sprague Dawley rats (4.8 L/h/kg) (33).

Plasma Protein Binding Studies

The protein binding of CM398 was evaluated in vitro

using an ultra-ﬁltration method at concentrations of 1 and

10 μM. The desired concentrations (1 and 10 μM) were

obtained by spiking the required volume of stock solutions in

rat plasma. Spiked organic content was ≤ 0.5% v/v. The

spiked plasma samples were placed in Centrifree® devices

(YM-30, Millipore, Bedford, USA) and incubated at 37°C for

30 min. The samples were then centrifuged for 10 min at

1000 g, and ultraﬁltrates were collected. Spiked plasma and

plasma ﬁltrate samples were processed and analyzed using

the UPLC-MS/MS method as described in supporting infor-

mation. Nonspeciﬁc binding was also determined and incor-

porated in the calculation of plasma protein binding values.

The fraction unbound (f

) and plasma protein binding were

calculated as described in Eqs. 7 and 8, respectively (37).

¼ C

= 1−NSBðÞ=C

½ ð7Þ

Protein binding ¼ 1− f

ðÞ100 ð8Þ

Where C

is the initial concentration, C

is the concentration

in ﬁltrate or free compound, NSB is the nonspeciﬁc binding

fraction, and f

is the percent unbound drug concentration.

Pharmacokinetic Studies in Rats

Oral (20 mg/kg) and intravenous (1 mg/kg) pharmacoki-

netic (PK) studies of CM398 were evaluated in male Sprague

Dawley rats ( N 0 5, each). Right jugular vein cannulated rats

(225 ± 25 g) were purchased from Envigo (Indianapolis,

USA). Prior to the PK study, the animals were quarantined

in the University of Mississippi vivarium for 48 h with a 12-h

dark/light cycle and allowed access to standard feed and

water ad libitum. All animal experiments were performed in

accordance with the University of Mississippi Institutional

Animal Care and Use Committee (IACUC) pre-approved

protocol. Animals were housed in individual Nalgene

metabolic cages with mesh ﬂoor and receptacles for urine

and feces. Rodent feed was removed from the metabolic

cages 12–18 h prior to oral (P.O.) dosing, and feed was again

provided 4 h post-dose. Animals in the (I.V.) administration

study were allowed constant access to standard feed. All

animals always had access to water ad libitum. Solution

formulations of CM398 were prepared in water (5 mg/mL)

and normal saline (1 mg/mL) for P.O. and I.V. PK studies,

respectively. The formulation for the I.V. study was ﬁltered

through 0.2 μmsyringeﬁlter. Both formulations were

analyzed for CM398 content. The I.V. solutions were

administered via caudal vein. The P.O. solution was admin-

istered via oral gavage. Blood samples were collected using

the indwelling cannula. An initial blood volume of 0.05 mL

was withdrawn to clear the line of heparinized saline. Using a

fresh syringe, 0.15 mL of blood was withdrawn and placed in

heparin coated micro-centrifuge tube. The cannula was then

ﬂushed with 0.20 ml of heparinized saline. For the P.O. study,

blood samples were taken pre-dose and at 0.17, 0.33, 0.5, 0.75,

1, 2, 4, 6, 8, 12, and 24 h post-dose. For the I.V. study, blood

samples were taken pre- dose and at 0.083, 0.17, 0.33, 0.5, 1, 2,

4, 6, 8, 12, and 24 h post-dose. Rat plasma was separated by

centrifugation of blood at 4000g for 10 min at 4°C and stored

at − 80°C refrigerator until analysis. Plasma samples were

processed and analyzed using an UPLC-MS/MS method as

described in supporting information. Plasma concentration-

time data and PK parameters are represented as mean ±

standard error of the mean (SEM). Peak plasma concentra-

tion (C

max

) and time to reach C

max

) were obtained

directly from the concentration-time data plot. Concentration

vs. time data was subjected to noncompartmental analysis

using Phoenix WinNonlin (version 6.3; Certara Inc, Missouri,

USA). The area under plasma concentration-time curve

(AUC) of CM398 was calculated using the linear trapezoidal

method. Clearance (CL) was calculated as a ratio of dose and

AUC. Absolute oral bioavailability (%F) was calculated

using Eq. 9.

%F ¼ Dose

intravenous

 AUC

lastðÞoral

=Dose

oralðÞ

 AUC

lastðÞintravenous



 100

ð9Þ

In Vivo Characterization in a Model of Inﬂammatory Pain

and Statistical Analysis

Thirty-ﬁve 10 weeks old male CD-1 mice obtained from

the Charles River Laboratories, Wilmington, Massachusetts,

USA ,were used in the formalin assay. Mice were housed ﬁve

to a cage in a temperature and humidity-controlled room at

the University of Florida vivarium on a 12:12-h light/dark

cycle (lights off at 19:00 h) with free access to food and water

except during experimental sessions. All procedures were

pre-approved and carried out in accordance with the UF

Institutional Animal Care and Use Committee as speciﬁed by

the 2011 National Institutes of Health Guide for the Care and

Use of Laboratory Animals. Animal studies are reported in

compliance with the ARRIVE guidelines (38,39). Sample

sizes (i.e., number of animals) were predetermined by power

analysis, and animals were assigned to groups randomly. Drug

94 Page 5 of 11

The AAPS Journal (2020) 22:94

treatment experiments were conducted in a blinded fashion.

No animals were excluded from statistical analysis.

After seven mice each received a 10-min pre-treatment

with vehicle (saline, 0.9% as a control group), morphine

(23.2 μmol/kg, i.p. as a positive control), or CM398 (0.23,

2.32, or 23.2 μmol/kg, i.p.), formalin (10 μL of a 10%

solution) was injected into the hind paw as described

previously (40,41). Paw-licking duration (in s) during 5 min

intervals was recorded for 70 min. The summated response

during the ﬁnal 60 min was analyzed, consistent with

nociception attributed to Phase II inﬂammation (41,42).

All paw-licking data for formalin testing are reported as

summed paw licking as area under the curve (AUC) by each

animal across the 60-min measured response, ± SEM. Mor-

phine data vs. saline was analyzed by Student ’s t test. CM398

data was analyzed by a one-way ANOVA (factor:dose) with

signiﬁcant results between groups further analyzed with

Tukey’s multiple comparisons post hoc test. The data and

statistical analysis comply with the recommendations on

experimental design and analysis in pharmacology (43 ). All

data are presented as mean ± SEM, with a signiﬁcance set at

P<0.05, denoted by the asterisk (*).

RESULT

Chemistry

The straightforward synthetic scheme for CM398 is

outlined in Fig. 2. Treatment of 1-ﬂuoro-2-nitro-benzene ( 1)

with 40% aqueous methylamine at room temperature gave

N-methyl-2-nitroaniline (2),whichwassubjectedtohydro-

genation (H

) in presence of 10% palladium on activated

charcoal to afford N

-methylbenzene-1,2-diamine (3)

(25,44,45). The later int ermediate was subje cted to ring

closure using 1,1′-carbonyldiimidazole (CDI) in anhydrous

tetrahydrofuran (THF) and heated to 65°C to obtain 1-

methyl-2-benzimidazolinone (4). Treatment of compound 4

with 1,4-dibromobutane in the presence of anhydrous

potassium carbonate in anhydrous dimethylformamide

(DMF) gave 5. The bromo derivative ( 5) was then coupled

with 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline in the

presence of potassium carbonate in DMF to afford the

target compound (CM398). Spectral analysis and high-

resolution mass spectra of CM398 were consistent with its

assigned structure.

Competition Binding Assays

The binding afﬁnity of CM398 for sigma-1 and sigma-2

receptor subtypes is summarized in Table I. CM398 possess

subnanomolar afﬁnities for the sigma-2 receptor and over

1000-fold selectivity for the sigma-1 receptor. Moreover,

binding afﬁnities versus other off-target proteins, including

monoamine transporter and other neurotransmitter recep-

tors, were also reported (Table I). The receptor binding

proﬁle of CM398 is indicative of its highly preference for the

sigma-2 compared with the sigma-1 and to the other non-

sigma proteins (selectivity ratio > 1000). Furthermore, CM398

resulted highly selective also for the norepinephrine trans-

porter r eceptor (Ki > 1,000 nM) and for the serotonin

transporter receptor (500-fold); on the other hand, CM398

displayed signiﬁcant afﬁnity for the dopamine transporter (Ki

0 32.90 nM). However, because dopamine transmission is not

strictly related to nociception but rather it may reinforce the

noradrenergic effects to inhibit certain type of pain (46),

CM398 represented a suitable probe for the scope of this

study.

Fig. 2. Synthesis of CM398. Reagents and conditions: a CH

O, 2 h, r.t. b 10% Pd/C, H

(1 atm), MeOH, 2 h. c CDI,

THF, 65°C, 18 h. d 1,4-dibromobutane, K

, DMF, 60°C, 2 h. e 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, K

DMF, 60°C, 1 h

94 Page 6 of 11 The AAPS Journal (2020) 22:94

Metabolic Stability in Rat Liver Microsomes

It is important to understand the fate of a discovery

compound undergoing hepatic metabolism; therefore, meta-

bolic stability of CM398 was performed in rat liver micro-

somes. In vitro metabolic stability method i s able to

determine the metabolic depletion of a compound by

cytochrome P450 enzyme s in a closed system without

considering the physiological factors like blood ﬂow or drug

binding within the matrix (35). The in vitro half-life of

CM398, when incubated in rat liver microsomes, was found

to be 0.10 ± 0 .01 h. The CL

int,H

derived from in vitro half-life

and scaling factors is 12.8 ± 0. 3 L/h/kg. Calc ulated value of

umic

was 0.63, which also suggests that only 37% of the

compound in liver will be available for metabolism by the

liver enzymes. In vitro CL

int,H

was also successfully extrap-

olated to hepatic clearance and extraction ratio. Compound

CM398 exhibited both high hepatic clearance (CL

,4.6±

0.0 L/h/Kg) and extraction ratio (0.96 ± 0.01). The high

extraction ratio suggests that only approximately 4% of

CM398 can escape unchanged after a single pass through

the liver following its phase I metabolism.

Plasma Protein Binding Studies

The reversible binding of a drug to plasma proteins is

an important determinant of its PK and pharmacodynamics

characteristics. The protein binding of CM398 was evalu-

ated in vitro using ultra-ﬁltration method. The

concentration-dependent F

ofCM398wasstudiedinrat

plasma. At concentrations of 1 and 10 μM, the F

values

were 6.7 ± 1.1 and 5.6 ± 0.1%, respectively. Average plasma

protein binding of CM398 was found to be 93.8 ± 0.9%.

Non-speciﬁc binding of CM398 to Centrifree® devices was

less than 5.0%.

Pharmacokinetic Studies in Rats

The PK properties of CM398 were studied following the

P.O. (20 mg/kg) and I.V. (1 mg/kg) administration of the

compound to male Sprague Dawley rats. After dosing of

CM398, close and continuous visual monitoring of the

animals revealed that there was no severe acute toxicity

response, as non e of the animals showed any signs of

behavioral or neurological toxicity during the entire study

period. The plasma concentration-time proﬁles are presented

in Fig. 3. PK parameters were estimated using non-

compartmental analysis with Phoenix WinNonlin and are

presented in Table II. CM398 shows a very rapid absorption

as C

max

(2052.8 ± 252.8 ng/mL) occurred at 0.17 ± 0.00 h

max

) post-dose. Oral a bsorption is so fast, that the

absorption phase cannot be captured. Absolute oral bioavail-

ability was calculated to be 29.0%, indicating adequate total

exposure after the oral dose. Total body CL (2.1 ± 0.1 L/h/kg)

of CM398 was lower than the total hepatic blood ﬂow (4.8 L/

h/kg) (33) in rats, indicating negligible extra-hepatic elimina-

tion of the compound. The volume of distribution (V

, 5.3 ±

0.9 L/kg) of CM398 was larger than the total blood volume of

rats (0.085 L/kg) (34), showing extra-vascular distribution of

the compound. After P.O. and I.V. dosing, the mean half- life

1/2

) was found to be 1.9 ± 0.2 and 1.7 ± 0.3 h, respectively.

In Vivo Characterization

The analgesic effects in vivo of CM398 were tested using

the formalin model of inﬂammatory pain in mice. Mice (n 0 7)

were pretreated wit h vehicle (saline, 0 .9%), m orphine

(23.2 μmol/kg), or CM398 (0.23, 2.32, or 23.2 μmol/kg )

through the intraperitoneal (i.p.) route and 10 min later,

administered formalin (10 μL in the hind paw). Mice

pretreated with saline spent an average of 205.1 ± 32.6 s

Fig. 3. Mean plasma concentration-time proﬁle of CM398 in male Sprague Dawley rats

(N 0 5, each) after a single oral (20 mg/kg) and intravenous (1 mg/kg) administration. Bar

represents SEM

94 Page 7 of 11The AAPS Journal (2020) 22:94

licking the injected hindpaw. Mice pretreated with morphine

spent signiﬁcantly less time licking their formalin-treated

hindpaw (205.1 ± 32.6 s; p 0 0.0002, Student’s t test). Pretreat-

ment with CM398 dose dependently reduced the time spent

licking the hind paw (Fig. 4), demonstrating a signiﬁcant

antinociceptive effect doses of 2.32 and 23.2 μmol/kg, i.p.

(F(3, 24) 0 7.66, p 0 0.0009; one-way ANOVA with Tukey’s

post hoc test).

DISCUSSION

Previous research and preclinical studies have eluci-

dated the role of the sigma-1 receptor in modulating

nociception (8,47). In particular, the high expression of

sigma-1 receptors in speciﬁc areas of the brain such as

dorsal spinal cord, thalamus, periaqueductal gray,

basolateral amygdala, and rostroventral medulla (48), as

well as in peripheral tissues (especially dorsal root ganglia

neurons) (49), clearly suggests its involvement in pain

modulation.

From a pharmacological point of view, inhibition of

sigma-1 receptor produces a decrease of nociception stimuli

through attenuated expression of pain behaviors in several

animal models, including mechanical hypersensitivity (cap-

saicin-induced test), inﬂammatory pain (formalin assay),

and neuropathic pain models (SNI in mice) (50). Moreover,

these ﬁndings were s upported by previous studies which

used sigma-1 receptor KO mice (51–53).

In a more recent study, Sahn et al. have tested the effect

of IT injection of different sigma-1 and sigma-2 receptor

ligands in the mouse SNI model (24). As a result, both the

sigma-1 and the sigma-2/Tmem97 preferring ligands pro-

duced antinociceptive effects, which were signiﬁcantly

different from vehicle, whereas the moderate selective

sigma-2 receptor agonist, siramesine, showed only a modest

inhibitory effect o n mechanical hypersensitivity (24). These

results led to further investigations into the putative role of

sigma-2 receptors in the modulation of pain. In fact, unlike

the results obtained for sigma-1 receptor antagonists, which

are consistent with previously reported literature, those

obtained by using slightly preferring sigma-2 receptor

ligands did not completely clarify if a mixed sigma-1/

sigma-2 or a selective sigma-2 compound might be beneﬁ-

cial to treat pain. Unfortunately, as is often the case, the

lack of very selective ligands for the protein target adds

complexity for elucidating their function. From this per-

spective, the main goal of this work was the preliminary

characterization of a highly selective sigma-2 receptor

ligand w ith drug-like properties, serving as a lead candidate

for further preclinical and clinical development.

The radioligand binding assays revealed that CM398

displayed subnanomolar afﬁnities (K i

0 0.43 nM) for the

sigma-2 receptor and very low afﬁnities (Ki 0 560 nM) for

the sigma-1 receptor, thus, to the best of our knowledge,

CM398 represents the most selective sigma-2 receptor ligand

reported to date with regard to sigma-1/sigma-2 selectivity

ratio (1000-fold) (54). The binding properties of CM398 were

consistent with those of a recently reported set of structurally

related benzimidazolone-based analogs (25). Structure-

afﬁnity relationships (SARs) studies suggested that the

presence of both the benzimidazolone as a scaffold and the

6,7-disubstituted tetrahydroisoquinolin e as a cyclic amine

fragment was optimal in term of sigma binding proﬁle.

Speciﬁcally, replacement of the 1-(4-ﬂuorophenyl)piperazine

(CM397, Fig.1) with the 6,7-dimethoxy-1,2,3,4-

tetrahydroisoquinoline moiety signiﬁcantly increased the

afﬁnity for the sigma-2 receptor (Ki 0 2.5 and 0.43 nM) and

the selectivity over the sigma-1 receptor subtype (179-fold

and > 1000 fold, respectively).

CM398 also possessed negligible afﬁnities for D

, 5-HT

NMDA, opioid receptors, and norepinephrine transporters

(1000–10,000 nM). CM398 did demonstrate notable afﬁnity for

dopamine (Ki 0 32.90 nM) and serotonin transporters (Ki 0

244.2 nM), but these are still 76-fold and > 500-fold over the

Table II. Pharmacokinetic parameters of CM398 after oral dose and

intravenous administration in male Sprague Dawley rats

Parameters Oral Intravenous

max

(ng/mL)

max

(h)

AUC

0 → t

(ng h/L)

Vd (L/kg)

2052.8 ± 252.8

0.17 ± 0.00

2733.6 ± 293.4

6.2 ± 0.6

–

471.3 ± 23.6

5.3 ± 0.9

T1/2 (h) 1.9 ± 0.2 1.7 ± 0.3

CL (L/h/kg) 2.2 ± 0.2 2.1 ± 0.1

Bioavailability (%) 29.0 –

Each value represents the average of five rats dosed oral (20 mg/kg)

and intravenous (1 mg/kg); values are mean ± SEM.

AUC area under the plasma concentration-time curve, CL clearance,

max

plasma peak concentration, T

max

time to C

max

1/2

elimination

half-life, V

volume of distribution

Fig. 4. Antinociceptive effects of CM398 in the formalin test. Values

represent summed time licking across 60 min testing period following

5% formalin injection into the mouse hindpaw. n 0 7 (each); *p 0 0.02

vs. vehicle response; one-way ANOVA with Tukey’s post hoc test for

CM398 , or p 0 0.0002 vs. vehicle response; Student’s t test for

morphine

94 Page 8 of 11 The AAPS Journal (2020) 22:94

afﬁnity for sigma-2 receptors. As mentioned above, the in vitro

pharmacologic proﬁle of CM398 was well suited to unveil the

utility of sigma-2 ligands as potential therapeutic for pain

treatment, especially due to the lack of afﬁnity with those

target proteins mainly involved in pain relief mechanisms, for

instance, opioid receptors, noradrenaline transporters, D

,and

NMDA receptors (55–59).

In regard to the pharmacokinetic evaluation, CM398

demonstrated adequate oral exposure, extravascular distribu-

tion, and negligible extra hepatic elimination in rats indicating

satisfactory pharmacokinetic properties to support its further

development as a pharmacological tool and potential orally

active drug candidate.

Finally, we decided to test the analgesic properties of

CM398 in a tonic pain stimuli induced by formalin injection

rather than allodynia elicited by chronic nerve injuries. The

formalin test is a standard animal model of inﬂammation-

induced nociception and frequently included in the battery of

behavioral pharmacology tests of pain (60).The2.32and23.2

μmol/kg i.p. doses of CM398 in mice produced a signiﬁcant

reduction of the time spent licking as compared with saline,

through attenuation of the localized inﬂammatory pain

produced after injection of formalin in the paw of mice.

These results suggest that targeting sigma-2 r eceptor with a

highly selective ligand, like CM398, may be effective in

alleviating inﬂammatory pain. Notably, this initial testing was

limitedtomalemicetobeconsistentwiththeprevioustesting

(24). Although sex differences have not been demonstrated

in antinociception studies involving sigma-1 receptors

(51,52), future studies are needed to examine possible sex

effects on sigma-2 receptor-mediated a ntinociception. Al-

though the main goal of this work was limited to the

preliminary evaluation of the pharmacological proﬁle of a

potent and highly selective sigma-2 receptor ligand, collec-

tively, this preliminary data offers support for further,

extensive characterization.

CONCLUSION

In summary, we synthesized and characterized CM398, a

highly selective sigma-2 receptor ligand, with the highest

sigma-1/sigma-2 selectivity ratio known to date. Moreover,

CM398 acted as a sigma-2 preferring ligand versus other non-

sigma receptors involved in pain modulation, p articularly

opioid receptors, NMDA, and norepinephrine transporters.

The metabolism studies perf ormed on CM398 showed

suitable drug-like properties for the development of an orally

active drug. Despite the favorable pharmacological proﬁle of

CM398, additional evaluation of analgesic effects in a full

battery of behavioral models examining the various modal-

ities of pain is needed. The data provided herein represent

further support for the development of sigma-2 receptors

ligands as an alternative pain medication.

ACKNOWLEDGMENTS

This study was supported, in part, by grants from the

National Institutes of Health NIDA R01 DA023205 (CRM,

RRM); NIMH P20 GM104932 (CRM, BAA); and from the

Department of Defense CMRMP PR161310/P1 (CRM, JPM).

REFERENCES

1. Colloca L, Ludman T, Bouhassira D, Baron R, Dickenson AH,

Yarnitsky D, et al. Neuropathic pain. Nat Rev Dis Primers.

2017;3:17002.

2. Yan YY, Li CY, Zhou L, Ao LY, Fang WR, Li YM. Research

progress of mechanisms and drug therapy for neuropathic pain.

Life Sci. 2017;190:68–77.

3. NIH NIoNDaS-. Peripheral Neuropathy Fact Sheet 2018

[July 06, 2018]. Available from: https://www.ninds.nih.gov/Dis-

orders/Patient-Caregiver-Education/ Fact-Sheets/Pe ripheral-

Neuropathy-Fact-Sheet.

4. Cruccu G, Truini A. A review of neuropathic pain: from

guidelines to clinical practice. Pain Ther. 2017;6(Suppl 1):35–42.

5. Calandre EP, Rico-Villademoro s F, Slim M. Alpha(2)delt a

ligands, gabapentin, pregabalin and mirogabalin: a review of

their clinical pharmacology and therapeutic use. Expert Rev

Neurother. 2016;16(11):1263–77.

6. Yaksh TL, Wallace MS. Opioids, analgesia, and pain manage-

ment. In: Goodman and Gilman’s the Pharmacological Basis of

Therapeutics. New York: McGraw-Hill Medical; 2011. p. 481–

526.

7. Bouhassira D, Attal N. Emerging therapies for neuropathic

pain: new molecules or new indications for old treatments?

Pain. 2018;159(3):576–82.

8. Merlos M, Burgueno J, Portillo-Salido E, Plata-Salaman CR,

Vela JM. Pharmacological modulation of the sigma 1 receptor

and the treatment of pain. Adv Exp Med Biol. 2017;964:85–107.

9. Kim FJ. Introduction to sigma proteins: evolution of the concept

of sigma receptors. Handb Exp Pharmacol. 2017;244:1–11.

10. Hanner M, Moebius FF, Flandorfer A, Knaus HG, Striessnig J,

Kempner E, et al. Puriﬁcation, molecular cloning, and expres-

sion of the mammalian sigma1-binding site. Proc Natl Acad Sci

U S A. 1996;93(15):8072–7.

11. Schmidt HR, Zheng S, Gurpinar E, Koehl A, Manglik A, Kruse

AC. Crystal structure of the human sigma1 receptor. Nature.

2016;532(7600):527–30.

12. Alon A, Schmidt HR, Wood MD, Sahn JJ, Martin SF, Kruse

AC. Identiﬁcation of the gene that codes for the sigma2

receptor. Proc Natl Acad Sci U S A. 2017;114(27):7160–5.

13. Diaz JL, Cuberes R, Berrocal J, Contijoch M, Christmann U,

Fernandez A, et al. Synthesis and biological evaluation of the 1-

arylpyrazole class of sigma(1) receptor antagonists: identiﬁca-

tion of 4-{2-[5-methyl-1-(napht halen-2-yl)-1H-pyrazol-3-

yloxy]ethyl}morpholine (S1RA, E-52862). J Med Chem.

2012;55(19):8211–24.

14. Lan Y, Chen Y, Cao X, Zhang J, Wang J, Xu X, et al. Synthesis

and biological evaluation of novel sigma-1 receptor antagonists

based on pyrimidine scaffold as agents for treating neuropathic

pain. J Med Chem. 2014;57(24):10404–23.

15. Romeo G, Prezzavento O, Intagliata S, Pittalà V, Modica MN,

Marrazzo A, et al. Synthesis, in vitro and in vivo characteriza-

tion of new benzoxazole and benzothiazole-based sigma recep-

tor ligands. Eur J Med Chem. 2019;174:226–35.

16. Cirino TJ, Eans SO, Medina JM, Wilson LL, Mottinelli M,

Intagliata S, et al. Characterization of sigma 1 receptor

antagonist CM-304 and its analog, AZ-66: novel therapeutics

against Allodynia and induced pain. Front Pharmacol.

2019;10:678.

17. Vidal-Torres A, de la Puente B, Rocasalbas M, Tourino C, Bura

SA, Fernandez-Pastor B, et al. Sigma-1 receptor antagonism as

opioid adjuvant strategy: enhancement of opioid antinociception

without increasing adverse effects. Eur J Pharmacol.

2013;711(1–3):63–72.

18. Abadias M, Escriche M, Vaque A, Sust M, Encina G. Safety,

tolerability and pharmacokinetics of single and multiple doses of

a novel sigma-1 receptor antagonist in three randomized phase I

studies. Br J Clin Pharmacol. 2013;75(1):103–17.

19. James ML, Shen B, Zavaleta CL, Nielsen CH, Mesangeau C,

Vuppala PK, et al. New positron emission tomography (PET)

radioligand for imaging sigma-1 receptors in living subjects. J

Med Chem. 2012;55(19):8272–82.

94 Page 9 of 11The AAPS Journal (2020) 22:94

20. Shen B, Park JH, Hjornevik T, Cipriano PW, Yoon D, Gulaka

PK, et al. Radiosynthesis and ﬁrst-in-human PET/MRI evalua-

tion with clinical-grade [(18)F]FTC-146. Molecular imaging and

biology : MIB : the ofﬁcial publication of the academy of. Mol

Imaging. 2017;19(5):779–86.

21. Shen B, Behera D, James ML, Reyes ST, Andrews L, Cipriano

PW, et al. Visualizing nerve injury in a neuropathic pain model

with [(18)F]FTC-146 PET/MRI. Theranostics. 2017;7(11):2794–

805.

22. Hjornevik T, Cipriano PW, Shen B, Park JH, Gulaka P, Holley

D, et al. Biodistribution and radiation dosimetry of (18)F-FTC-

146 i n huma ns. J Nucl Med Off Publ Soc Nucl Med.

2017;58(12):2004–9.

23. Zamanillo D, Romero L, Merlos M, Vela JM. Sigma 1 receptor:

a new therapeutic target for pain. Eur J Pharmacol. 2013;716(1–

3):78–93.

24. Sahn JJ, Mejia GL, Ray PR, Martin SF, Price TJ. Sigma 2

receptor/Tmem97 agonists produce long lasting antineuropathic

pain effects in mice. ACS Chem Neurosci. 2017;8(8):1801–11.

25. Intagliata S, Alsharif WF, Mesangeau C, Fazio N, Seminerio

MJ, Xu YT, et al. Benzimidazolone-based selective σ2 receptor

ligands: synthesis and pharmacological evaluation. Eur J Med

Chem. 2019;165:250–7.

26. Mesangeau C, Narayanan S, Green AM, Shaikh J, Kaushal N,

Viard E, et al. Conversion of a highly selective sigma-1 receptor-

ligand to sigma-2 receptor preferring ligands with anticocaine

activity. J Med Chem. 2008;51(5):1482–6.

27. Nicholson HE, Alsharif WF, Comeau AB, Mesange au C,

Intagliata S, Mottinelli M, et al. Divergent cytotoxic and

metabolically stimulativ e functions of sigma-2 receptors:

structure- activity relationships of 6-acetyl-3-(4-(4-(4-

ﬂuorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol- 2(3H)-one

(SN79) derivatives. J Pharmacol Exp Ther. 2018;7.

28. Robson MJ, Turner RC, Naser ZJ, McCurdy CR, O'Callaghan

JP, Huber JD, et al. SN79, a sigma recept or antagonist,

attenuates methamphetamine-induced astrogliosis through a

blockade of OSMR/gp130 signaling and STAT3 phosphoryla-

tion. Exp Neurol. 2014;254:180–9.

29. Katz JL, Hiranita T, Kopajtic TA, Rice KC, Mesangeau C,

Narayanan S, et al. Blockade of cocaine or sigma receptor

agonist self administration by subtype-selective sigma receptor

antagonists. J Pharmacol Exp Ther. 2016;358(1):109–24.

30. Obeng S, Patel A, Burns M, Intagliata S, Mottinelli M, Reeves

ME, et al. The sigma1 receptor antagonist CM304 potentiates

the antinociceptive but not the discriminative stimulus effects of

the cannabinoid receptor agonist THC in rats. FASEB J.

2020;34(S1):1.

31. Patel A, Obeng S, Burns M, Intagliata S, Mottinelli M, Reeves

ME, et al. The sigma1 receptor antagonist CM304 enhances the

antinociceptive effects of the cannabinoid receptor agonists, but

not Mu-o pioid receptor full agonists in mice. FASEB J.

2020;34(S1):1.

32. Matsumoto RR, McCracken KA, Pouw B, Zhang Y, Bowen

WD. Involvement of sigma receptors in the behavioral effects of

cocaine: evidence from novel ligands andantisense

oligodeoxynucleotides. Neuropha rmacology. 2002;42(8):1043–

55.

33. Peters SA. Physiol ogical ly-based phar macokinetic (PBPK)

modeling and simulations: principles, methods, and applications

in the pharmaceutical industry: John Wiley & Sons; 2012.

34. Davies B, Morris T. Physiological parameters in laboratory

animals and humans. Pharm Res. 1993;10(7):1093–5.

35. Khojasteh SC, Wong H, Hop CE. Drug metabolism and

pharmacokinetics quick guide: Springer Science & Business

Media; 2011.

36. Hallifax D, Houston JB. Binding of drugs to hepatic micro-

somes: comment and assessment of current prediction

methodology with recommendation for improvement. Drug

Metab Dispos. 2006;34(4):724–6.

37. Lee K-J, Mower R, Hollenbeck T, Castelo J, Johnson N,

Gordon P, et al. Modulation of nonspeciﬁc binding in ultraﬁl-

tration protein binding studies. Pharm Res. 2003;20(7):1015–21.

38. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG.

Improving bioscience research reporting: the ARRIVE guidelines

for reporting animal research. PLoS Biol. 2010;8(6):e1000412.

39. McGrath JC, Lilley E. Implementing guidelines on reporting

research using animals (ARRIVE etc.): new requirements for

publication in BJP. Br J Pharmacol. 2015;172(13):3189–93.

40. Wheeler-Aceto H, Porreca F, Cowan A. The rat paw formalin

test: comparison of noxious agents. Pain. 1990;40(2):229–38.

41. Gong N, Huang Q, Chen Y, Xu M, Ma S, Wang Y-X. Pain

assessment using the rat and mouse formalin tests. Bio-protocol.

2014;4(21):e1288.

42. Cheng HY, Pitcher GM, Laviolette SR, Whishaw IQ, Tong KI,

Kockeritz LK, et al. DREAM is a critical transcriptiona l

repressor for pain modulation. Cell. 2002;108(1):31–43.

43. Curtis MJ, Bond RA, Spina D, Ahluwalia A, Alexander SP,

Giembycz MA, et al. Experimental design and analysis and their

reporting: new guidance for publication in BJP. Br J Pharmacol.

2015;172(14):3461–71.

44. Intagliata S, Modica MN, Pittala V, Salerno L, Siracusa MA,

Cagnotto A, et al. New N- and O-arylpiperazinylalkyl pyrimi-

dines and 2-methylquinazolines derivatives as 5-HT7 and 5-

HT1A receptor ligands: synthesis, structure-activity relation-

ships, and molecular modeling studies. Bioorg Med Chem.

2017;25(3):1250–9.

45. Modica MN, Intagliata S, Pittala V, Salerno L, Siracusa MA,

Cagnotto A, et al. Synthesis and binding properties of new long-

chain 4-substituted piperazine derivatives as 5-HT(1)a and 5-

HT(7) receptor ligands. Bioorg Med Chem Lett.

2015;25(7):1427–30.

46. Obata H. Analgesic mechanisms of antidepressants for neuro-

pathic pain. Int J Mol Sci. 2017;18(11).

47. Merlos M, Romero L, Zamanillo D, Plata-Salaman C, Vela JM.

Sigma-1 receptor and pain. Handb Exp Pharmacol.

2017;244:131–61.

48. Alonso G, Phan V, Guillemain I, Saunier M, Legrand A, Anoal

M, et al. Immunocytochemical localizatio n of the sigma(1)

receptor in the adult rat central nervous system. Neuroscience.

2000;97(1):155–70.

49. Bangaru ML, Weihrauch D, Tang QB, Zoga V, Hogan Q, Wu

HE. Sigma-1 receptor expression in sensory neurons and the

effect of painful peripheral nerve injury. Mol Pain. 2013;9:47.

50. Romero L, Zamanillo D, Nadal X, Sanchez-Arroyos R, Rivera-

Arconada I, Dordal A, et al. Pharmacological properties of

S1RA, a new sigma-1 receptor antagonist that inhibits neuro-

pathic pain and activity-induced spinal sensitization. Br J

Pharmacol. 2012;166(8):2289–306.

51. Entrena JM, Cobos EJ, Nieto FR, Cendan CM, Gris G, Del

Pozo E, et al. Sigma-1 receptors are essential for capsaicin-

induced mechanical hypersensitivity: studies with selective

sigma-1 ligands and sigma-1 knockout mice. Pain.

2009;143(3):252–61.

52. Cendan CM, Pujalte JM, Portillo-Salido E, Montoliu L,

Baeyens JM. Formalin-induced pain is reduced in sigma(1)

receptor knockout mice. Eur J Pharmacol. 2005;511(1):73–4.

53. de la Puente B, Nadal X, Portillo-Salido E, Sanchez-Arroyos R,

Ovalle S, Palacios G, et al. Sigma-1 receptors regulate activity-

induced spinal sensitization and neuropathic pain after periph-

eral nerve injury. Pain. 2009;145(3):294–303.

54. Nastasi G, Miceli C, Pittalà V, Modica MN, Prezzavento O,

Romeo G, et al. S2RSLDB: a comprehensive manually curated,

internet-accessible database of the sigma-2 receptor selective

ligands. J Cheminformatics. 2017;9:3.

94 Page 10 of 11 The AAPS Journal (2020) 22:94