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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2018 Aug 17;175(19):3784–3796. doi: 10.1111/bph.14448

Analgesic effects of ASP3662, a novel 11β‐hydroxysteroid dehydrogenase 1 inhibitor, in rat models of neuropathic and dysfunctional pain

Tetsuo Kiso 1,✉, Toshihiro Sekizawa 1, Hiroshi Uchino 1, Mina Tsukamoto 1, Shuichiro Kakimoto 1
PMCID: PMC6135787  PMID: 30006998

Abstract

Background and Purpose

Glucocorticoids are a major class of stress hormones known to participate in stress‐induced hyperalgesia. Although 11β‐hydroxysteroid dehydrogenase 1 (11β‐HSD1) is a key enzyme in the intracellular regeneration of glucocorticoids in the CNS, its role in pain perception has not been assessed. Here, we examined the effects of ASP3662, a novel 11β‐HSD1 inhibitor, on neuropathic and dysfunctional pain.

Experimental Approach

The enzyme inhibitory activities and pharmacokinetics of ASP3662 were examined, and its antinociceptive effects were evaluated in models of neuropathic pain, fibromyalgia and inflammatory pain in Sprague‐Dawley rats.

Key Results

ASP3662 inhibited human, mouse and rat 11β‐HSD1 but not human 11β‐HSD2, in vitro. ASP3662 had no significant effect on 87 other possible targets (enzymes, transporters and receptors). ASP3662 inhibited in vitro conversion of glucocorticoid from its inactive to active form in extracts of rat brain and spinal cord. Pharmacokinetic analysis in Sprague‐Dawley rats showed that ASP3662 has CNS‐penetrability and long‐lasting pharmacokinetic properties. Single oral administration of ASP3662 ameliorated mechanical allodynia in spinal nerve ligation (SNL) and streptozotocin‐induced diabetic rats and thermal hyperalgesia in chronic constriction nerve injury rats. ASP3662 also restored muscle pressure thresholds in reserpine‐induced myalgia rats. Intrathecal administration of ASP3662 was also effective in SNL rats. However, ASP3662 had no analgesic effects in adjuvant‐induced arthritis rats.

Conclusions and Implications

ASP3662 is a potent, selective and CNS‐penetrable inhibitor of 11β‐HSD1. The effects of ASP3662 suggest that selective inhibition of 11β‐HSD1 may be an attractive approach for the treatment of neuropathic and dysfunctional pain, as observed in fibromyalgia.

Abbreviations

AIA

adjuvant‐induced arthritis

CFA

complete Freund's adjuvant

CCI

chronic constriction nerve injury

GR

glucocorticoid receptor

11β‐HSD

11β‐hydroxysteroid dehydrogenase

SNL

spinal nerve ligation

RIM

reserpine‐induced myalgia

STZ

streptozotocin

Introduction

Chronic stress is widely known to exacerbate pain in a wide range of conditions (Blackburn‐Munro and Blackburn‐Munro, 2001; Abdallah and Geha, 2017). Glucocorticoids are a major class of stress hormones that have been reported to participate in stress‐induced hyperalgesia (Alexander et al., 2009). The enzyme http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2763 converts inactive glucocorticoids (http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5171 in humans, 11‐dehydrocorticosterone in rodents) to active glucocorticoids (http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2868 in humans, http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5171 in rodents) in specific tissues, notably the liver and adipose tissues (Moisan et al., 1990; Lakshmi et al., 1991). In contrast, the type 2 isozyme (11β‐HSD2), which is mainly expressed in the kidneys, catalyses the conversion of active glucocorticoids to inactive glucocorticoids. 11β‐HSD1 has been proposed as a potential therapeutic target for the treatment of diabetes (Kotelevtsev et al., 1997; Masuzaki et al., 2001), cognitive disorders (Seckl and Walker, 2001; Sandeep et al., 2004; Seckl, 2004), depression (Carroll et al., 1981; Thomson et al., 2007) and other neurological disorders (Zhang et al., 2005; Veen et al., 2009). However, the role of 11β‐HSD1 in pain perception has not been assessed.

Glucocorticoids exert their effect via http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=625. Central GRs are reported contributors to the mechanisms of neuropathic pain (Wang et al., 2004, 2005). Intrathecal administration of a GR antagonist or GR antisense oligonucleotide to a chronic constriction nerve injury (CCI) rat model of neuropathic pain attenuated pain behaviour (Wang et al., 2004). 11β‐HSD1 is expressed not only in peripheral tissues but also in the CNS, in both the brain and the spinal cord (Moisan et al., 1990; Monder and Lakshmi, 1990). Inhibition of 11β‐HSD1 in the CNS could prevent an increase in active glucocorticoid levels in the CNS and potentially lead to the amelioration of neuropathic pain.

Fibromyalgia is a musculoskeletal syndrome characterized by chronic widespread pain, tenderness to palpation and various other concomitant symptoms. Fibromyalgia patients also experience widespread reduced muscle pressure threshold. The pathophysiology of fibromyalgia has been linked to the dysfunction of biogenic amine‐mediated, pain control pathways in the CNS (Arnold, 2006; Ablin et al., 2008). The reserpine‐induced myalgia (RIM) rat model for fibromyalgia was established in our laboratory to mimic the symptoms experienced by patients with fibromyalgia, such as manifestation of chronic pain and a dysfunctional biogenic amine system (Nagakura et al., 2009). Because glucocorticoids affect pain levels in fibromyalgia patients (Fischer et al., 2016), there could be a role for 11β‐HSD1 in the pain associated with fibromyalgia.

To clarify the role of 11β‐HSD1 on neuropathic and dysfunctional pain, we studied a novel inhibitor of 11β‐HSD1, 4‐{5‐[2‐(4‐chloro‐2,6‐difluorophenoxy)propan‐2‐yl]‐4‐methyl‐4H‐1,2,4‐triazol‐3‐yl}‐3‐fluorobenzamide (ASP3662; inset Figure 1). We examined the in vitro profile and pharmacokinetics of ASP3662 and its effects in neuropathic pain models [spinal nerve ligation (SNL), CCI and streptozotocin (STZ)‐induced diabetic rats] and a fibromyalgia model (RIM rats).

Figure 1.

Figure 1

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Chemical structure of ASP3662 (inset) and the inhibitory effect of ASP3662 on 11β‐HSD1 in enzyme assays. To determine the Ki values, the effects of ASP3662 at 6, 10, 15 nM on human 11β‐HSD1 (A); 3, 6, 8 nM on mouse 11β‐HSD1 (B); and 10, 15, 20 nM on rat 11β‐HSD1 (C) were examined. Representative Lineweaver–Burk plots show that ASP3662 competitively inhibited all tested species of 11β‐HSD1. S, substrate concentration; V, reaction velocity.

Methods

Animals

All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Astellas Pharma Inc. (Ibaraki, Japan), which has been awarded Accreditation Status by the Association for Assessment and Accreditation of Laboratory Animal Care International. All efforts were made to minimize the number of animals used and their suffering. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). Male Sprague‐Dawley (SD) rats weighing 150–315 g (Charles River Laboratories Japan, Yokohama, Japan or Japan SLC, Hamamatsu, Japan) were used. The animals were housed in groups of one to three per cage, under a light–dark cycle (lights on: 07:30–19:30), with free access to food and water.

Administration of compounds

For in vitro experiments, ASP3662 was dissolved in DMSO. For in vivo experiments, ASP3662 was suspended and diluted in 0.5% methylcellulose solution or the solid dispersion form of ASP3662 was suspended in distilled water. ASP3662 was given in a volume of 5 mL·kg−1. For intrathecal administration, ASP3662 was dissolved and diluted in 20% 2‐hydroxypropyl‐β‐cyclodextrin, and 10 μL was administered to each animal. The dose of ASP3662 was calculated as the free form. Diclofenac was suspended in 0.5% methylcellulose solution.

In vitro 11β‐HSD1 enzyme assays

We constructed pET28a expression vectors carrying human (NM_181755), mouse (NM_008288) and rat (NM_017080) His‐tagged 11β‐HSD1 cDNA. The recombinant 11β‐HSD1 protein for each species was expressed in E. coli and extracted and purified by Ni‐NTA affinity chromatography and anion exchange chromatography. The purified enzymes were stored at −20°C until use. To assay 11β‐HSD1 activity, 11β‐HSD1 protein was added to assay buffer (10 μL) containing 80 μM NADPH, cortisone (human and mouse: 200–1335 nM, rat: 600–4005 nM) and 10 mM phosphate buffer with or without ASP3662 (in DMSO, final 1%) and incubated for 7 min at 37°C. The reagents of the HTRF cortisol assay kit with 400 μM carbenoxolone were added to the reaction mixture. Time‐resolved fluorescence was measured at 620 and 665 nm after excitation at 320 nm using the ARVOHTS (Perkin Elmer, MA, USA). The specific signal was calculated from the ratio of the signal at 665 nm to that at 620 nm and was inversely proportional to the concentration of cortisol in the sample. The cortisol concentration was calculated from six calibration curves constructed for six concentrations of cortisone (200, 333.5, 500, 666.5, 1000 and 1335 nM) because the specific signal was affected by unreacted cortisone. Ki values of ASP3662 were determined from concentration‐dependent inhibition curves constructed by SAS software. The geometric mean and 95% confidence interval of the Ki values were calculated from data from three independent experiments.

In vitro 11β‐HSD2 enzyme assays

The expression vector pcDNA3.1(+) carrying human 11β‐HSD2 cDNA (NM_000196) was transfected into HEK293‐EBNA cells using LipofectAmine 2000 transfection regent (Invitrogen, CA, USA) according to the manufacturer's instructions. The cell pellets were suspended in buffer (20 mM Tris–HCl, 1 mM EDTA, 300 mM NaCl, 10% glycerol, pH 8.0) and homogenized using a Potter–Elvehjem tissue homogenizer. The cell homogenate was centrifuged at 20 400× g for 2 min, and the supernatant was collected. This fraction was used as human 11β‐HSD2 enzyme and stored at −80°C until use. To assay 11β‐HSD2 activity, human 11β‐HSD2 enzyme was added to assay buffer (10 μL) containing 25 nM cortisol, 200 μM NAD and 40 mM Tris HCl buffer (pH 8.0) with or without ASP3662 and incubated for 90 min at 37°C. The cortisol concentration was calculated from a calibration curve, as described for the 11β‐HSD1 assay. To normalize the raw data, all data were expressed as a percentage of 0% and 100% inhibition. Measurements were performed in triplicate. Mean (±SEM) % inhibition by ASP3662 were calculated from data from three independent experiments using the Statistical Analysis System (SAS Institute Japan, Tokyo, Japan).

Selectivity assay

Binding affinity or inhibitory activity of 1 μM ASP3662 to a variety of receptors, ion channels, transporters and enzymes were measured at Sekisui Medical (Ibaraki, Japan), using proprietary assays.

In vitro conversion of cortisone to cortisol in the brain and spinal cord

Rats were killed, and the prefrontal cortex and spinal cord were collected. Pieces of tissue (about 100 mg) were placed into the wells of a 24‐well plate, and 500 μL of PBS was added. The tissues were minced using a pair of scissors into 2–3 mm square cubes. ASP3662 was added, and the plate was incubated for 30 min at 37°C and 5% CO2. After adding 1 μM cortisone, the HTRF cortisol assay kit was used to measure the concentration of cortisol in the supernatant of each well over time.

SNL model

SNL surgery was conducted according to a previous report (Kim and Chung, 1992). Animals were anaesthetized with pentobarbital (50 mg·kg−1), and the left paraspinal muscles were separated from the spinous processes. The L6 transverse process was removed using a pair of rongeurs to expose the spinal nerves. The left L5 and L6 spinal nerves were tightly ligated with silk thread, and the wound was sutured. The sham group was subjected to all surgical operations except the nerve ligation. The mechanical paw withdrawal threshold was determined using the von Frey hair test. The plantar surface of the hindpaw was stimulated by touching with von Frey hairs, and the 50% withdrawal response threshold was calculated using the up‐down method (Chaplan et al., 1994). Six days after the operation, rats were selected and randomized to four groups (for vehicle and three doses of ASP3662) based on their mechanical paw withdrawal threshold. The next day, ASP3662 or vehicle was orally administered, and paw withdrawal thresholds were measured 2 h after administration. The measurement was performed by an experimenter blinded to drugs being administered. The ED50 value and 95% confidence limits were calculated according to the linear regression method by defining the withdrawal threshold of the left hindpaw in the vehicle‐treated and sham‐operated groups as 0% and 100% respectively.

Intrathecal administration of ASP3662

Intrathecal catheter insertion surgery was conducted at the same time as SNL surgery. A catheter made from a 32‐gauge polyurethane tube and polyethylene tubes was inserted into the intrathecal space through the L5–6 intervertebral space. The catheter was passed through the subcutaneous space and exited through the skin in the back of the neck. ASP3662 and/or corticosterone, or vehicle was administered through the intrathecal catheter seven or more days after the operation. Paw withdrawal thresholds were measured at 15, 30 and 60 min after administration. Each rat was used twice at 6‐ or 7‐day intervals.

CCI model

CCI operation of the sciatic nerve was conducted according to a previous report (Bennett and Xie, 1988). Animals were anaesthetized with pentobarbital (50 mg·kg−1), and the left common sciatic nerve was exposed at the level of the mid‐thigh through the biceps femoris. Chromic gut 4.0 sutures were affixed to form four loose ligatures around the sciatic nerve. The sham group was subjected to all surgical operations except the nerve ligature. In addition to the mechanical paw withdrawal threshold, the thermal withdrawal latency was determined using the plantar test (Ugo Basile, Comerio, Italy). The plantar surface of the rat hindpaw was stimulated by noxious radiant heat, and the latency from the initial heat stimulation to paw withdrawal was recorded. On day 13, rats were selected and randomized to four groups based on their paw withdrawal threshold or thermal withdrawal latency. On day 14, ASP3662 or vehicle was orally administered, and paw withdrawal thresholds or paw withdrawal latencies were measured 2 h after administration. The measurement was performed by experimenters blinded to drugs being administered.

STZ model

Rats were fasted from the evening prior to the day of STZ injection. Access to food was resumed after the injection. STZ diluted in citrate buffer solution was administered via the tail vein at a dose of 45 mg·kg−1. Non‐STZ (normal) rats received vehicle by the same route. Twelve days after STZ administration, plasma glucose levels were measured using the Antsense III blood glucose meter (Horiba, Kyoto, Japan), and rats with plasma glucose levels below 3 mg·mL−1 were excluded from the study. Seven weeks after STZ administration, paw withdrawal thresholds as determined by the von Frey test were measured. Rats were habituated to acrylic test cages with a wire grid floor for at least 20 min. An electronic von Frey Anesthesiometer Model 2390 (IITC Life Science, Woodland Hills, CA, USA) was used to stimulate the plantar surface of the rats' hindpaws. The force was gradually increased until a withdrawal response was elicited. The amount of force required to elicit a withdrawal response (withdrawal threshold) was determined twice for each hindpaw. The mean of the four values was calculated for each rat. Rats were selected and randomized to four groups based on their withdrawal threshold. ASP3662 or vehicle was orally administered, and paw withdrawal thresholds were measured 2 h after administration. The measurement was performed by an experimenter blinded to drugs being administered.

RIM model

The RIM model was generated according to a previous report (Nagakura et al., 2009). Reserpine was dissolved in glacial acetic acid and diluted in a final concentration of 0.5% acetic acid in distilled water. Reserpine (1 mg·mL−1) or vehicle (sham group) was subcutaneously injected into the back of the neck once a day for three consecutive days. After the last injection of reserpine on day 4, the pre‐drug muscle pressure threshold was measured. Rats were placed inside a sock, and the right hindlimb was positioned such that incremental pressure could be applied to the mid‐gastrocnemius muscle using a Randall–Selitto apparatus. Linearly increasing mechanical force was applied until a hindlimb withdrawal response was elicited. The mean of three trials was calculated for each session. Rats were selected and randomized to four groups based on their muscle pressure threshold. On day 5 after the last injection of reserpine, ASP3662 or vehicle was orally administered, and muscle pressure thresholds were measured 2 h after administration. The measurements were performed by an experimenter blinded to drugs being administered. The ED50 value and 95% confidence limits were calculated according to the linear regression method by regarding the 50% muscle pressure threshold in the vehicle‐treated and sham‐operated groups as 0% and 100% respectively.

Adjuvant‐induced arthritis (AIA) model

The reduction of spontaneous locomotor activity in the model of AIA (Matson et al., 2007) was used as an index of nociception. Animals were anaesthetized with isoflurane, and 50 μL of CFA or saline was injected bilaterally into the tibia–femur joints. After 2 days, the rearing behaviour was automatically measured using an activity monitoring system (Supermex; Muromachi Kikai, Tokyo, Japan). Rats were placed into an open field, and the number of rearing behaviours was recorded every 5 min for 15 min. ASP3662 or vehicle was orally administered 2 h before measurement, and diclofenac was orally administered 1 h before measurement.

Rotarod test

Motor coordination was measured using an accelerating rotarod apparatus (model LE8500, Panlab, Barcelona, Spain) that accelerated from 4 to 40 r.p.m. in 5 min, as reported previously (Murai et al., 2016). Each rat was subjected to three training sessions, and those that remained on the rod for over 90 s were selected and randomized for testing. In the test session, ASP3662 or vehicle was orally administered and time spent on the rod was measured twice 2 h after administration. The mean of two trials was calculated for each animal.

Acute pain

Rats were randomized based on their body weight. The hot‐plate test was performed using a hot‐plate device (MK‐350D, Muromachi Kikai). We measured the latency of a nociceptive response (licking, jumping, shaking or lifting) and withdrawal behaviour (55°C, cut‐off time: 20 s). A tail pinch test was performed by attaching a clamp to the base of the tail and measuring the latency of a nociceptive response (vocalization, escape reaction or shrinking). The cut‐off time was set at 300 s. The effect of ASP3662 or vehicle was evaluated 2 h after administration. As a positive control, the effect of morphine (5 mg·kg−1 s.c.) was also examined 30 min after administration.

Analysis of plasma and brain concentration of ASP3662

We measured ASP3662 concentrations in the plasma and brain of normal SD rats, given a single oral dose of ASP3662 at 0.3, 1 or 3 mg·kg−1. To evaluate the absolute oral bioavailability, a separate set of male SD rats were given a single intravenous dose of ASP3662 at 0.3 mg·kg−1. Plasma and brain samples were collected at each time point after drug administration. To prevent hydrolysis of ASP3662 in plasma, 1/100 of 2.5% dichlorvos solution was added to the blood collection tube. Homogenates were prepared from brains. The concentration of ASP3662 in plasma and brain was determined using a validated high‐performance LC–MS/MS method. Pharmacokinetic parameters were calculated from the mean plasma or brain concentrations for each dosage by non‐compartmental analysis using WinNonlin Professional Version 4.1 software (Pharsight, Cary, NC, USA).

Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2018). Data are expressed as mean ± SEM or SD. Statistical differences between the vehicle‐treated and drug‐treated groups were assessed using Student's t‐test for two groups, while one‐way ANOVA with post hoc Dunnett's multiple comparisons test was used for multiple groups. Time‐course studies for in vitro conversion of cortisone to cortisol were analysed using two‐way repeated measures ANOVA followed by a Bonferroni post hoc test. P < 0.05 was regarded as statistically significant.

Materials

ASP3662 was synthesized by Astellas Pharma Inc. (Tokyo, Japan) (Yoshimura et al., 2010). Corticosterone and STZ were purchased from Wako Pure Chemical Industries (Osaka, Japan). Reserpine was purchased from Nacalai Tesque (Kyoto, Japan). Morphine hydrochloride was purchased from Daiichi Sankyo Propharma (Tokyo, Japan). Cortisol assay kit HTRF was purchased from Cisbio Bioassays (Codolet, France). Cortisone, cortisol (hydrocortisone), carbenoxolone, complete Freund's adjuvant (CFA) and diclofenac were purchased from Sigma‐Aldrich (St. Louis, MO, USA).

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017a, 2017b)

Results

Effects of ASP3662 on 11β‐HSD1/2 enzymes and on other targets

To examine the selectivity of ASP3662 for 11β‐HSD1, we examined the effects of ASP3662 in enzyme assays for 11β‐HSD1 and 11β‐HSD2 and selectivity assays for other potential targets. ASP3662 demonstrated competitive inhibition of human, mouse and rat 11β‐HSD1, with Ki values of 5.3 (95% CI: 2.5–11), 2.6 (95% CI: 1.4–4.9) and 23 nM (95% CI: 11–47) respectively (Figure 1). ASP3662 at concentrations up to 30 μM did not inhibit human 11β‐HSD2. The maximum inhibition by ASP3662 was 6.5% at 0.1 μM, but no concentration dependency was observed.

ASP3662 (1 μM) had no appreciable binding affinity to or inhibition (less than 50% inhibition) of the following 87 other possible targets: adenosine receptors (A1, A2a), adrenoceptors (α1A, α1B, α2A, α2B, α2C, β1, β2), angiotensin receptors (AT1, AT2), bradykinin B2 receptor, calcium channels (L type, N type), cannabinoid receptors (CB1, CB2), cholecystokinin receptors (CCK‐A, CCK‐B), corticotrophin‐releasing factor receptor 1 (CRF1), dopamine receptors (D1, D2 short, D3, D4.2, D5), dopamine transporter (DAT), oestrogen receptor, endothelin receptors (ETA, ETB), GABAA receptors (agonist site, BZ central, BZ peripheral), GABA receptor chloride channel, GABAB receptor, GABA transporter, GR, glutamate receptors (AMPA, kainate, NMDA agonist site, NMDA glycine site, phencyclidine site, polyamine site), glycine receptor, histamine receptors (H1, H2, H3), potassium channels (KATP, SKCa), LT receptors (LTB4, LTD4), melatonin MT1 receptor, mineralocorticoid receptor, muscarinic receptors (M1, M2, M3, M4, M5), sodium channel (site 2), neurokinin receptors (NK1, NK2, NK3), noradrenaline transporter (NET), nicotinic receptor, opiate receptors (δ, κ, μ, ORL1), oxytocin receptor, PAF receptor, progesterone PR‐B receptor, 5‐HT receptors (5HT1A, 5HT2A, 5HT2B, 5HT2C, 5HT3, 5HT4, 5HT5A, 5HT6, 5HT7), 5‐HT transporter (SERT), sigma receptors (σ1, σ2), testosterone receptor, vasopressin V1B receptor, VIP receptor 1 (VIP1), AChE and MAOs (MAO‐A, MAO‐B).

Effect of ASP3662 on the conversion of cortisone to cortisol in the brain and spinal cord

We examined the effect of ASP3662 on the conversion of glucocorticoid from its inactive to active form in the brain and spinal cord in in vitro assays using brain and spinal cord tissues. After adding 1 μM cortisone, the concentration of cortisol increased over time in wells containing prefrontal cortex (Figure 2A) or spinal cord (Figure 2B) samples. ASP3662 (0.03–10 μM) concentration‐dependently inhibited the increase in cortisol for both prefrontal cortex and spinal cord samples, and 10 μM ASP3662 almost completely inhibited the conversion.

Figure 2.

Figure 2

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Inhibitory effect of ASP3662 on the in vitro conversion of cortisone to cortisol in the brain and spinal cord of SD rats. The concentration of cortisol in wells containing prefrontal cortex (A) and spinal cord (B) tissues was measured after adding 1 μM cortisone. ASP3662 (0.03–10 μM) was added 30 min before cortisone. Data shown are means ± SEM; n = 11 rats. *P < 0.05, significantly different from control (0 μM ASP3662); two‐way repeated‐measures ANOVA with Bonferroni post hoc test.

Pharmacokinetics in rats after single intravenous or oral administration of ASP3662

The mean plasma ASP3662 concentration 0.1 h after single intravenous administration at 0.3 mg·kg−1 was 131.3 ng·mL−1; the concentration thereafter declined bi‐exponentially with a terminal t 1/2 of 3.8 h (Figure 3A). Mean plasma ASP3662 concentrations peaked within 2 h after single oral administration at 0.3, 1 and 3 mg·kg−1 and remained almost constant for up to 4 h post‐dose before declining. The t 1/2 ranged from 3.6 to 4.8 h (Figure 3B). The pharmacokinetic parameters are shown in Table 1. Mean brain ASP3662 concentrations reached maximum within 2 h post‐dose and declined thereafter with t 1/2 ranging from 3.7 to 4.8 h (Figure 3C). The mean brain/plasma concentration ratio at each time point ranged from 0.5 to 0.7, except at 0.25 h when it ranged from 0.3 to 0.4.

Figure 3.

Figure 3

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Pharmacokinetic properties of ASP3662 after single intravenous or oral administration to SD rats. (A) The plasma concentration of ASP3662 after a single intravenous administration at 0.3 mg·kg−1 was determined using a validated LC–MS/MS method. (B) The plasma concentration of ASP3662 after a single oral dose of ASP3662 at 0.3, 1 or 3 mg·kg−1. (C) The brain concentration of ASP3662 after a single oral dose of ASP3662 at 0.3, 1 or 3 mg·kg−1. Data shown are means ± SD; n =3.

Table 1.

Plasma concentration and pharmacokinetic parameters of ASP3662 after single intravenous or oral administration of ASP3662 to rats

Route Dose (mg·kg−1) C max (ng·mL−1) t max (h) t 1/2 (h) Bioavailability (%)
Intravenous (i.v.) 0.3 – – 3.8 –
Oral (p.o.) 0.3 82.4 2.00 4.8 122.4
1 261.5 (3.2) 0.25 4.2 122.7
3 751.8 (9.1) 1.00 3.6 128.8
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These pharmacokinetic parameters were calculated using mean plasma concentrations (n = 3). Dashes (–) mean not applicable. Values in parentheses indicate the ratio of the C max at the respective dose to that at 0.3 mg·kg−1 p.o.

Effects of ASP3662 in rat models of neuropathic pain

The effect of ASP3662 on neuropathic pain was examined using SNL and CCI nerve ligation models and a STZ‐induced diabetic neuropathy model. One week after SNL, the mechanical paw withdrawal thresholds of the ipsilateral (operated‐side) hindpaw in the vehicle‐treated group were clearly lower than those in the sham‐operated group (mechanical allodynia). Single oral administration of ASP3662 at doses of 0.1 and 0.3 mg·kg−1 significantly raised mechanical thresholds in SNL rats 2 h after administration (Figure 4A). The improvement rate in mechanical thresholds at 0.3 mg·kg−1 was 80%, and the ED50 value (95% confidence limit) was 0.087 mg·kg−1 (0.049–0.14). Intrathecal administration of ASP3662 (0.03, 0.1 and 0.3 μg per animal) dose‐dependently reduced the mechanical allodynia in the ipsilateral hindpaw (Figure 4B). The effect of 0.1 and 0.3 μg ASP3662 reached statistical significance 60 min after administration (Figure 4C). The effect of ASP3662 was diminished by co‐administration of 10 μg corticosterone (Figure 4D).

Figure 4.

Figure 4

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Analgesic effect of ASP3662 on mechanical allodynia in SNL rats. (A) One week after the SNL operation, mechanical allodynia was confirmed by a reduction in the paw withdrawal threshold (PWT). PWT was measured 2 h after a single oral administration of ASP3662 at 0.03, 0.1 or 0.3 mg·kg−1 or vehicle. Each bar represents the mean ± SEM of PWT for 12 rats. (B) The analgesic effect of intrathecal administration of ASP3662 at 0.03, 0.1 or 0.3 μg per animal. (C) Dose‐dependency 60 min after intrathecal administration of ASP3662. (D) The effect of corticosterone at 10 μg per animal on the analgesic effect of ASP3662 at 0.3 μg per animal. Data shown are the means ± SEM of PWT for 7–18 rats. *P < 0.05, significantly different from vehicle‐treated group; one‐way ANOVA with post hoc Dunnett's multiple comparisons test.

Two weeks after the CCI operation, in addition to mechanical allodynia, thermal withdrawal latency of the hindpaw on the operated‐side was clearly shorter in vehicle‐treated CCI compared to sham‐operated rats (thermal hyperalgesia). Single oral doses of ASP3662 at 0.3 and 1 mg·kg−1 significantly prolonged thermal withdrawal latencies in CCI rats 2 h after administration (Figure 5B). The improvement rate at 1 mg·kg−1 was 61%. In contrast, ASP3662 did not affect mechanical allodynia in CCI rats (Figure 5A).

Figure 5.

Figure 5

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Analgesic effect of ASP3662 on mechanical allodynia and thermal hyperalgesia in CCI rats. (A) Two weeks after the CCI operation, mechanical allodynia was confirmed by a reduction in the paw withdrawal threshold (PWT). PWT was measured 2 h after oral administration of ASP3662 at 0.1, 0.3 or 1 mg·kg−1 or vehicle. Each bar represents the mean ± SEM of PWT for 8–10 rats. (B) Thermal hyperalgesia was confirmed by a reduction in the paw withdrawal latency (PWL). PWL was measured 2 h after oral administration of ASP3662 at 0.1, 0.3 or 1 mg·kg−1 or vehicle. Data shown are the means ± SEM of PWL for 9–10 rats. *P < 0.05, significantly different from vehicle‐treated group; one‐way ANOVA with post hoc Dunnett's multiple comparisons test.

Induction of mechanical allodynia in STZ rats was confirmed by a reduction in the paw withdrawal thresholds 7 weeks after STZ injection. Single oral administration of ASP3662 at doses of 0.3 and 1 mg·kg−1 significantly ameliorated mechanical allodynia in the hindpaws of STZ rats 2 h after administration (Figure 6A). The improvement rate at 1 mg·kg−1 was 52%.

Figure 6.

Figure 6

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Analgesic effect of ASP3662 on mechanical allodynia in STZ‐induced diabetic rats and muscle pain in RIM rats. (A) Seven weeks after STZ administration, mechanical allodynia was confirmed by a reduction in the paw withdrawal threshold (PWT). PWT was measured 2 h after oral administration of ASP3662 at 0.1, 0.3 or 1 mg·kg−1 or vehicle. Each bar represents the mean ± SEM of PWT for 12 rats. (B) Repeated administration of reserpine caused a decrease in the muscle pressure threshold (MPT). MPT was measured 2 h after administration of ASP3662 at 0.1, 0.3 or 1 mg·kg−1 or vehicle. Data shown are means ± SEM of MPT for eight rats. *P < 0.05, significantly different from vehicle‐treated group; one‐way ANOVA with post hoc Dunnett's multiple comparisons test.

Effect of ASP3662 in a rat model of dysfunctional pain

The effect of ASP3662 on dysfunctional pain was examined using the RIM model of fibromyalgia. Repeated administration of reserpine caused a reduction in the muscle pressure threshold. Single oral administration of ASP3662 at doses of 0.1, 0.3 and 1 mg·kg−1 significantly restored the muscle pressure threshold in RIM rats 2 h after administration (Figure 6B). The improvement rate at 1 mg·kg−1 was 82%, and the ED50 value (95% confidence interval) was 0.31 mg·kg−1 (0.26–0.37). The analgesic effect of ASP3662 sustained for 24 h (data not shown).

Effect of ASP3662 in a rat model of inflammatory pain

The effect of ASP3662 on inflammatory pain was examined using an AIA model. Two days after the CFA injection into the joints, bilateral inflammation of the knee joints decreased the rearing behaviour of rats to about one‐third of that observed in vehicle‐treated controls. Single oral administration of diclofenac at 1 mg·kg−1 significantly restored the rearing behaviour in AIA rats with an improvement rate of 65% (Figure 7). In contrast, ASP3662 (0.1, 0.3 and 1 mg·kg−1) had no effect on rearing behaviour.

Figure 7.

Figure 7

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Effect of ASP3662 on rearing behaviour in AIA rats. Two days after the CFA injection into the joints, rearing behaviour during a 15 min observation period was decreased. Rearing behaviour was measured 2 h after oral administration of ASP3662 at 0.1, 0.3 or 1 mg·kg−1 or vehicle, or 1 h after oral administration of diclofenac at 1 mg·kg−1. Data shown are the means ± SEM from 5–10 rats. *P < 0.05, significantly different from vehicle‐treated group; Student's t‐test.

Effects of ASP3662 on acute pain in rats

The effect of single oral administration of ASP3662 (0.1, 0.3 and 1 mg·kg−1) on acute pain was evaluated in rats using the hot‐plate test and tail pinch test. ASP3662 at doses up to 1 mg·kg−1 did not affect latency in the hot‐plate test (Figure 8A). ASP3662 at 1 mg·kg−1 significantly increased latency in the tail pinch test, but the effect was much weaker than that observed for morphine (Figure 8B).

Figure 8.

Figure 8

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Effect of ASP3662 on acute pain. (A) The latency of a nociceptive response (licking, jumping, shaking or lifting) or withdrawal behaviour was measured in rats (55°C, cut‐off time: 20 s) in the hot‐plate test. (B) The latency of a nociceptive response (vocalization, escape reaction or shrinking) following the clamping of the base of the tail was measured in rats (cut‐off time 300 s). The effect of ASP3662 or vehicle was evaluated 2 h after administration. As a positive control, the effect of morphine (5 mg·kg−1 s.c.) was also examined 30 min after administration. Data shown are the means ± SEM of the latency from eight rats. # P < 0.05, significantly different from vehicle‐treated group; Student's t‐test. *P < 0.05, significantly different from vehicle‐treated group; one‐way ANOVA with post hoc Dunnett's multiple comparisons test.

Effect of ASP3662 on motor coordination in rats

The effect of single oral administration of ASP3662 (0.3, 1 and 3 mg·kg−1) on motor coordination was evaluated in rats using an accelerating rotarod test. ASP3662 at doses up to 3 mg·kg−1 did not affect time spent on the rod (Figure 9).

Figure 9.

Figure 9

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Effect of ASP3662 on motor coordination. Motor coordination was assessed using an accelerating rotarod apparatus that accelerated from 4 to 40 r.p.m. in 5 min. ASP3662 at 0.3, 1 or 3 mg·kg−1 or vehicle was orally administered and time spent on the rod was measured 2 h after administration. Data shown are means ± SEM of time spent on the rod from eight rats.

Discussion

Our results showed that ASP3662 is a potent competitive inhibitor of human, mouse and rat 11β‐HSD1. The Ki values calculated in this study may be useful for determining the dosage of ASP3662 in clinical and non‐clinical studies. In contrast, ASP3662 had no inhibitory activity on 11β‐HSD2, even at concentrations of 30 μM. We concluded that ASP3662 is a highly selective inhibitor of 11β‐HSD1 but not 11β‐HSD2. In addition, ASP3662 had no appreciable binding affinity to or inhibition of the 87 off‐targets tested, which included receptors, ion channels, transporters and enzymes. These results indicate that ASP3662 is a potent and selective 11β‐HSD1 inhibitor.

11β‐HSD1 is expressed in the CNS including the cortex and spinal cord, and its enzymic activity has been reported in these tissues (Moisan et al., 1990; Monder and Lakshmi, 1990). Immunostaining shows that 11β‐HSD1 is predominantly localized to neurons and to a lesser extent to glial cells (Sakai et al., 1992). In the present study, we confirmed the inhibitory effect of ASP3662 on the conversion of glucocorticoids from their inactive to active form, in homogenates of the brain and spinal cord in vitro in the presence of cortisone. If ASP3662 can penetrate the blood–brain barrier and reach the brain or spinal cord, we predict that it would be effective for preventing local increases in active glucocorticoids.

The results of our pharmacokinetics study of ASP3662 in rats indicate that the C max increased in an almost dose‐proportional fashion. In addition, bioavailability of this compound was almost 100%. Therefore, both the pharmacokinetics and the oral absorption of ASP3662 in rats are very favourable. The brain/plasma concentration ratio at each time point indicates that ASP3662 exhibited CNS‐penetrability.

We showed that single oral administration of ASP3662 ameliorated mechanical allodynia in SNL and STZ rats. In CCI rats, ASP3662 did not affect mechanical allodynia but ameliorated thermal hyperalgesia. Our previous study examining the desensitization of C‐fibres with http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2491, an ultrapotent http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2486 analogue, showed that C‐fibres predominantly mediate thermal hyperalgesia but only partially mediate mechanical allodynia in CCI rats (Murai et al., 2014). Resiniferatoxin reportedly inhibits mechanical allodynia in SNL rats (Koh et al., 2016) but not in STZ rats (Bishnoi et al., 2011). Mechanical allodynia in STZ rats is reportedly mediated by small diameter nociceptive nerve fibres (Field et al., 1999), suggesting that capsaicin‐insensitive C‐fibres might mediate mechanical allodynia in STZ rats. These results suggest that ASP3662 at least inhibits capsaicin‐sensitive and capsaicin‐insensitive C‐fibre‐mediated pain. The effects of ASP3662 on A‐fibres are unknown and the selectivity of ASP3662 for various nerve fibre types should be examined in further studies

The larger inflammatory component in CCI compared to SNL rats may also explain the ineffectiveness of ASP3662 on mechanical allodynia in CCI rats (Cui et al., 2000; Bridges et al., 2001a, b). Ineffectiveness of ASP3662 on inflammatory pain in this study may be associated with the results in CCI rats mentioned above. Our findings in SNL and CCI nerve ligation models and STZ‐induced diabetic neuropathy rat models, which are widely used and well‐characterized animal models of neuropathic pain and painful diabetic neuropathy, respectively, suggest that ASP3662 is a promising candidate for the treatment of neuropathic pain. Responder rates for the neuropathic pain drug http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5484 are not very high. For instance, responder rates for 50% pain reduction are 26.5–52% for pregabalin‐treated patients (300–600 mg·day−1) compared with 7.5–24% for placebo (Stacey and Swift, 2006). Our previous work showed that pregabalin and duloxetine are effective against mechanical allodynia but not thermal hyperalgesia in CCI rats (Murai et al., 2014, 2016). The present results show that ASP3662 is effective against thermal hyperalgesia but not mechanical allodynia in CCI rats. The effect of ASP3662 on neuropathic pain therefore differs from that of pregabalin, suggesting its potential effectiveness for those patients who do not respond to pregabalin.

We also showed the effectiveness of intrathecal administration of ASP3662 on mechanical allodynia in SNL rats, which was diminished by co‐administration of excess corticosterone. ASP3662 inhibited rat 11β‐HSD1 activity in an enzyme assay and the generation of active glucocorticoids in spinal cord tissue in vitro and exhibited CNS‐penetrability in a pharmacokinetics study. These results suggest that the spinal cord is one site of action for ASP3662 and that the analgesic effect arises from the prevention of increases in local levels of active glucocorticoids.

We further demonstrated that single oral administration of ASP3662 produced analgesia in RIM rats. As RIM rats are expected to contribute to the evaluation of the efficacy of drugs for fibromyalgia (Nagakura et al., 2009; DeSantana et al., 2013), our results suggest that ASP3662 is a promising candidate for the treatment of fibromyalgia.

Many studies have reported the potential of 11β‐HSD1 inhibitors in the treatment of Type 2 diabetes (Hollis and Huber, 2011; Sun et al., 2011). In the present study, we showed new evidence that 11β‐HSD1 inhibitors may also be effective for treating neuropathic pain and dysfunctional pain, such as that observed in fibromyalgia. However, there is some concern that 11β‐HSD1 inhibition may exacerbate acute inflammation because glucocorticoids attenuate acute inflammation (Chapman et al., 2013). In fact, 11β‐HSD1‐deficient mice exhibit worse acute inflammation compared to wild type (Coutinho et al., 2012). However, our study in AIA rats showed that ASP3662 neither decreased nor increased rearing behaviours. This suggests that inhibition of 11β‐HSD1 does not exacerbate inflammatory pain in this condition. The discrepancy between the previous and current results may be due to the different roles of 11β‐HSD1 in inflammatory conditions. Inhibition of 11β‐HSD1 reportedly reduces some chronic inflammation (Chapman et al., 2013; Kipari et al., 2013). Given that the effect on inflammation is uncertain, clinical trials of 11β‐HSD1 inhibitors should carefully monitor its effect on inflammation.

We also examined the effect of ASP3662 on acute pain. Although ASP3662 administration did not affect latency in the hot‐plate test, it was associated with a slightly increased latency in the tail pinch test. However, the effect was much weaker than that observed with morphine, suggesting that the analgesic effect of ASP3662 on acute pain is very weak compared to its effect on chronic pain, such as neuropathic and dysfunctional pain. GRs have been shown to be expressed in the spinal dorsal horn, which has a role in pain transmission (Cintra et al., 1993), and GR antagonists attenuate neuropathic pain at the level of the spinal cord (Takasaki et al., 2005). ASP3662 can penetrate the CNS and prevent the conversion of glucocorticoids from their inactive to active form in the spinal cord, suggesting that 11β‐HSD1 inhibitors may also ameliorate chronic pain by preventing increases in local levels of active glucocorticoids but has a minimal effect on inflammatory and acute pain. To our knowledge, this is the first report to demonstrate the antinociceptive effect of a CNS‐penetrable 11β‐HSD1 inhibitor. Further studies should elucidate the detailed antinociceptive mechanism of action of 11β‐HSD1 inhibitors.

Previous studies have reported that acute stress induces analgesia, which involves glucocorticoids (McEwen and Kalia, 2010), while chronic stress induces hyperalgesia in many cases (Gamaro et al., 1998; Imbe et al., 2006). In addition, acute stress in a nerve ligation model increases mechanical allodynia, and a GR antagonist ameliorates this stress‐induced augmentation of pain (Alexander et al., 2009). Stress reportedly exacerbates pain in fibromyalgia patients (Fischer et al., 2016). Therefore, 11β‐HSD1 inhibitors may also indirectly decrease chronic pain by inhibiting stress‐induced pain augmentation.

Active glucocorticoids are released from the adrenal glands and their circulating levels are regulated by a feedback mechanism of the hypothalamic–pituitary–adrenal (HPA) axis (Harno and White, 2010). 11β‐HSD1 inhibitors decrease the generation of active glucocorticoids in the liver, adipose tissue and other tissues (Moisan et al., 1990; Lakshmi et al., 1991). Our pharmacokinetics study showed that plasma and brain concentrations of ASP3662 were reduced but not eliminated 24 h after administration. There is therefore a concern that continued reduction of active glucocorticoids by ASP3662 in certain tissues may affect the feedback mechanisms of the HPA axis and cause increased levels of active glucocorticoids. However, evidence suggests that inhibition of 11β‐HSD1 only minimally affects the HPA axis and circulating glucocorticoid levels in animals and humans (Harno and White, 2010). Nevertheless, the effect of ASP3662 on the HPA axis should be carefully monitored in toxicity tests in animals and clinical studies.

Existing drugs for neuropathic pain and fibromyalgia, such as pregabalin, frequently cause side effects such as somnolence and dizziness in humans and induce motor coordination deficits in rats at analgesic doses (Tzellos et al., 2010; Ogawa et al., 2012). ASP3662 had no significant effect on performance on the rotarod test at analgesic doses in neuropathic and dysfunctional pain models, suggesting that inhibition of 11β‐HSD1 is not associated with such side effects and would therefore be an attractive approach for treating chronic pain.

In conclusion, ASP3662 is a potent and selective inhibitor of 11β‐HSD1 with CNS‐penetrability and long‐lasting efficacy. ASP3662 exhibits analgesic effects in various animal pain models and does not induce motor coordination dysfunction at analgesic doses. These results suggest that ASP3662 is a promising candidate for use in the treatment of neuropathic and dysfunctional pain, such as that observed in fibromyalgia, with a wide safety margin.

Author contributions

T.K. and S.K. contributed to the conception of the study. T.K., T.S., H.U. and S.K. designed the study. T.K., T.S., M.T. and H.U. acquired the data. T.K. and H.U. contributed to the analysis or interpretation of the data. T.K. drafted the manuscript. T.K., T.S., H.U., M.T. and S.K. revised the content of the manuscript. T.K., T.S., H.U., M.T. and S.K. approved the final version of the manuscript.

Conflict of interest

The authors are all employees of Astellas Inc.

Declaration of transparency and scientific rigour

This http://onlinelibrary.wiley.com/doi/10.1111/bph.13405/abstract acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Acknowledgements

We would like to thank Dr Kayoko Mihara for her valuable comments, and Dr Eiji Yoshimi, Dr Kouhei Inamura, Dr Keisuke Tamaki, Dr Kenichi Kakefuda, Takayasu Gotoh, Hiroko Yamamoto, Yukiko Funatsu, Masanobu Iino and Ritsuko Matsuda for their expert technical assistance. We also thank Koji Ishibashi and Hayato Kaneko for analysis of ASP3662 concentrations and Noriyuki Kawano for the synthesis of ASP3662.

Kiso, T. , Sekizawa, T. , Uchino, H. , Tsukamoto, M. , and Kakimoto, S. (2018) Analgesic effects of ASP3662, a novel 11β‐hydroxysteroid dehydrogenase 1 inhibitor, in rat models of neuropathic and dysfunctional pain. British Journal of Pharmacology, 175: 3784–3796. 10.1111/bph.14448.

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