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Research ArticleNeuropharmacology

Selective Cathepsin S Inhibition with MIV-247 Attenuates Mechanical Allodynia and Enhances the Antiallodynic Effects of Gabapentin and Pregabalin in a Mouse Model of Neuropathic Pain

Ellen Hewitt, Thomas Pitcher, Biljana Rizoska, Karin Tunblad, Ian Henderson, Britt-Louise Sahlberg, Urszula Grabowska, Björn Classon, Charlotte Edenius, Marzia Malcangio and Erik Lindström
Journal of Pharmacology and Experimental Therapeutics September 2016, 358 (3) 387-396; DOI: https://doi.org/10.1124/jpet.116.232926

Abstract

Cathepsin S inhibitors attenuate mechanical allodynia in preclinical neuropathic pain models. The current study evaluated the effects when combining the selective cathepsin S inhibitor MIV-247 with gabapentin or pregabalin in a mouse model of neuropathic pain. Mice were rendered neuropathic by partial sciatic nerve ligation. MIV-247, gabapentin, or pregabalin were administered alone or in combination via oral gavage. Mechanical allodynia was assessed using von Frey hairs. Neurobehavioral side effects were evaluated by assessing beam walking. MIV-247, gabapentin, and pregabalin concentrations in various tissues were measured. Oral administration of MIV-247 (100-200 µmol/kg) dose-dependently attenuated mechanical allodynia by up to approximately 50% reversal when given as a single dose or when given twice daily for 5 days. No behavioral deficits were observed at any dose of MIV-247 tested. Gabapentin (58-350 µmol/kg) and pregabalin (63-377 µmol/kg) also inhibited mechanical allodynia with virtually complete reversal at the highest doses tested. The minimum effective dose of MIV-247 (100 µmol/kg) in combination with the minimum effective dose of pregabalin (75 µmol/kg) or gabapentin (146 µmol/kg) resulted in enhanced antiallodynic efficacy without augmenting side effects. A subeffective dose of MIV-247 (50 µmol/kg) in combination with a subeffective dose of pregabalin (38 µmol/kg) or gabapentin (73 µmol/kg) also resulted in substantial efficacy. Plasma levels of MIV-247, gabapentin, and pregabalin were similar when given in combination as to when given alone. Cathepsin S inhibition with MIV-247 exerts significant antiallodynic efficacy alone, and also enhances the effect of gabapentin and pregabalin without increasing side effects or inducing pharmacokinetic interactions.

Introduction

Neuropathic pain is likely to be multifactorial, and preclinical data from animal models of neuropathic pain suggest that, in addition to spinal neuronal excitability, spinal microglial cells are also activated. Microglia cells are recognized as resident macrophages in the central nervous system and can release neuromodulatory transmitters that influence neuronal firing. Peripheral nerve injury has been shown to increase the density of spinal microglia cells and alter microglial gene expression (McMahon and Malcangio, 2009). These changes occur in response to the increased peripheral sensory afferent input into the dorsal horn (Hathway et al., 2009; Suter et al., 2009). Changes in microglial density and phenotype correlate with tactile allodynic behavior in rats subjected to partial sciatic nerve injury (Coyle, 1998) or spared nerve injury (Suter et al., 2009) and in mice subjected to partial sciatic nerve ligation (PNL) (Staniland et al., 2010). Taken together, the data suggest that inhibiting microglial activation may be beneficial in the treatment of neuropathic pain.

The cysteine protease cathepsin S plays a role in maintaining microglia activity in pain states (Clark and Malcangio, 2012). Cathepsin S is expressed by spinal microglia and becomes more abundant in response to nerve injury since the number of microglia increases (Clark et al., 2007). Cathepsin S is released from microglia in response to nociceptive neurotransmitters such as ATP (Clark et al., 2010), and inhibitors of cathepsin S reverse mechanical allodynia and hyperalgesia in rodent models of neuropathic pain (Clark et al., 2007; Irie et al., 2008). The proposed target substrate for cathepsin S in the spinal cord is neuronal fractalkine. Dorsal horn preparations ex vivo show that the liberation of soluble fractalkine is dependent upon cathepsin S (Clark et al., 2009), and mechanical allodynia evoked by spinal administration of exogenous cathepsin S is abolished in mice lacking the fractalkine CX3CR1 receptor (Clark et al., 2007).

Gabapentin and pregabalin exert analgesic effects in neuropathic pain states in humans, such as postherpetic neuralgia (Stacey and Glanzman, 2003; Gore et al., 2007) and diabetic neuropathy (Backonja et al., 1998). The effects of gabapentin and pregabalin do not appear to be mediated by GABA receptors, but rather via the inhibition of calcium currents mediated by calcium channels containing the α2δ-1 subunit (Fink et al., 2002; Sutton et al., 2002). Blockade of these channels leads to reduced release of pronociceptive neurotransmitters and attenuation of postsynaptic excitability in the spinal dorsal horn (Fehrenbacher et al., 2003; Takasusuki and Yaksh, 2011).

Despite the efficacy displayed on clinical conditions, gabapentin and pregabalin have several drawbacks. Common side effects of treatment with gabapentin and pregabalin include dizziness and sedation (Dworkin et al., 2010), and treatment with gabapentin and pregabalin provide a substantial benefit in only a minority of patients (Moore et al., 2011). Since neuropathic pain is multifactorial because of multiple pathways, enhanced pain relief is more likely to be achieved by combining compounds that affect pain transmission via different mechanisms of action (Raffa et al., 2010).

MIV-247 is a selective, orally active inhibitor of cathepsin S that is designed for the treatment of neuropathic pain. MIV-247 data have previously been presented in abstract form at the 15th World Congress on Pain in 2014 (Buenos Aires, Argentina) and the 5th International Congress on Neuropathic Pain 2015 (Nice, France). The current study characterizes MIV-247 and evaluates the effect of combining MIV-247 with gabapentin or pregabalin in a mouse model of mechanical allodynia evoked by peripheral nerve injury.

Materials and Methods

Enzyme Assays.

Cathepsin S from all species and cathepsin K were recombinant human enzymes expressed in Baculovirus, purified and activated in-house. Purified human cathepsin L, trypsin, chymotrypsin, and human neutrophil elastase were obtained from Calbiochem (San Diego, CA). Purified human cathepsin B and H were obtained from Athens Research Technology (Athens, GA). Purified human cathepsin V was obtained from R&D Systems (Minneapolis, MN).

For cathepsins S and V, the substrate used was boc-Val-Leu-Lys-AMC; for nonrodent cathepsin K, the substrate was H-D-Ala-Leu-AMC; for mouse cathepsin K, the substrate was Z-Leu-Arg-AMC; for cathepsin L, the substrate was H-D-Val-Leu-Lys-AMC; for cathepsin B, the substrate was Z-Arg-Arg-AMC; for cathepsin H, the substrate was H-Arg-AMC; for trypsin, the substrate was Boc-Gln-Gly-Arg-AMC; for chymotrypsin, the substrate was Succ-Ala-Ala-Pro-Phe-AMC; and for human neutrophil elastase, the substrate was Succ-Ala-Ala-Pro-Val-AMC. All substrates were from Bachem (Bubendorf, Switzerland).

For cathepsin S, the buffer was 100 mmol/l sodium phosphate, 100 mmol/l NaCl, 1 mmol/l dithiotreitol (DTT), and 0.1% polyethylene glycol (PEG) 4000, pH 6.5. For cathepsin K, the buffer was 100 mmol/l sodium phosphate, 5 mmol/l EDTA, 1 mmol/l DTT, and 0.1% PEG 4000, pH 6.5. For cathepsin L, the buffer was 100 mmol/l sodium acetate, 1 mmol/l EDTA, 1 mmol/l DTT, and 0.1% PEG 4000, pH 5.5. For cathepsin B, the buffer was 50 mmol/l sodium phosphate, and 1 mmol/l EDTA, pH 6.25. For cathepsin H, the buffer was 100 mmol/l Tris-acetate, and 1% PEG4000, pH 7.5. For cathepsin V, the buffer was 25 mmol/l sodium acetate and 2.5 mmol/l EDTA, pH 5.5. For trypsin, chymotrypsin, and human neutrophil elastase, the buffer was 50 mmol/l HEPES, 100 mmol/l NaCl, 10 mmol/l CaCl2, and 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, pH 8.0.

Assays were carried out in white polystyrene 96-well plates in a final volume of 100 µl. Substrate concentrations were 10–100 µmol/l, and enzyme concentrations were 0.1–5 nmol/l. Compounds were added in DMSO in the range 1 nmol/l to 100 µmol/l at a final DMSO concentration of 1%.

Plates were read in a Fluoroskan Ascent (Thermo Labsystems, Helsinki, Finland) in kinetic mode, with excitation and emission filters of 390 and 460 nm, respectively. Rates were determined by linear regression of the fluorescence/time data in Excel (Microsoft, Redmond, WA). Rates were fitted by nonlinear regression to either the competitive inhibition equation, with the substrate concentration fixed at the value in the assay and the Km fixed to the value previously determined, or the IC50 equation using Prism software (version 6; GraphPad Software, La Jolla, CA) to obtain dissociation constant (Ki) or IC50 values, respectively.

In one set of experiments, the reversibility of MIV-247 binding to human recombinant cathepsin S was evaluated using the dilution method. Cathepsin S activity (20 nmol/l) was evaluated in the presence of 200 nmol/l MIV-247 (full inhibition expected). A 100-fold dilution of 20 nmol/l cathepsin S and 200 nmol/l MIV-247 into a cathepsin S enzyme assay was performed and the steady state was measured. This was compared with the steady-state rate by adding 0.2 nmol/l cathepsin S to an assay containing 2 nmol/l MIV-247. A comparable steady-state rate in the dilution experiments would indicate full reversibility.

Selectivity.

The selectivity of MIV-247 was also tested against a panel of 273 receptors, ion channels, transporters, and enzymes at Eurofins PanLabs (Taipei, Taiwan). MIV-247 was evaluated at a concentration of 10 µmol/l. Responses were defined as significant if more than 50% inhibition occurred.

Animals.

All experiments were carried out in accordance with the UK Home Office Regulations (Animal Scientific Procedures Act, 1986). Male C57BL/6 mice (25-30 g), obtained from Harlan UK (Bicester, UK), were used in partial nerve ligation and beam-walking experiments. The animals were housed in groups of five under a 12-hour light/dark cycle (lights on at 7:00 AM) with food and water provided ad libitum. Animals were allowed to habituate for at least 5 days prior to surgery or experiments.

Male C57BL/6 mice (20-30 g), obtained from Taconic (Ejby, Denmark), were used in the pharmacokinetic (PK) studies. The animals were housed (maximum of five animals/cage) with food and water provided ad libitum. Fluorescent lighting with a 12-hour light/dark cycle was used, and the animals were allowed to habituate for at least 5 days prior to the experiments.

Surgery—Partial Nerve Ligation.

Mice were anesthetized with an isoflurane 2%/O2 mixture maintained during surgery via a nose cone. After surgical preparation, the common sciatic nerve was exposed at the middle of the thigh by blunt dissection through biceps femoris. Proximal to the sciatic trifurcation, about 7 mm of nerve was freed of adhering tissue, and one-third to one-half of the dorsal aspect of the nerve was ligated (5-0 silk) (according to Seltzer et al., 1990, as adapted to mice by Malmberg and Basbaum, 1998), then the incision was closed in layers.

Assessment of Mechanical Withdrawal Thresholds.

Mechanical thresholds of the ipsilateral (left) paw and contralateral (right) paw were assessed before surgery, and on days 3, 5, 7, and 10 after surgery to ascertain the development of mechanical allodynia (assessed as the difference in thresholds between the contralateral and ipsilateral paws). Animals were transferred to the experimental room on the test day and allowed to acclimatize in acrylic cubicles (8 × 5 × 10 cm) atop a wire mesh grid for up to 60 minutes prior to testing. Static mechanical withdrawal thresholds were assessed by applying calibrated von Frey hairs (flexible nylon fibers of increasing diameter that exert defined levels of force; Touch Test; Stoelting, Kiel, WI) to the plantar surface of the hind paw until the fiber bent. The hairs were held in place for 3 seconds or until the paw was withdrawn, the latter defining a positive response. Starting with a stimulus strength of 0.07 g, hairs were applied according to the “up-down” method within a range of 0.008–1.0 g, and from this 50% paw withdrawal thresholds (PWTs) were calculated (Dixon, 1980; Chaplan et al., 1994). All experiments were carried out by an observer blinded to drug treatments.

Animals were considered allodynic when they displayed a response of 0.1 g or less, normal responses are from 0.6 to 1 g. From day 11 after surgery, approximately 80% of the mice developed allodynia and were randomized into groups. PWTs were assessed before and at 1, 3, and 6 hours after dosing of the compound or vehicle.

The raw 50% PWT data were used for statistical analysis (see below). For simplicity and comparison, the effects of compounds were expressed as the percentage of reversal using the following formula:

Embedded Image(1)

Beam Walking.

The risk of pregabalin, gabapentin, or MIV-247 exerting possible dizziness or ataxia behavior was assessed in normal (noninjured) C57BL/6 mice using a beam-walking test (Stanley et al., 2005). The beam-walking apparatus consisted of a 1.5-m-long wooden beam that was elevated 1 m above the floor, and the test was conducted in a light-attenuated room. A switch-activated source of bright light (60-W tungsten bulb) was located at the start end of the beam and served as avoidance stimuli (approximately 520 lux), whereas a dark box at the other end represented a goal box to reach (approximately 18 lux). Animals were acclimatized to the beam for 5 days with two trials conducted per day. On the test day, animals that crossed the beam in less than 10 seconds, and with no more than one foot slip were selected. The animals were then randomly divided into subgroups. Animals were assessed before and at 1, 3, and 6 hours after dosing. A maximum score of 20 seconds and 10 foot slips was assigned to those mice that did not cross the beam or fell off it.

PK Data.

PK data were generated in two separate studies where C57BL/6 male mice were administered MIV-247, pregabalin, gabapentin, a combination of MIV-247 and pregabalin, or a combination of MIV-247 and gabapentin. Three mice were included in each group, and the doses were 100, 75, and 146 µmol/kg for MIV-247, pregabalin, and gabapentin, respectively. The dose volume was 5 ml/kg for each compound.

Compounds.

MIV-247 was synthesized by Medivir AB (Huddinge, Sweden), formulated in 20% hydroxypropyl-β-cyclodextrin in water, and administered via oral gavage at a dose volume of 5 ml/kg at doses up to 200 µmol/kg. The molecular structure of MIV-247 is shown in Fig. 1. The synthesis description of MIV-247 can be found in the Supplemental Material.

Fig. 1.
Fig. 1.

Molecular structure of MIV-247.

Gabapentin was purchased from Toronto Research Chemicals Inc (Toronto, Canada), dissolved in distilled water, and administered via oral gavage at a dose volume of 5 ml/kg at doses up to 584 µmol/kg.

Pregabalin was purchased from Tocris Bioscience (Bristol, UK), dissolved in distilled water, and administered via oral gavage at a dose volume of 5 ml/kg at doses up to 377 µmol/kg.

Measurement of MIV-247, Gabapentin, and Pregabalin Concentrations.

In some cases, blood was withdrawn from euthanized neuropathic mice immediately after the experiments (i.e., ∼6 hours postdose) by heart puncture into prechilled heparinized tubes. Blood samples were immediately put on ice prior to centrifugation. Plasma was prepared by centrifugation for 10 minutes at approximately 1000g at +4°C within 10 minutes after sampling. Plasma samples were stored at −20°C prior to analysis. Brain and spinal cord were dissected and snap frozen in liquid nitrogen, and stored at −20°C prior to analysis.

In the PK studies, seven blood samples (∼20 µl) were drawn from the lateral saphenous vein of each mouse at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, and 7 hours postdose. The blood was collected into lithium heparin-coated tubes, placed on wet ice, and protected from light prior to centrifugation (3600 rpm; rotor A-4-44; Eppendorf, Mt. Laurel, NJ) at approximately 4°C within 30 minutes of collection. Plasma samples were stored at −20°C prior to analysis. In addition, in some PK experiments, brain and spinal cord were dissected out at 3–3.5 hours postdose, snap frozen in liquid nitrogen, and stored at −20°C prior to analysis.

Determination of compound concentrations in plasma and tissue homogenates was performed using liquid chromatography-tandem mass spectrometry. Brain samples were homogenized in 0.9% NaCl in a volume four times the frozen weight using Ultra Turrax (13,500 rpm/min) for 20 seconds. Spinal cord samples were homogenized in 0.9% NaCl in a volume four times the frozen weight using a TissueLyser (50 oscillations/min; Qiagen, Hilden, Germany) for 60 seconds. The homogenized samples were stored at −20°C until further analysis.

Extraction of MIV-247 from brain and spinal cord tissue samples was performed by the addition of four parts of acetonitrile to one part homogenate, with mixing and sonication for 15 minutes. The samples were centrifuged (for 10 minutes at 20,000g and 10°C), and the supernatants were injected into the analytical column (2.1 × 50 mm, 2.6 µm; Kinetex C18; Phenomenex, Torrance, CA). Plasma was deproteinized by adding four parts acetonitrile to one part plasma, mixing, and performing centrifugation (for 10 minutes at 20,000g and 10°C). The supernatants were injected into the analytical column. Mass analysis was carried out using Agilent Technologies (Wilmington, DE) 6460 Triple Quad liquid chromatography-mass spectrometry (LC/MS) with electrospray ionization in a positive mode. MIV-247 was monitored by using the transition from m/z 438 to m/z 418 at a fragmentor voltage of 135 V and collision energy of 5 V. The limit of detection for MIV-247 was 0.025 µmol/l.

Extraction of gabapentin from brain and spinal tissue homogenate was performed by the addition of five parts 70% methanol to 1 part homogenate, sonication of the homogenate for 15 minutes, and centrifugation (for 10 minutes at 20,000g and 10°C). The supernatants were filtered through filter cups (30,000 nominal molecular weight limit; Amicon Ultrafree-MC; Merck KGaA, Darmstadt, Germany) for 20 minutes at 5000g, and the ultrafiltrates were injected into the analytical column (3 × 150 mm, 3.5 µm; Zorbax SB-Phenyl; Agilent Technologies). Plasma was deproteinized by adding one part acetonitrile to one part plasma, mixing, and performing centrifugation (for 10 minutes at 20,000g and 10°C). The supernatants were diluted with two parts of water prior to injection into the analytical column. Mass analysis was carried out using Agilent Technologies 6460 Triple Quad LC/MS with electrospray ionization in a positive mode. Gabapentin was monitored by using the transition from m/z 172 to m/z 154 at a fragmentor voltage of 100 V and a collision energy of 10 V. The limit of detection for gabapentin was 0.025 µmol/l.

Extraction of pregabalin from brain and spinal tissue homogenate was performed by the addition of five parts 70% methanol to one part homogenate, sonication for 15 minutes, and centrifugation (for 10 minutes at 20,000g and 10°C). Plasma was deproteinized by adding 5 parts 70% methanol to one part plasma, mixing, and performing centrifugation (for 10 minutes at 20,000g and 10°C). The remaining supernatants of the tissue and plasma were injected into the analytical column (4.6 × 100 mm; Synergi 4u Polar; Phenomenex). Mass analysis was carried out using Agilent Technologies 6460 Triple Quad LC/MS with electrospray ionization in a positive mode. Pregabalin was monitored using the transition from m/z 160 to m/z 142 at a fragmentor voltage of 80 V and a collision energy of 5 V. The limit of detection for pregabalin was 0.025 µmol/l.

PK Calculations.

The PK data analysis was performed using the software WinNonlin version 5.3 (Pharsight Corporation, Mountain View, CA). The PK data for MIV-247, pregabalin, and gabapentin were analyzed using noncompartmental methodology.

The following PK parameters were reported:

  • The area under the plasma concentration versus time curve from time 0 to time t (AUC0-t) was calculated by the log/linear trapezoidal method. The last sampling time point was 7 hours postdose. AUC0-t was extrapolated to infinity (AUC0-inf) by adding Clast/λz, where Clast is the last measurable plasma concentration and λz is the terminal elimination rate constant.

  • Maximal plasma concentration (Cmax).

  • Time at maximal concentration (Tmax).

  • The terminal half-life (t1/2).

The t1/2 was calculated as In2/λz, where λz is the elimination rate constant. The t1/2 was calculated only if at least three data points could be included in the regression and the r2 for the regression was >0.80.

Statistical Analysis.

Mechanical thresholds in neuropathic pain studies were expressed as 50% PWTs in grams and statistically analyzed by repeated-measure (RM) two-way ANOVA followed by Tukey test or Dunnett’s test. Beam-walking data were expressed as foot slips (number), and were statistically analyzed by RM two-way ANOVA followed by Tukey test. In partial nerve ligation studies, data are given as the mean ± S.E.M., whereas PK data are given as the mean ± S.D., with n reflecting the number of individual mice. In enzyme studies in vitro, Ki values are given as the geometric mean ± confidence interval.

Results

Characterization of MIV-247 Pharmacology In Vitro.

MIV-247 had a mean Ki value of 2.1 nmol/l for human cathepsin S and was highly selective versus other related cathepsins (Table 1). MIV-247 was also a potent inhibitor of mouse and cynomolgus monkey cathepsin S, with Ki values of 4.2 and 7.5 nmol/l, respectively (Table 1), while having moderate potency against mini-pig and dog cathepsin S. In the rat, there is an important difference in the S2 pocket between strains. In humans, cathepsin S residue 137 is a glycine, whereas this residue is a cysteine (G137C) in wild-type Sprague-Dawley and Wistar rat strains commonly used in preclinical research (Irie et al., 2008). This probably explains the 10,000-fold loss in potency on rat cathepsin S (Table 1).

View this table:
TABLE 1

Potency of MIV-247 against various enzymes

The reversibility of MIV-247 binding to human cathepsin S was evaluated. A 100-fold dilution of 20 nmol/l cathepsin S and 200 nmol/l MIV-247 into a cathepsin S enzyme assay resulted in a steady-state rate of 1.16 ∆F/s (change in fluorescence per second), which is close to the steady-state rate of 1.18 ∆F/s obtained by adding 0.2 nmol/l cathepsin S to an assay containing 2 nmol/l MIV-247. Thus, the dilution experiments conclude that the inhibition of cathepsin S by MIV-247 was rapid and reversible.

The selectivity of MIV-247 was also tested against a panel of receptors, ion channels, transporters, and enzymes. No significant responses were noted for MIV-247 against 273 different targets at a concentration of 10 µmol/l (Medivir; data on file).

Taken together, the data generated in vitro suggest that MIV-247 is a potent, selective, and reversible human cathepsin S inhibitor with similar potency against mouse cathepsin S while displaying 10,000 lower potency against rat cathepsin S. Hence, mice were the chosen species for in vivo pharmacology.

Effect of MIV-247 on PNL-Evoked Allodynia.

Single oral dosing of MIV-247 attenuated mechanical allodynia in mice in a dose-dependent manner from 100 to 200 µmol/kg (Fig. 2A). A dose of 100 µmol/kg MIV-247 transiently attenuated mechanical allodynia, whereas a dose of 200 µmol/kg MIV-247 resulted in sustained significant efficacy lasting at least 6 hours. The maximal antiallodynic effect of 200 µmol/kg MIV-247 (52% reversal of allodynia, P < 0.01) was reached at 6 hours postdose. A higher dose of 500 µmol/kg MIV-247 resulted in similar, but not higher, antiallodynic efficacy (data not shown). Contralateral thresholds were not affected by MIV-247 at any dose (Fig. 2B). Repeated oral dosing of MIV-247 (twice daily for 5 days) also attenuated mechanical allodynia in a dose-dependent manner when measured at 3 hours postdose in the morning (Fig. 2C). Significant reversal (ranging between 36% and 49%) was observed compared with vehicle on all days after oral administration of MIV-247 at a dose of 200 µmol/kg. The lower dose of 100 µmol/kg MIV-247 also affected mechanical allodynia, and a significant effect was recorded on day 3 of the study (32% reversal, P < 0.05).

Fig. 2.
Fig. 2.

Effect of oral MIV-247 on mechanical allodynia in the mouse PNL model. Ipsilateral (A) and contralateral (B) PWTs (50% PWT) after single oral dosing and ipsilateral thresholds (C) (50% PWT) after repeat oral dosing twice daily for 5 days. Data are from 3 hours postdose in the morning. Data are reported as the mean ± S.E.M., n = 8 in each group. *P < 0.05, **P < 0.01, ***P < 0.001, RM two-way ANOVA followed by Dunnett’s test.

Effect of Pregabalin and Gabapentin on PNL-Evoked Allodynia.

Single oral dosing of pregabalin and gabapentin attenuated mechanical allodynia in a dose-dependent manner (Fig. 3, A and B). Doses of 188 and 377 µmol/kg (equivalent to 30 and 60 mg/kg) pregabalin resulted in sustained efficacy lasting at least 6 hours (Fig. 3A), but a dose of 63 µmol/kg (10 mg/kg) was without effect. A dose of 350 µmol/kg gabapentin (60 mg/kg) resulted in sustained efficacy, whereas a dose of 175 µmol/kg (30 mg/kg) gabapentin resulted in transient efficacy lasting 1 hour (Fig. 3B). The lowest dose of gabapentin (58 µmol/kg, 10 mg/kg) was without effect. The contralateral thresholds were not affected by pregabalin or gabapentin (data not shown).

Fig. 3.
Fig. 3.

Effect of oral administration of pregabalin and gabapentin on mechanical allodynia in the mouse PNL model. PWTs (50% PWT) after treatment with pregabalin (A) and gabapentin (B). Data are reported as the mean ± S.E.M., n = 8 in each group. *P < 0.05, **P < 0.01, ***P < 0.001, RM two-way ANOVA followed by Dunnett’s test.

Effect of MIV-247 and Pregabalin in Combination on PNL-Evoked Allodynia.

Minimum effective doses of MIV-247 and pregabalin were evaluated alone and in combination (Fig. 4A). MIV-247 (100 µmol/kg) and pregabalin (75 µmol/kg), when given alone, transiently attenuated mechanical allodynia. The effect of MIV-247 was nonsignificant (22% and 29% reversal at 1 hour and 3 hours, respectively), whereas the effect of pregabalin was significant at 1 hour postdose (28% reversal, P < 0.05) but not at 3 hours postdose (32% reversal, P > 0.05). When combining these compounds at the indicated doses, significant and prolonged antiallodynic efficacy was observed with 51%, 81%, and 33% reversal at 1 hour, 3 hours, and 6 hours postdose, respectively.

Fig. 4.
Fig. 4.

Effect of oral administration of MIV-247 and pregabalin alone or in combination on mechanical allodynia in the mouse PNL model. (A) PWTs (50% PWT) in response to 100 µmol/kg MIV-247, 75 µmol/kg pregabalin, or a combination of the two doses. (B) PWTs (50% PWT) in response to 50 µmol/kg MIV-247, 38 µmol/kg pregabalin, or a combination of the two doses. Data are reported as the mean ± S.E.M., n = 8 in each group. *P < 0.05, ***P < 0.001, RM two-way ANOVA followed by Dunnett’s test.

Subeffective doses of MIV-247 and pregabalin were evaluated alone and in combination (Fig. 4B). Neither MIV-247 (50 µmol/kg) nor pregabalin (38 µmol/kg), when given alone, had any effect on mechanical allodynia. When combining these compounds at the indicated doses, significant antiallodynic efficacy with 35% reversal was observed at 3 hours postdose.

Effects of MIV-247 and Gabapentin in Combination on PNL-Evoked Allodynia.

Minimum effective doses of MIV-247 and gabapentin were evaluated alone and in combination (Fig. 5A). MIV-247 (100 µmol/kg) and gabapentin (146 µmol/kg), when given alone, transiently attenuated allodynia 1–3 hours postdose. The effects of MIV-247 were not statistically significantly different from those in controls (22% reversal, P < 0.05), whereas the effect of gabapentin was significant only at the 3-hour postdose time point (34% reversal, P < 0.05). When combining these compounds at the indicated doses, significant antiallodynic efficacy was observed for up to 8 hours postdose with 48%, 85%, 61%, and 35% reversal at 1, 3, 6, and 8 hours postdose, respectively. The effect of the combination had subsided at 24 hours postdose.

Fig. 5.
Fig. 5.

Effect of oral administration of MIV-247 and gabapentin alone or in combination on mechanical allodynia in the mouse PNL model. (A) PWTs (50% PWT) in response to 100 µmol/kg MIV-247, 146 µmol/kg gabapentin, or a combination of the two doses. (B) PWTs (50% PWT) in response to 50 µmol/kg MIV-247, 73 µmol/kg pregabalin, or a combination of the two doses. Data are reported as the mean ± S.E.M., n = 8 in each group. *P < 0.05, **P < 0.01, ***P < 0.001, RM two-way ANOVA followed by Dunnett’s test.

Subeffective doses of MIV-247 and gabapentin were evaluated alone and in combination (Fig. 5B). MIV-247 (50 µmol/kg) and pregabalin (73 µmol/kg) had no significant effects on allodynia when given alone, as expected. When combining these compounds at the indicated doses, significant antiallodynic efficacy with 56%, 68%, and 41% reversal at 1 hour, 3 hours, and 6 hours, respectively, postdose was seen.

Effects of Minimum Effective Doses of MIV-247 and Pregabalin on Beam Walking.

As expected, 584 µmol/kg (100 mg/kg) gabapentin, serving as a positive control, induced a significant increase in the number of foot slips at 1 hour and 3 hours postdose in normal, noninjured mice (Fig. 6A). Minimal effective doses of MIV-247 (100 µmol/kg), pregabalin (75 µmol/kg), or their combination did not affect the number of slips compared with vehicle (Fig. 6A). Hence, the enhanced antiallodynic effects exerted by combining MIV-247 and pregabalin were most likely not associated with behavioral changes consistent with dizziness or ataxia.

Fig. 6.
Fig. 6.

(A) Effect of oral administration of MIV-247 (100 µmol/kg) and pregabalin (75 µmol/kg) alone or in combination on beam walking (foot slips). (B) Effect of MIV-247 (100 µmol/kg) and gabapentin (146 µmol/kg) alone or in combination on beam walking (foot slips). Data are reported as the mean ± S.E.M., n = 10 in each group. ***P < 0.001, RM two-way ANOVA followed by Tukey test. High-dose gabapentin (584 µmol/kg) was used as a positive control.

Effects of Minimum Effective Doses of MIV-247 and Gabapentin on Beam Walking.

As expected, 584 µmol/kg (100 mg/kg) gabapentin, used as a positive control, induced a significant increase in the number of foot slips at 1 hour and 3 hours postdose (Fig. 6B). Minimal effective doses of MIV-247 (100 µmol/kg), gabapentin (146 µmol/kg), or their combination did not affect the number of slips compared with vehicle (Fig. 6B). Hence, the enhanced antiallodynic effects exerted by combining MIV-247 and gabapentin were most likely not associated with behavioral changes consistent with dizziness or ataxia.

Concentrations of MIV-247, Pregabalin and Gabapentin When Given Alone or in Combination.

The plasma concentrations of MIV-247 and pregabalin when given alone or in combination at minimal effective doses in satellite, noninjured mice are shown in Fig. 7A. The corresponding values for MIV-247 and gabapentin are shown in Fig. 7B. The data show that combining MIV-247 with either pregabalin or gabapentin does not alter plasma exposures of either compound when compared with separate administration of the compounds. The PK parameters for these experiments are given in Table 2.

Fig. 7.
Fig. 7.

(A) Plasma levels of pregabalin when given alone (75 µmol/kg, open circles) or when given together with MIV-247 (100 µmol/kg, closed circles); and plasma levels of MIV-247 when given alone (100 µmol/kg, open squares) or when given together with pregabalin (75 µmol/kg, closed squares). (B) Plasma levels of gabapentin when given alone (146 µmol/kg, open circles) or when given together with MIV-247 (100 µmol/kg, closed circles); and plasma levels of MIV-247 when given alone (100 µmol/kg, open squares) or when given together with gabapentin (146 µmol/kg, closed squares). Data are reported as the mean ± S.E.M., n = 3.

View this table:
TABLE 2

PK parameters of MIV-247, pregabalin, and gabapentin

Although not systematically measured in every experiment, the data in hand suggest that the concentrations of the different compounds in the brain and spinal cord did not appear to differ when given either alone or in combination. Table 3 shows plasma, brain, and spinal cord concentrations of MIV-247 and pregabalin at 3.5 hours postdose when given at subeffective doses to satellite mice. Table 4 shows plasma, brain, and spinal cord concentrations of MIV-247 and gabapentin at 3 hours postdose when given at minimum effective doses to satellite mice, and concentrations at 6 hours postdose when given to neuropathic mice. Similar concentrations were present when compounds were given separately or in combination. The ratios between brain and plasma or spinal cord and plasma concentrations were similar for MIV-247, pregabalin, and gabapentin when administered alone or in combination.

View this table:
TABLE 3

Plasma, brain, and spinal cord levels of MIV-247 and pregabalin

View this table:
TABLE 4

Plasma, brain, and spinal cord levels of MIV-247 and gabapentin

Discussion

The current study demonstrates in a mouse model of neuropathic pain that cathepsin S inhibition per se, using the orally active, highly potent and selective inhibitor MIV-247, attenuates mechanical allodynia without any detectable behavioral side effects in mice. Enhanced antiallodynic efficacy was observed when combining MIV-247 with gabapentin or pregabalin without evoking additional side effects and without any detectable PK interactions. Hence, combination of the selective cathepsin S inhibitor MIV-247 with pregabalin or gabapentin represents a means of increasing both efficacy and the therapeutic window.

MIV-247 was slightly more potent than gabapentin at reversing mechanical allodynia, while being slightly less potent than pregabalin when given orally. A dose of 100 µmol/kg MIV-247 was required for a minimum antiallodynic effect versus 146 µmol/kg gabapentin and 75 µmol/kg pregabalin. The rank order of potency was different when comparing the plasma levels required for minimal efficacy. Minimal effective doses of MIV-247 gave rise to plasma levels of 8–12 µmol ⋅ l−1 ⋅ h, whereas the corresponding exposures for gabapentin and pregabalin were 68–69 and 86–92 µmol ⋅ l−1 ⋅ h, respectively. Interestingly, a Cmax of approximately 16 µmol/l gabapentin is reached in humans at 3 hours postdose in response to administration of 300 mg of gabapentin (Chang et al., 2014), whereas a Cmax of approximately 40 µmol/l pregabalin is reached in humans at 1 hour postdose in response to administration of 200 mg of pregabalin (Brodie et al., 2005). This is similar to the plasma levels of gabapentin and pregabalin reached in our study at minimum effective doses (Table 2), suggesting that the exposure of gabapentin in the current study is of clinical relevance. It is thus tempting to speculate that the same degree of efficacy could be reached at lower plasma levels of gabapentin or pregabalin when given together with MIV-247, thereby widening the therapeutic window (see discussion below). However, clinical studies will be needed to prove a role for cathepsin S in neuropathic pain, and the mechanical allodynia endpoint used in the current study is not the only clinical symptom in patients. Assuming that the central nervous compartment is the main site of action, then MIV-247 is even more potent in comparison since approximately 10–20% of MIV-247 enters the central nervous system (measured in brain and spinal cord), whereas approximately 50% of pregabalin/gabapentin reached the CNS. Indeed, Table 4 demonstrates that subeffective threshold doses of MIV-247 gave rise to 0.1–0.2 µmol/l concentrations in the spinal cord and brain, while corresponding levels for pregabalin were 2–3 µmol/l. This superior potency is perhaps not surprising since MIV-247 has a Ki of 4.2 nmol/l against mouse cathepsin S, whereas gabapentin is estimated to have an affinity of approximately 100 nmol/l (depending on splice variant) against the α2δ-1 subunit of voltage-gated calcium channels (Lana et al., 2014).

Although MIV-247 was more potent (based on plasma levels) in this model of neuropathic pain, gabapentin and pregabalin displayed higher maximal efficacy. Gabapentin and pregabalin completely reversed mechanical allodynia, whereas MIV-247 resulted in a maximal 50% reversal, which was not further increased by raising the dose or by repeated dosing twice daily for 5 days. However, the doses of gabapentin and pregabalin required for maximal efficacy were close to doses associated with side effects, which could confound antiallodynic effects. Indeed, in separate beam-walking experiments, 350 µmol/kg gabapentin treatment significantly reduced the time to cross the beam, indicating signs of dizziness (data not shown). By contrast, although 200 µmol/kg MIV-247 reversed allodynia by only 50%, this was achieved without any obvious side effects. MIV-247 has been run in the neurobehavioral Irwin test battery in mouse at up to 1000 µmol/kg without any side effects being noted (Medivir; data on file).

It is unknown why an approximately 50% reversal appears to be the maximal attainable effect in response to cathepsin S inhibition in this model. Interestingly, this is virtually identical to the results from others (Clark et al., 2007; Irie et al., 2008) who have demonstrated that cathepsin S inhibitors produced approximately 50% reversal of mechanical hyperalgesia in a similar neuropathic pain model in rats. The maximal effect in the current study was probably not due to saturable PK limitations since gradually higher doses of MIV-247 did give rise to higher exposures in the central nervous system and plasma without increasing efficacy. Hence, it appears as if at least a part of the developed mechanical allodynia in this model is independent of cathepsin S.

The first set of experiments demonstrated that gabapentin and pregabalin are agents with low potency (high plasma and CNS levels required) and a narrow therapeutic window but with an apparent high maximal efficacy. By contrast, MIV-247 was a highly potent agent with a wide therapeutic window while reaching a half-maximal effect. In addition, the agents most likely have different mechanisms of action, with gabapentin/pregabalin reducing the release of nociceptive transmitters from the central terminals of primary afferent fibers, whereas MIV-247 is presumed to reduce the facilitation of nociceptive transmission indirectly in the dorsal horn by inhibiting microglia activity. Considering these two distinct profiles, it was of interest to combine the two agents.

The magnitude of antiallodynic efficacy was markedly increased when combining minimum effective doses of gabapentin/pregabalin and MIV-247 compared with when either agent was given alone. No significant effects on beam walking were detected in response to the combinations. Since we were able to achieve improved efficacy without detectable side effects, we combined the two agents at lower subeffective doses. Interestingly, despite neither agent having any detectable efficacy when given alone, their combined effect was significant. For the MIV-247/pregabalin combination at subeffective doses, antiallodynic efficacy was significant at 3 hours postdose, whereas the subeffective MIV-247/gabapentin combination resulted in significant efficacy at several time points postdose. Furthermore, the compounds did not seem to achieve their improved efficacy by interfering with the other compounds PK since plasma and CNS exposures after the experiment appeared similar when given in combination and when given alone. Thus, it is highly likely that the improved efficacy is due to positive pharmacological interactions.

When combining two agents in preclinical pharmacological studies as was done herein, a frequently asked question is whether the combined effect is additive or synergistic. Often, investigators will construct so-called isobolograms by plotting dose levels of the two agents on the x- and y-axes, respectively (for review, see Tallarida, 2006). Although the question is interesting, for instance in a clinical study where only a few approved dosages of each compound can be given, we feel it is not relevant whether given oral doses of agents are synergistic or additive in preclinical studies like the current one. In preclinical studies, endless combinations can be given, and the actual given dose is thus not of interest; the question should rather be whether the pharmacological effects per se may cause additive or synergistic effects. For that analysis to be made, one needs to know the actual concentration of test agent reaching the anticipated site of action. Since measuring compound levels in plasma or target tissue is seldom (if ever) measured in preclinical combination studies, a detailed analysis of whether two pharmacological actions are synergistic is beyond the scope of the current study. Instead, we considered that it was more relevant to evaluate whether a given combination enhanced efficacy without enhancing side effects or exposures of the agents. This seems to be achievable by combining agents like pregabalin and gabapentin with MIV-247 in the current study.

The preclinical data generated so far suggest that a major substrate for cathepsin S in the dorsal horn of the spinal cord is the chemokine fractalkine. Cathepsin S liberates soluble fractalkine, and mechanical allodynia evoked by spinal administration of exogenous cathepsin S is abolished in mice lacking the CX3CR1 receptor, which mediates fractalkine signaling (Clark et al., 2007, 2009). However, it is possible that the inhibition of cathepsin S in the periphery may also contribute and increase the effects of pregabalin and gabapentin. Recently, cathepsin S has been shown to activate PAR2 receptors (Reddy et al., 2010; Elmariah et al., 2014; Zhao et al., 2014), and cathepsin S-induced colonic hyperalgesia in the periphery was suggested to be mediated via PAR2 receptors (Cattaruzza et al., 2011). Less is known about PAR2 receptors in the spinal cord, but specific PAR2 receptor agonists cause mechanical and spinal hyperalgesia when administered intrathecally (Alier et al., 2008), possibly via the inhibition of inhibitory GABAergic neurotransmission (Huang et al., 2011).

Given the multimechanistic nature of neuropathic pain, it is unlikely that one agent will be optimally effective clinically. Although many studies have evaluated antinociceptive drugs in combination on pain-related endpoints in preclinical models, few have compared therapeutic interactions together with possible PK and adverse effect interactions. The current study shows that MIV-247 exerts antiallodynic efficacy without any detectable neurobehavioral side effects in a mouse model of neuropathic pain and also enhances the antiallodynic effect of gabapentin and pregabalin without enhancing side effects or compound exposures. Cathepsin S inhibition offers a new mechanism of action for the treatment of neuropathic pain, either alone or in combination with established therapeutics such as gabapentin and pregabalin.

Authorship Contributions

Participated in research design: Hewitt, Pitcher, Rizoska, Tunblad, Malcangio, and Lindström

Conducted experiments: Pitcher, Henderson, and Sahlberg

Contributed new reagents or analytic tools: Grabowska and Classon

Performed data analysis: Pitcher, Tunblad, Henderson, Sahlberg, and Lindström

Wrote or contributed to the writing of the manuscript: Hewitt, Pitcher, Rizoska, Tunblad, Edenius, Malcangio, and Lindström

Footnotes

    • Received February 19, 2016.
    • Accepted June 20, 2016.

  • E.H. and T.P. contributed equally to this work.

  • E.H., B.R., K.T., I.H., B.-L.S., U.G., B.C., C.E., and E.L. were employees at Medivir at the time of the studies. T.P. was sponsored by Medivir. M.M. was a consultant for Medivir.

  • dx.doi.org/10.1124/jpet.116.232926.

  • ↵Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

Abbreviations

ANOVA
analysis of variance
AMC
7-amino-4-methylcoumarin
AUC0-inf
area under the plasma concentration versus time curve from time 0 to time t extrapolated to infinity
AUC0-t
area under the plasma concentration versus time curve from time 0 to time t
Cmax
Maximal plasma concentration
CX3CR1
chemokine (C-X3-C-motif) receptor 1
∆F/s
change in fluorescence per second
DMSO
dimethylsulfoxide
DTT
dithiotreitol
Ki
dissociation constant
λz
elimination rate constant
LC-MS
liquid chromatography-mass spectrometry
PAR
protease activated receptor
PEG
polyethylene glycol
PK
pharmacokinetic
PNL
partial sciatic nerve ligation
PWT
paw withdrawal threshold
RM
repeated-measure
t1/2
half-life
Tmax
Time at maximal concentration

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Research ArticleNeuropharmacology

Antiallodynic Effects in Response to Cathepsin S Inhibition

Ellen Hewitt, Thomas Pitcher, Biljana Rizoska, Karin Tunblad, Ian Henderson, Britt-Louise Sahlberg, Urszula Grabowska, Björn Classon, Charlotte Edenius, Marzia Malcangio and Erik Lindström
Journal of Pharmacology and Experimental Therapeutics September 1, 2016, 358 (3) 387-396; DOI: https://doi.org/10.1124/jpet.116.232926
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