Inhibition of AAK1 Kinase as a Novel Therapeutic Approach to Treat Neuropathic Pain

Abstract

To identify novel targets for neuropathic pain, 3097 mouse knockout lines were tested in acute and persistent pain behavior assays. One of the lines from this screen, which contained a null allele of the adapter protein-2 associated kinase 1 (AAK1) gene, had a normal response in acute pain assays (hot plate, phase I formalin), but a markedly reduced response to persistent pain in phase II formalin. AAK1 knockout mice also failed to develop tactile allodynia following the Chung procedure of spinal nerve ligation (SNL). Based on these findings, potent, small-molecule inhibitors of AAK1 were identified. Studies in mice showed that one such inhibitor, LP-935509, caused a reduced pain response in phase II formalin and reversed fully established pain behavior following the SNL procedure. Further studies showed that the inhibitor also reduced evoked pain responses in the rat chronic constriction injury (CCI) model and the rat streptozotocin model of diabetic peripheral neuropathy. Using a nonbrain-penetrant AAK1 inhibitor and local administration of an AAK1 inhibitor, the relevant pool of AAK1 for antineuropathic action was found to be in the spinal cord. Consistent with these results, AAK1 inhibitors dose-dependently reduced the increased spontaneous neural activity in the spinal cord caused by CCI and blocked the development of windup induced by repeated electrical stimulation of the paw. The mechanism of AAK1 antinociception was further investigated with inhibitors of α2 adrenergic and opioid receptors. These studies showed that α2 adrenergic receptor inhibitors, but not opioid receptor inhibitors, not only prevented AAK1 inhibitor antineuropathic action in behavioral assays, but also blocked the AAK1 inhibitor-induced reduction in spinal neural activity in the rat CCI model. Hence, AAK1 inhibitors are a novel therapeutic approach to neuropathic pain with activity in animal models that is mechanistically linked (behaviorally and electrophysiologically) to α2 adrenergic signaling, a pathway known to be antinociceptive in humans.

Copyright © 2016 The Author(s).

Figures

Fig. 1.

AAK1 knockout mice have an antinociceptive phenotype in the formalin assay and SNL model. (A) Hind paw flinches in formalin phase I and phase II from wild-type (WT) and AAK1 knockout (AAK1 KO) mice (n = 25–56 per group, male and female mice distributed equally between groups). ***P < 0.001 versus WT by two-way repeated measures analysis of variance, followed by Bonferroni’s post hoc test. (B) Distance traveled in open field testing of WT (n = 15) and AAK1 KO mice (n = 20). Male and female mice were used and distributed equally between groups. P = 0.27 unpaired t test. (C) Mechanical allodynia measured using manual von Frey (mVF) fibers after SNL surgery for WT (n = 15) and AAK1 KO mice (n = 22). Male and female mice were used and distributed equally between groups. ***P < 0.001 versus WT by two-way repeated measures analysis of variance, followed by Bonferroni’s post hoc test.

Fig. 2.

Chemical structure of AAK1 inhibitors. (A) LP-935509. (B) BMT-090605. (C) LP-922761. (D) BMT-124110 (E) LP-927443. (F) BMS-901715.

Fig. 3.

AAK1 inhibitor LP-935509 recapitulates the knockout phenotype in mice. (A) Mouse phase II formalin responses after oral vehicle, gabapentin (GBP) (200 mg/kg), or LP-935509 (10, 30, or 60 mg/kg) (n = 8–10 male mice per group). **P < 0.01 versus Veh by one-way analysis of variance, followed by Dunnett’s t test. (B) Mechanical allodynia over time postdose [doses as in (A)] in mice with SNL injury after oral vehicle, GBP, or LP-935509 (n = 9–13 male mice per group). ***P < 0.001 versus vehicle by repeated measures analysis of variance, followed by Dunnett’s t test. (C) Open field locomotor activity of mice at 30 minutes after oral [doses as in (A)] vehicle, GBP, or LP-935509 (n = 7 male mice per group). P = 0.27 by one-way analysis of variance.

Fig. 4.

AAK1 inhibitor LP-935509 efficacy in SNL model follows plasma exposure. Mouse SNL mechanical allodynia responses after oral vehicle, gabapentin (GBP) (200 mg/kg), or LP-935509 (3, 10, or 30 mg/kg) (n = 4–7 male mice per group). *P < 0.05 and ***P < 0.001 versus vehicle by repeated measures analysis of variance, followed by Dunnett’s t test.

Fig. 5.

AAK1 inhibitor LP-935509 efficacy in SNL model does not change with repeat dosing. Mouse SNL mechanical allodynia responses with twice-daily dosing over 5 consecutive days with oral vehicle, gabapentin (200 mg/kg), or LP-935509 (3, 10, or 30 mg/kg) (n = 4–7 male mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001 versus vehicle by repeated measures analysis of variance, followed by Dunnett’s t test.

Fig. 6.

AAK1 inhibitor LP-935509 is antinociceptive in multiple rat models of neuropathic pain, but not acute pain (A–D). CCI-operated rats were tested at 3 hours after oral vehicle (0), LP-935509 (at indicated doses), or gabapentin (GBP; 100 mg/kg) in assays of (A) thermal hyperalgesia, (B) cold allodynia, (C) mechanical allodynia using Manual von Frey, or (D) mechanical hyperalgesia (n = 7–8 male rats per group). For comparison, sham-operated animals (Sham) are included. (E) Naive rats were testing in the accelerating rotarod assay, dosed as in Fig. 4, (n = 5–10 male rats per group). (F) STZ-injured rats were tested for mechanical allodynia, dosed as in Fig. 4, (n = 7–8 male rats per group). For comparison, sham-injected animals (Sham) are included. (G and H) Naive rats were tested in assays of acute pain, including the following: (G) hot plate and (H) tail flick at 1.5 hours after LP-935509 (30 mg/kg, Per Os po) or 0.5 hour after morphine (5 mg/kg, s.c.) (n = 7–8 male rats per group). ***P < 0.001 versus vehicle, or †††P < 0.001 versus sham controls by one-way analysis of variance, followed by Bonferroni’s post hoc test; ###P < 0.001 versus vehicle by paired t test.

Fig. 7.

AAK1 inhibitors cause antinociception by inhibiting AAK1 in the spinal cord. (A) Mice with SNL injury were tested for thermal hyperalgesia 2 hours after oral vehicle, LP-922761 (poorly brain-penetrant AAK1 inhibitor), or gabapentin (GBP) at indicated doses (n = 7–8 male rats per group). (B) Rats with CCI surgery were tested for thermal hyperalgesia 15 minutes after intrathecal vehicle (0), BMT-090605 (0.3–3 µg/rat), or clonidine (Clon, 3 µg) (n = 7–8 male rats per group). For comparison, sham-operated animals (Sham) are included. **P < 0.01 ***P < 0.001 versus vehicle/0, †††P < 0.001 versus Sham by one-way analysis of variance, followed by Bonferroni’s post hoc test.

Fig. 8.

AAK1 inhibitors decrease neural activity in pain-related circuits. (A) CCI rat spontaneous activity over time of a single spinal dorsal horn neuron showing inhibition by LP-935509 (1 mg/kg, i.v.). Arrow indicates time of LP-935509 injection (inset shows response to vehicle). (B) Dose-dependent inhibition by LP-935509 (0.3–3 mg/kg, i.v.) on spontaneous activity of spinal dorsal horn neurons in CCI male rats (responders, n = 3/4 at 1 mg/kg, n = 4/6 at 3 mg/kg). *P < 0.05 versus vehicle (0) by one-way analysis of variance, followed by Dunnett’s t test. (C) Absence of effect of thoracic T8–T10 transection on LP-935509 (1 mg/kg, i.v.) inhibition of spontaneous activity over time in a single spinal dorsal horn neuron of a CCI male rat (arrow indicates time of injection). (D) Effect of LP-935509 (1 mg/kg, i.v.) on normalized spontaneous activity of CCI male rats (n = 4) with thoracic T8–10 transection. *P < 0.05 versus baseline by unpaired t test. (E) Naive wide-dynamic range neuron spikes evoked by hind limb repetitive electrical stimulation (16 pulses, 2 ms, 2.0 mA) with effect of systemic vehicle or LP-935509 (1–3 mg/kg, i.v.) across pulses (n = 4–8 male rats per group). (F) Systemic inhibition by LP-935509 (0.3–3 mg/kg, i.v.) of electrically evoked windup in spinal wide-dynamic range neurons from naive male rats (responders, n = 5/8 at 1 mg/kg, n = 4/4 at 3 mg/kg). *P < 0.05 versus vehicle (0) by one-way analysis of variance, followed by Dunnett's t test. (G) Naive rat spinal wide-dynamic range neuron spikes across pulses (as in Fig. 6E) with effect of microiontophoretically-applied BMT-124110 (30 mM, pH 4, +10–30 nA, 3 minutes) (n = 10 male rats per group). (H) Spinal inhibition by BMT-124110 (as in Fig. 6G) of electrically evoked windup in dorsal horn wide-dynamic range neurons from naive male rats (responders, n = 10/14). *P < 0.05 by unpaired t test.

Fig. 9.

α2 adrenergic antagonist blocks AAK1 inhibition–induced antinociception in CCI and STZ rats. (A) STZ-treated rats were tested for mechanical allodynia using electronic von Frey 1.5 hours after vehicle (per os), LP-935509 (30 mg/kg, per os), or morphine (3 mg/kg, s.c.) delivered in the presence (+) or absence (−) of naloxone (1 mg/kg, s.c.) given 30 minutes prior to the other agents (n = 8 male rats per group). For comparison, sham-treated (CTL) animals are included. ***P < 0.001 versus vehicle by one-way analysis of variance, followed by Bonferroni’s post hoc test. (B) CCI-operated rats were tested for thermal hyperalgesia 30 minutes after tizanidine (1 mg/kg, i.p.), or 1.5 hours after either vehicle (per os) or LP-935509 (30 mg/kg, po) all delivered in the presence (+) or absence (−) of yohimbine (1 mg/kg, i.p.) given 100 minutes before testing (n = 6–11 male rats per group). CTL as in (A). ***P < 0.001 versus (−) yohimbine pair by one-way analysis of variance, followed by Bonferroni’s post hoc test. (C) STZ-treated rats were tested for mechanical allodynia with CTL and dosing as in (B) (n = 8 male rats per group). ***P < 0.001 versus (−) yohimbine as in (B).

Fig. 10.

α2 adrenergic antagonist blocks AAK1 inhibition–induced reduction in spontaneous neuronal activity in CCI rats. (A) CCI rat spontaneous activity time course of two individual spinal dorsal horn neurons treated with LP-935509 (1 mg/kg, i.v.; filled arrow) without (top trace) or with (bottom trace) pretreatment of yohimbine (0.3 mg/kg, i.v.; open arrow). (B) CCI rat group data for spontaneous activity with design and dosing as in (A) plus baseline and yohimbine controls (n = 6–8 male rats per group). *P < 0.05 versus baseline by one-way analysis of variance, followed by Dunnett's t test.