Sustained blockade of neurotrophin receptors TrkA, TrkB and TrkC reduces non-malignant skeletal pain but not the maintenance of sensory and sympathetic nerve fibers

Abstract

Current therapies for treating skeletal pain have significant limitations as available drugs (non-steroidal anti-inflammatory drugs and opiates) have significant unwanted side effects. Targeting nerve growth factor (NGF) or its cognate receptor tropomysin receptor kinase A (TrkA) has recently become an attractive target for inhibition of adult skeletal pain. Here we explore whether sustained administration of a selective small molecule Trk inhibitor that blocks TrkA, TrkB and TrkC kinase activity with nanomolar affinity reduces skeletal pain while allowing the maintenance of sensory and sympathetic neurons in the adult mouse. Twice-daily administration of a Trk inhibitor was begun 1 day post fracture and within 8 h of acute administration fracture pain-related behaviors were reduced by 50% without significant sedation, weight gain or inhibition of fracture healing. Following administration of the Trk inhibitor for 7 weeks, there was no significant decline in the density of unmyelinated or myelinated sensory nerve fibers, sympathetic nerve fibers, measures of acute thermal pain, acute mechanical pain, or general neuromuscular function. The present results suggest that sustained administration of a peripherally selective TrkA, B and C inhibitor significantly reduces skeletal pain without having any obvious detrimental effects on adult sensory and sympathetic nerve fibers or early fracture healing. As with any potential therapeutic advance, understanding whether the benefits of Trk blockade are associated with any risks or unexpected effects will be required to fully appreciate the patient populations that may benefit from this therapeutic approach.

Figure 1. Major NGF/Trk axis targets to attenuate chronic pain

Current strategies for targeting NGF or its cognate receptor TrkA include; monoclonal antibodies that sequester NGF (1), monoclonal antibodies that targe t TrkA and prevent NGF from binding to TrkA (2), small molecule TrkA antagonist therapy (3) and the focus of the current study, a small molecule kinase inhibitor of Trk's (4). The pan-Trk therapy is a small molecule inhibitor demonstrating nanomolar cellular antagonism of TrkA (6.5nM), TrkB (8.1nM), and TrkC (10.6nM) and a high level of selectivity over a panel of kinase and non-kinase receptors (supplemental material Table S1 and S2). Schematic drawing adapted from Pezet and McMahon.

Figure 2. Chronic administration of a Trk inhibitor significantly reduces bone fracture pain-related behaviors

The time spent guarding (A) and number of spontaneous flinches (B) of the fractured limb over a 2-minute observation period were used as measures of ongoing pain following mid-diaphyseal fracture of the left femur. The Trk inhibitor ARRY-470 (30 mg/kg, p.o.) was administered twice daily, 1 hour prior to behavioral analysis beginning on day 1 post fracture. Note that ARRY-470 significantly reduced both ongoing fracture pain-related behaviors 2, 4, 7, 10, and 14 days following fracture. ARRY-470 (30 mg/kg, p.o.) was administered once at time 0 and again at 4 hours following the initial dose on day 4 post-fracture. The time spent guarding the fractured limb over a 2-minute observation period was used as a measure of ongoing pain 0, 1, 2, 4, 5, 6, 8 and 24 hours after mid-diaphyseal fracture of the left femur (C). Administration of the Trk inhibitor, ARRY-470, (30 mg/kg, p.o.) significantly reduced ongoing fracture pain 4, 5, 6, 8 and 24 hours following initial dose and achieved approximately 50% pain reduction by 8 hours following initial dose. Results represent the mean ± SEM using an n≥7 for each experimental group.

Figure 3. Effects of sustained ARRY-470 treatment on calcified callus and bone bridging measures of bone healing are limited

Measures of bone healing were assessed using radiographic analysis of the calcified callus area formed at the site of fracture and bone bridging of the fractured femur. Bone bridging scores were assigned based on the appearance of bridging across the left and right peripheries of the callus (1 point each) and the left and right cortices of the fractured femur (1 point each). Although there was a small but statistically significant increase in callus area in the ARRY-470-treated fracture vs. the vehicle-treated fracture group 14, 17, 21, 35, 42, and 49 days post-fracture (A-F, G), there was no significant difference in the overall fracture site bone bridging score following 48 consecutive days of twice-daily ARRY-470 administration (A-F, H). Results represent the mean ± SEM using an n≥7 for each experimental group.

Figure 4. Sensory and sympathetic nerve fiber density in the skin of the hind paw is maintained following 48 days of sustained administration of the Trk inhibitor

(A-F) Representative confocal images of sensory nerve fibers in hind paw skin sections immunohistochemically labeled with antibodies against PGP 9.5, CGRP or NF200 following 48 consecutive days of ARRY-470 administration (30 mg/kg, p.o., bid). CGRP- labeled nerve fibers were primarily localized in the epidermis and upper dermis, NF200- labeled nerve fibers were localized in the dermis, while PGP 9.5-labeled nerve fibers were localized to the epidermis (epidermal nerve fibers; ENFs), upper and lower dermis, sweat glands, and subepidermal neural plexus (SNP). Note that chronic administration of ARRY-470 in femur fractured mice did not significantly reduce the number of PGP 9.5 (A, B), TH (C, D), CGRP (E, F), or NF200 (G, H) labeled nerve fibers following 48 days of sustained administration as compared to vehicle treated mice. Results represent the mean ± SEM using an n≥6 for each experimental group.

Figure 5. Quantitation of the effects of sustained ARRY-470 treatment on normal hind paw skin nerve fiber density

(A-D) Hind paw skin sections were stained for PGP9.5, CGRP, NF200 sensory or TH sympathetic nerve fibers following 48 days of sustained ARRY-470 administration (30 mg/kg, p.o., bid) and nerve fiber density was quantified using confocal microscope image analysis. Following confocal image acquisition the total length of nerve fibers labeled with each marker in the glabrous skin was determined within a defined densitometric threshold applied to all sections analyzed. Chronic administration of ARRY-470 in non-fractured mice for 48 days did not significantly reduce the number of PGP9.5 (A), CGRP (B), NF200 (C), or TH (D) labeled nerve fibers, as compared to vehicle-treated mice. Results represent the mean ± SEM using an n≥6 for each experimental group.

Figure 6. Sustained ARRY-470 treatment does not influence behavioral responses of the skin to noxious thermal or mechanical stimuli, general neuromuscular function, or total body weight

(A-C) Nociceptive responses were measured using mechanical stimulation (von Frey filament analysis) and thermal stimulation (thermal analgesia analysis). General neuromuscular function was measured using rotorod analysis. Chronic administration of ARRY-470 (30 mg/kg, p.o., bid) in femur-fractured mice for 48 days did not significantly reduce measures of general neuromuscular function (A) the response to mechanical (B) or thermal (C) stimulation, as compared to pin + fracture + vehicle mice. In addition, chronic administration of ARRY-470 in non-fractured mice did not significantly affect total body weight, as compared to pin + fracture + vehicle mice (D). Results represent the mean ± SEM using an n≥3 for each experimental group.