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REVIEW ARTICLE
NGF/TrkA Signaling as a Therapeutic Target
for Pain
Munetaka Hirose, MD, PhD*; Yoshihiro Kuroda, PhD
†
; Eri Murata, VMD, MS
‡
*Department of Anesthesiology and Pain Medicine, Hyogo College of Medicine, Hyogo;
†
Department of Pharmaceutical Health Care, Faculty of Pharmaceutical Sciences, Himeji
Dokkyo University, Hyogo;
‡
Department of Anesthesiology and Reanimatology, Faculty of
Medical Sciences, University of Fukui, Fukui, Japan
&Abstract: Nerve growth factor (NGF) was first discov-
ered approximately 60 years ago by Rita Levi-Montalcini as
a protein that induces the growth of nerves. It is now
known that NGF is also associated with Alzheimer’s disease
and intractable pain, and hence, it, along with its high-
affinity receptor, tropomyosin receptor kinase (Trk) A, is
considered to be 1 of the new targets for therapies being
developed to treat these diseases. Anti-NGF antibody and
TrkA inhibitors are known drugs that suppress NGF/TrkA
signaling, and many drugs of these classes have been
developed thus far. Interestingly, local anesthetics also
possess TrkA inhibitory effects. This manuscript describes
the development of an analgesic that suppresses NGF/TrkA
signaling, which is anticipated to be 1 of the new methods
to treat intractable pain. &
Key Words: local anesthetic, nerve growth factor, NGF,
pain, tropomyosin receptor kinase, protein kinase, TrkA, anti-
NGF antibody, tanezumab, review
INTRODUCTION
Levi-Montalcini, who continually conducted research on
the growth of nerve fibers, discovered that mouse
sarcomas transplanted into chicken embryos secrete a
factor into the blood which induces sensory and sympa-
thetic nerve growth.
1,2
Furthermore, it was demonstrated
that sympathetic neurons become denatured when an
antiserum against this factor is injected into newborn
mammals.
3
This factor, indispensable for the prenatal
growth of sensory and sympathetic nerves, was named
“nerve growth factor (NGF).” A tissue sample isolated
from a mouse submandibular gland revealed that NGF is
composed of 118 amino acid residue sequences.
4
More-
over, it was discovered that there are 2 NGF receptors, 1
with high affinity and the other with low affinity for
NGF,
5
and these were later named tropomyosin receptor
kinase (Trk) A and p75 neurotrophin receptor
(p75NTR), respectively. Subsequently, aside from
NGF, brain-derived neurotrophic factor (BDNF), neu-
rotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5)
were discovered as other factors involved in nerve
growth. These were collectively termed “neurotrophic
factors.” Thereafter, the receptors for each of these
factors, TrkB and TrkC, which have very similar amino
acid sequences as TrkA, were discovered. TrkB was
shown to be the receptor for BDNF and NT-4/5, while
TrkC was indicated as the receptor for NT-3.
6
Currently, NGF is known not only for its function in
prenatal nerve growth, but also for its significant role in
pain and immune function in adults. This manuscript
Address correspondence and reprint requests to: Munetaka Hirose,
MD, PhD, Department of Anesthesiology and Pain Medicine, Hyogo
College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501,
Japan. E-mail: mhirose@hyo-med.ac.jp.
Conflict of Interests: The authors have no conflict of interests or
financial ties to disclose.
Submitted: February 15, 2015; Revision accepted: June 15, 2015
DOI. 10.1111/papr.12342
©2015 World Institute of Pain, 1530-7085/15/$15.00
Pain Practice, Volume , Issue , 2015 –
describes the development of analgesics that target
NGF/TrkA signaling.
TRKA ACTIVATION BY NGF
TrkA is expressed in various organs and tissues, such as
the peripheral nervous system, central nervous system,
immune tissue, digestive tract, adrenal cortex, prostate,
uterus, kidney, and skin.
6,7
NGF binds to TrkA on the
cell membrane, which possesses tyrosine kinase activity
that phosphorylates tyrosine in amino acid residues. As
shown in Figure 1, the activation loop of TrkA is
wedged in the center of the enzymatic activity site in an
inactivate state, and this prevents the adenosine triphos-
phate (ATP) from entering the site, consequently
suppressing the tyrosine kinase activity.
8
When the
NGF dimer binds to the TrkA dimer,
9
the activation
loop is released from the center of the enzymatic activity
site, following which TrkA autophosphorylates (pY)
tyrosine residues (Y676, Y680, Y681) on the contralat-
eral activation loop with ATP
8
(Figures 1 and 2). This
activated form of TrkA phosphorylates other intracel-
lular matrix proteins, which then trigger the intracellu-
lar signal transduction system of NGF/TrkA signaling,
transmitting signals into the nucleus.
In particular, NGFs that act on the peripheral
nociceptive neuron terminals bind to TrkA on the cell
membrane, which are then taken up by endosomes and
are subsequently transported in a retrograde manner
through the axon to the dorsal root ganglia cell bodies.
There, the downstream intracellular signal transduction
system is activated, producing various types of
proteins.
10
NGF/TRKA SIGNALING AND PAIN
NGFs are involved in pain in 2 distinct ways. The first is
during the fetal period via the growth of nerve fibers that
transmit pain sensations, and the other is via the role
played during adulthood in inducing pain.
Congenital Insensitivity to Pain
When NGF-mediated nerve growth is absent during the
fetal period, insensitivity to pain develops. In 1976,
when it was already known that NGF is essential in the
formation of sensory and sympathetic nerves, the blood
levels of NGF were measured in patients with congenital
insensitivity to pain, who inherently do not sense pain.
However, this study did not investigate the cause of this
disorder.
11
With advances in molecular biology, it was subse-
quently discovered that a genetic mutation in TrkA was
the cause of congenital insensitivity to pain together
with anhidrosis. This is a disorder in which nociceptive
and sympathetic nerves are missing. It is an extremely
rare disorder where patients suffer repetitive injuries
because they do not feel pain, and develop high
temperatures upon exercising due to their inability to
sweat, both caused by defective tyrosine kinase activity
of TrkA.
12,13
In addition, reportedly, in a variant
congenital insensitivity to pain (hereditary sensory and
autonomic neuropathy type 5), where there is insensi-
tivity to pain but normal ability to sweat, a mutation in
the NGF gene
14
leads to a defect in the physiological
actions of prenatal NGF.
NGF-induced Pain in Adulthood
When NGF actions are continuously suppressed by
anti-NGF antibodies from the prenatal through the
neonatal period, the growth of sensory and sympa-
thetic nerves is completely inhibited. However, if NGF
actions are suppressed postnatally, only a portion of
nerve growth is inhibited.
15
This suggests a difference
in physiological actions of NGF between prenatal and
postnatal stages.
Due to its nerve-growing properties, NGF gained
interest in the late 1980s as a candidate target for a
therapeutic agent for central and peripheral nervous
system disorders, and a study that administered NGF
into animals was conducted.
16
This study revealed that
hyperalgesia develops when NGF is administered to
adult rats, following which NGF became known as 1 of
Figure 1. Activation of high-affinity receptor TrkA by NGF.
2HIROSE ET AL.
the chemical substances that induce pain during adult-
hood.
17
In a study that investigated the effects of NGF in
humans, its intravenous administration induced whole-
body muscle pain, and subcutaneous administration at
the same dose induced hyperalgesia of the skin at the
injection site in addition to whole-body muscle pain.
Muscle pain continued for nearly a week, and hyperal-
gesia of the skin persisted for several weeks.
18
Subsequently, the mechanism of action of NGF was
investigated. Physical pain can be divided into 2 types:
nociceptive and neuropathic pain. Nociceptive pain is
defined as “pain that arises from actual or threatened
damage to non-neural tissue and is due to the activation
of nociceptors,” and is generally interpreted as physio-
logical pain.
19
In contrast, neuropathic pain is defined as
“pain caused by a lesion or disease of the somatosensory
nervous system”
19
and is considered to be a pathological
pain that does not arise normally. Both types of pain can
develop into chronic pain that persists for a long period
of time, with treatments for such conditions being
difficult to determine. NGF, interleukins, and tumor
necrosis factor (TNF)-aare secreted by inflammatory
cells in injured tissue and by Schwann cells in damaged
nerves and are involved in both types of pain.
20,21
When
tissue injury occurs, NGF expression at the site of injury
increases.
22,23
NGFs secreted by inflammatory cells act
on TrkA located on the cell membrane of the sensory
nerve endings, phosphorylating other proteins associ-
ated with pain, which induces a conformational change
and increases the expression of these proteins. It is
believed that these in turn elicit peripheral sensitization
in the peripheral nerve and central sensitization in the
spinal cord, inducing the onset of a hypersensitive
reaction (hyperalgesia) in response to pain, together
with allodynia, a painful response to a stimulus that is
not normally painful.
24–26
In addition to neuropathic pain in the arm or leg
ipsilateral to peripheral nerve lesions, mirror image pain
also occurs in the contralateral sites.
27
NGF may be
involved in the mechanisms of mirror image pain
pathogenesis.
28
NGF as a Cause of Pain
It is curious why NGF, an essential protein for the
prenatal growth of sensory and sympathetic nerves,
plays a role in inducing pain postnatally. Although the
exact reason is unknown, it is possible that when an
inflammatory response occurs due to tissue damage,
NGF required for the repair of the injured peripheral
nerve, which occurs simultaneously with this inflamma-
tory response, acts on the surrounding sensory nerves
that are not injured, thereby inducing pain and protect-
ing the injured site. This may, consequently, promote the
repair of injured peripheral nerves.
In conditions in which the inflammatory response
continues and nociceptive pain is constant, such as with
autoimmune diseases, or in a disease state that induces
neuropathic pain, NGF triggers peripheral and central
sensitization, thereby causing hyperalgesia and allody-
nia. In such pathological conditions, an analgesic that
blocks NGF/TrkA signaling would be extremely useful.
NGF/TRKA SIGNALING AND THE DEVELOPMENT
OF ANALGESICS
In modern society, approximately 20 to 30% of adults
suffer from refractory pain such as chronic low back
Figure 2. Amino acid sequences of the
TrkA activation loop and TrkA activity
inhibitor IPTRK3.
TrkA and Pain 3
pain that cannot be suitably alleviated even with the use
of various analgesics. This is a large societal issue as it
leads to a decreased productivity.
29
The development of
novel analgesics is, therefore, desirable. The NGF/TrkA
signaling that is involved in both nociceptive and
neuropathic pain is considered to be 1 of the important
targets for such therapeutic development.
30–32
In
particular, clinical studies on anti-NGF antibody have
already been conducted, and its clinical use as a new
analgesic is anticipated in the future. Additionally, the
development of drugs that block TrkA activity is also
ongoing.
Classification of NGF/TrkA Inhibitors
Drugs that suppress NGF actions are classified into the
following 3 types
30,31
: (1) drugs that capture NGF; (2)
drugs that inhibit the binding of NGF and TrkA; and (3)
drugs that directly inhibit the enzymatic activity of TrkA
(Figure 3). Many NGF/TrkA inhibitors have been
developed in the past, as described below (Table 1).
33–48
Anti-NGF Antibodies. Anti-NGF antibody is the oldest
known NGF/TrkA inhibitor; it captures NGF. Its
efficacy in suppressing nociceptive pain was demon-
strated in the early 1990s,
33
and later, its analgesic
effects were shown in various experimental animal
models of nociceptive and neuropathic pain. Trunk burn
injury in rats is known to induce hyperalgesia at the
bottom of the hind paw in an area outside the region of
burn injury. It was later shown that NGF produced at
the burn site induces hyperalgesia at a distant location
and that this phenomenon can be suppressed by an anti-
NGF antibody.
49
Subsequently, anti-NGF antibody was clinically used
in humans, and a clinical study of tanezumab was
conducted in the USA to investigate its efficacy in
osteoarthritis of the knee and hip.
34
Although a long-
term analgesic effect was achieved with intravenous
administration of the drug every 2 months, the possible
development of side effects, including hyperesthesia,
hypalgesia, and exacerbation of osteoarthritis and
osteonecrosis, was noted and further study was discon-
tinued in 2010. Later, it was revealed that the exacer-
bation of osteoarthritis and osteonecrosis was
associated with the long-term usage combined with
nonsteroidal anti-inflammatory drugs (NSAIDs) and
with high doses of tanezumab.
50
Studies have resumed,
and anti-NGF antibody may eventually gain use as an
analgesic, not only for osteoarthritis, but also for
cancer
51,52
and postoperative pain.
53
TrkA Activity Inhibitors. As the enzymatic activity site
of TrkA is located intracellularly (Figure 1), it is
necessary for TrkA activity inhibitors to have the ability
to enter the cell (Figure 3). For this reason, these drugs
are either small molecules or require the addition of a
peptide that promotes cell membrane permeability (cell-
penetrating peptide).
The first reported cell-penetrating peptide was an
amino acid sequence (Tat: YGRKKRRQRRR) within a
segment of the acquired immune deficiency syndrome
(AIDS) virus protein
54
. One of the enigmatic properties
of this cell-penetrating peptide is that its addition to
large molecules, such as deoxyribonucleic acid (DNA),
oligonucleotides, peptides, and proteins, which cannot
normally cross the cell membrane, enables them to pass
through the cell membrane.
Previously, we created a peptide in which this Tat
peptide is bound to a TrkA activity-suppressing peptide
with a linker (e-aminocaproic acid: acp), to develop a
TrkA activity inhibitor (IPTRK3) (Figure 2).
42
As TrkA
activity-suppressing peptide has an amino acid sequence
that is partially equivalent to that of the TrkA activation
loop, it is thought to suppress TrkA activity by acting as
Figure 3. Site of action of NGF/TrkA signaling inhibitor.
Table 1. NGF/TrkA Signaling Inhibitors
Drugs capturing NGF Anti-NGF antibody (ABT-110, alpha-D11,
AMG403, fulranumab, Medi-578,
muMab911, REGN475, tanezumab)
33–35
TrkA-IgG
36
TrkAd5
37
Inhibitors of
NGF binding to TrkA
Anti-TrkA antibody (MNAC13)
38
ALE0540
39
PD90780
40
TrkA inhibitors ARRY-470
41
, ARRY-872
CT-327, CT-335, CT-340
IPTRK3
42–45
K252a
46,47
NMS-P626
TrkA antisense oligodeoxynucleotide
48
4HIROSE ET AL.
a decoy for the activation loop and embedding itself into
the center of the enzymatic activity site. IPTRK3 has
been reported to inhibit nociceptive pain induced by
Freund’s complete adjuvant in rats,
43
neuropathic pain
generated by partial sciatic nerve ligation in mice,
44
and
cancer pain caused by malignant melanoma inoculation
into mouse hind paws.
45
Subsequently, it was demon-
strated that IPTRK3 also suppresses the activity of
numerous protein kinases (Erk, Janus kinase (JAK), p38,
protein kinase C (PKC)) that are involved in pain
(unpublished data) (Figure 4), and the development of
small molecule TrkA activity inhibitors that more
selectively suppress TrkA activity is presently under-
way.
55
The ability of TrkA activity inhibitors to penetrate
the cell membrane makes it possible for them to be
taken up by the central nervous system. However, the
fact that NGF depletion in the central nervous system
can induce Alzheimer’s disease
56
and that activation of
NGF/TrkA signaling may suppress the onset of
Alzheimer’s disease
57
may be a significant deterrent
to the use of TrkA activity inhibitors. Surprisingly,
TrkA inhibitors have conversely been indicated to be a
potential therapeutic agent for Alzheimer’s disease.
58
Studies investigating the effects of long-term adminis-
tration of TrkA activity inhibitors on the central
nervous system are needed.
LOCAL ANESTHETICS AND NGF/TRKA SIGNALING
Although it is not well known, local anesthetics suppress
NGF/TrkA signaling. In cell culture experiments, where
neurite outgrowth occurred with the addition of NGF
into the petri dish, it was reported that 40 to 50 lMof
lidocaine suppresses this NGF-mediated neurite out-
growth.
59,60
Lidocaine is known to bind to the cytoplasmic linker
between domains III and IV of the sodium channel,
61
and the amino acid sequence of this linker is extremely
similar to the amino acid sequences of the activation
loops of the insulin receptor and of TrkA (Figure 5).
59,62
For this reason, lidocaine at a dose of ≥40 lMinhibits
Figure 4. Inhibitory action of IPTRK3 against the activity of
various protein kinases.
1163
1488
1489
1490
1158
1162
672 682
676 680681
linker of
sodium channel
Activation loop of
TrkA
A
ctivation loop of
insulin receptor
kinase
= autophosphorylation sites = basic amino acid
= acidic amino acid = neutral amino acid
Figure 5. Amino acid sequence
similarities in the sodium channel,
insulin receptor, and TrkA.
TrkA and Pain 5
the tyrosine kinase activity of both insulin receptors and
TrkA.
59,62,63
As lidocaine toxicity occurs at a blood concentration
of 5 lg/mL (20 lM), intravenously administered lido-
caine does not reach a concentration that suppresses
TrkA activity. However, with local injection of lido-
caine (1% lidocaine is equivalent to approximately
40 mM) in nerve blocks, even if the injected dose
becomes less concentrated after diffusion into the nerve
fibers (1% lidocaine after diffusion in the vicinity of the
nerves achieves a concentration of approximately 100 to
400 lM), it is still considered to adequately suppress
TrkA activity. This suggests that local anesthetics used
in nerve blocks not only inhibit sodium channels, but
also potentially suppress TrkA activity. Figure 6 shows
molecular interactions between TrkA and ATP and also
between TrkA and lidocaine using UCSF Chimera
version 1.10.1.
64
Lidocaine probably inhibits tyrosine
kinase activity of TrkA by blocking ATP-binding site of
TrkA and may suppress neuropathic pain or mirror
image pain.
22,23,28
NGF/TRKA SIGNALING AND CANCER PAIN
Activation of NGF/TrkA signaling induces tumor pro-
gression, and either NGF or TrkA is a therapeutic target
against cancer.
65,66
Therefore, NGF/TrkA inhibitors are
expected to be useful for both cancer pain and tumori-
genesis.
45,66
TrkA inhibitor, which decreases proliferation of
melanoma cells, suppresses melanoma-induced cancer
pain in mice.
45
In bone metastasis model of prostate
carcinoma or sarcoma, however, neither anti-NGF
antibodies nor TrkA inhibitors, which suppress cancer
pain in rodents, showed any inhibitory effects on tumor
growth.
41,51,67
Further investigations are needed for
potential therapeutic strategies using NGF/TrkA inhibi-
tors to suppress tumorigenesis in addition to cancer
pain.
CONCLUSION
NGF, a factor that possesses physiological features
indispensable to the growth of sensory and sympathetic
nerves prenatally, becomes a chemical substance that
produces pain postnatally. If tissue injury is associated
with a prolonged inflammatory response or if the
damaged nerve does not regenerate into its original
state, pathological pain ensues. In such situations,
analgesics that suppress NGF/TrKA signaling might be
considered to be effective therapy. For instance, NGF/
TrkA inhibitors could be administered in the perioper-
ative period to prevent refractory chronic postoperative
pain following surgeries that are prone to cause periph-
eral nerve damage, such as thoracotomy (associated
with chronic post-thoracotomy pain syndrome) and
mastectomy (associated with chronic postmastectomy
pain syndrome). Moreover, NGF/TrkA inhibitors are
also candidate therapeutic agents for clinical use in the
treatment of chronic pain caused by osteoarthritis and
cancer pain.
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