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 discovered 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 highaffinity 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, antiNGF antibody, tanezumab, review
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: [email protected]. 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, 1530-7085/16/$15.00 Pain Practice, Volume 16, Issue 2, 2016 175–182
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 sympathetic 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 Moreover, 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), neurotrophin-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
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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 triphosphate (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 contralateral activation loop with ATP8 (Figures 1 and 2). This activated form of TrkA phosphorylates other intracellular matrix proteins, which then trigger the intracellular 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 subsequently 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 insensitivity to pain but normal ability to sweat, a mutation in the NGF gene14 leads to a defect in the physiological actions of prenatal NGF. NGF-induced Pain in Adulthood
Figure 1. Activation of high-affinity receptor TrkA by NGF.
When NGF actions are continuously suppressed by anti-NGF antibodies from the prenatal through the neonatal period, the growth of sensory and sympathetic 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
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Figure 2. Amino acid sequences of the TrkA activation loop and TrkA activity inhibitor IPTRK3.
the chemical substances that induce pain during adulthood.17 In a study that investigated the effects of NGF in humans, its intravenous administration induced wholebody 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 hyperalgesia 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 physiological 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)-a are 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 associated 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 inflammatory response, acts on the surrounding sensory nerves that are not injured, thereby inducing pain and protecting 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 allodynia. 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
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Table 1. NGF/TrkA Signaling Inhibitors Drugs capturing NGF
Figure 3. Site of action of NGF/TrkA signaling inhibitor.
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 types30,31: (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 demonstrated 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 antiNGF 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 longterm analgesic effect was achieved with intravenous
Inhibitors of NGF binding to TrkA
TrkA inhibitors
Anti-NGF antibody (ABT-110, alpha-D11, AMG403, fulranumab, Medi-578, muMab911, REGN475, tanezumab)33–35 TrkA-IgG36 TrkAd537 Anti-TrkA antibody (MNAC13)38 ALE054039 PD9078040 ARRY-47041, ARRY-872 CT-327, CT-335, CT-340 IPTRK342–45 K252a46,47 NMS-P626 TrkA antisense oligodeoxynucleotide48
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 discontinued in 2010. Later, it was revealed that the exacerbation 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 cancer51,52 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 (cellpenetrating 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 protein54. 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
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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 demonstrated 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 underway.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 disease56 and that activation of NGF/TrkA signaling may suppress the onset of Alzheimer’s disease57 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 administration of TrkA activity inhibitors on the central nervous system are needed.
LOCAL ANESTHETICS AND NGF/TRKA SIGNALING
Figure 4. Inhibitory action of IPTRK3 against the activity of various protein kinases.
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 lM of lidocaine suppresses this NGF-mediated neurite outgrowth.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 lM inhibits
linker of sodium channel
1489 1488
Activation loop of insulin receptor kinase Activation loop of TrkA Figure 5. Amino acid sequence similarities in the sodium channel, insulin receptor, and TrkA.
1490
1158
676 672
1162 1163
680
681 682
= autophosphorylation sites
= basic amino acid
= acidic amino acid
= neutral amino acid
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A
B
Figure 6. Potential binding site of lidocaine in TrkA. (A) Interaction between TrkA and ATP. (B) Interaction between TrkA and lidocaine.
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 lidocaine does not reach a concentration that suppresses TrkA activity. However, with local injection of lidocaine (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 progression, 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 tumorigenesis.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 inhibitors 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 perioperative period to prevent refractory chronic postoperative pain following surgeries that are prone to cause peripheral 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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54. Nagahara H, Vocero-Akbani AM, Snyder EL, et al. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat Med. 1998;4:1449–1452. 55. Stachel SJ, Sanders JM, Henze DA, et al. Maximizing diversity from a kinase screen: identification of novel and selective pan-Trk inhibitors for chronic pain. J Med Chem. 2014;57:5800–5816. 56. Calissano P, Matrone C, Amadoro G. Nerve growth factor as a paradigm of neurotrophins related to Alzheimer’s disease. Dev Neurobiol. 2010;70:372–383. 57. Matrone C, Barbagallo APM, La Rossa LR, et al. APP is phosphorylated by TrkA and regulates NGF/TrkA signaling. J Neurosci. 2011;31:11756–11761. 58. Zhang Q, Descamps O, Hart MJ, et al. Paradoxical effect of TrkA inhibition in Alzheimer’s disease Models. J Alzheimers Dis. 2014;40:605–617. 59. Takatori M, Kuroda Y, Hirose M. Local anesthetics suppress nerve growth factor-mediated neurite outgrowth by inhibition of tyrosine kinase activity of TrkA. Anesth Analg. 2006;102:462–467. 60. Onizuka S, Shiraishi S, Tamura R, et al. Lidocaine treatment during synapse reformation periods permanently inhibits NGF-induced excitation in an identified reconstructed synapse of Lymnaea stagnalis. J Anesth. 2012;26:45–53. 61. Kuroda Y, Miyamoto K, Tanaka K, et al. Interactions between local anesthetics and Na+ channel inactivation gate peptides in phosphatidylserine suspensions as studied by 1HNMR spectroscopy. Chem Pharm Bull. 2000;48:1293–1298. 62. Hirose M, Kuroda Y, Sawa S, et al. Suppression of insulin signaling by a synthetic peptide KIFMK suggests the cytoplasmic linker between DIII-S6 and DIV-S1 as a local anaesthetic binding site on the sodium channel. Br J Pharmacol. 2004;142:222–228. 63. Hirose M, Martyn JAJ, Kuroda Y, Marunaka Y, Tanaka Y. Mechanism of suppression of insulin signaling with lignocaine. Br J Pharmacol. 2002;136:76–80. 64. Pettersen EF, Goddard TD, Huang CC, et al. UCSF Chimera-a visualization system for exploratory research and analysis. J Comput Chem. 2004;25:1605–1612. 65. Nakagawa A. Trk receptor tyrosine kinases: a bridge between cancer and neural development. Cancer Lett. 2001;169:107–114. 66. Wang W, Chen J, Guo X. The role of nerve growth factor and its receptors in tumorigenesis and cancer pain. Biosci Trends. 2014;8:68–74. 67. Mantyh WG, Jimenez-Andrade JM, Stake JI, et al. Blockade of nerve sprouting and neuroma formation markedly attenuates the development of late stage cancer pain. Neuroscience. 2010;171:588–598.
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 discovered 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 highaffinity 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, antiNGF antibody, tanezumab, review
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: [email protected]. 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, 1530-7085/16/$15.00 Pain Practice, Volume 16, Issue 2, 2016 175–182
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 sympathetic 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 Moreover, 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), neurotrophin-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
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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 triphosphate (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 contralateral activation loop with ATP8 (Figures 1 and 2). This activated form of TrkA phosphorylates other intracellular matrix proteins, which then trigger the intracellular 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 subsequently 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 insensitivity to pain but normal ability to sweat, a mutation in the NGF gene14 leads to a defect in the physiological actions of prenatal NGF. NGF-induced Pain in Adulthood
Figure 1. Activation of high-affinity receptor TrkA by NGF.
When NGF actions are continuously suppressed by anti-NGF antibodies from the prenatal through the neonatal period, the growth of sensory and sympathetic 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
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Figure 2. Amino acid sequences of the TrkA activation loop and TrkA activity inhibitor IPTRK3.
the chemical substances that induce pain during adulthood.17 In a study that investigated the effects of NGF in humans, its intravenous administration induced wholebody 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 hyperalgesia 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 physiological 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)-a are 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 associated 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 inflammatory response, acts on the surrounding sensory nerves that are not injured, thereby inducing pain and protecting 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 allodynia. 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
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Table 1. NGF/TrkA Signaling Inhibitors Drugs capturing NGF
Figure 3. Site of action of NGF/TrkA signaling inhibitor.
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 types30,31: (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 demonstrated 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 antiNGF 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 longterm analgesic effect was achieved with intravenous
Inhibitors of NGF binding to TrkA
TrkA inhibitors
Anti-NGF antibody (ABT-110, alpha-D11, AMG403, fulranumab, Medi-578, muMab911, REGN475, tanezumab)33–35 TrkA-IgG36 TrkAd537 Anti-TrkA antibody (MNAC13)38 ALE054039 PD9078040 ARRY-47041, ARRY-872 CT-327, CT-335, CT-340 IPTRK342–45 K252a46,47 NMS-P626 TrkA antisense oligodeoxynucleotide48
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 discontinued in 2010. Later, it was revealed that the exacerbation 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 cancer51,52 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 (cellpenetrating 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 protein54. 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
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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 demonstrated 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 underway.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 disease56 and that activation of NGF/TrkA signaling may suppress the onset of Alzheimer’s disease57 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 administration of TrkA activity inhibitors on the central nervous system are needed.
LOCAL ANESTHETICS AND NGF/TRKA SIGNALING
Figure 4. Inhibitory action of IPTRK3 against the activity of various protein kinases.
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 lM of lidocaine suppresses this NGF-mediated neurite outgrowth.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 lM inhibits
linker of sodium channel
1489 1488
Activation loop of insulin receptor kinase Activation loop of TrkA Figure 5. Amino acid sequence similarities in the sodium channel, insulin receptor, and TrkA.
1490
1158
676 672
1162 1163
680
681 682
= autophosphorylation sites
= basic amino acid
= acidic amino acid
= neutral amino acid
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A
B
Figure 6. Potential binding site of lidocaine in TrkA. (A) Interaction between TrkA and ATP. (B) Interaction between TrkA and lidocaine.
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 lidocaine does not reach a concentration that suppresses TrkA activity. However, with local injection of lidocaine (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 progression, 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 tumorigenesis.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 inhibitors 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 perioperative period to prevent refractory chronic postoperative pain following surgeries that are prone to cause peripheral 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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