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Review

Voltage-Gated Ion Channels


in the PNS: Novel Therapies
for Neuropathic Pain?
Gareth R. Tibbs,1 David J. Posson,1 and Peter A. Goldstein1,2,*
Neuropathic pain arises from injury to the nervous system. Conditions associated with neuropathic pain are diverse, and lesions and/or pathological changes
in the central nervous system (CNS) or peripheral nervous system (PNS) can
frequently, but not always, be identied. It is difcult to treat, with patients often
on multiple, different classes of medications, all with appreciable adverse side
effect proles. Consequently, there is a pressing need for the development of
new medications. The development of such therapeutics is predicated on a
clear understanding of the relevant molecular and cellular processes that
contribute to the development, and maintenance, of the neuropathic pain state.
One proposed mechanism thought to contribute to the ontogeny of neuropathic
pain is altered expression, trafcking, and functioning of ion channels
expressed by primary sensory neurons. Here, we will focus on three voltagegated ion channel families, CaV, HCN, and NaV, rst reviewing the preclinical
data and then the human data where it exists.
Neuropathic Pain
As dened by the International Association for the Study of Paini [1], pain is an unpleasant
sensory and emotional experience associated with actual or potential tissue damage; chronic
pain is pain that persists beyond the normal tissue healing time (usually 3 months); neuropathic
pain is pain caused by a lesion of, or damage to, the somatosensory nervous system (Figure 1A).
Chronic pain greatly impairs an individual's quality of life, is widely prevalent, and has signicant
cost. In the USA alone, at least 116 million adults suffer from chronic pain; the associated costs
exceed $500 billion/year [2]. Neuropathic pain accounts for 18% of patients with chronic pain.
Despite limited efcacy, opioid pain relievers (OPRs) are routinely prescribed for chronic pain; in
2012, 240 million prescriptions were written for OPRs in the USA (a 33% increase from 2001),
with a direct cost of $8.34 billion, a 110% increase over the same period [3]. Signicant risks
are involved with this therapy, including physical dependence, addiction, and fatal poisoning [4].
In the USA, approximately half of all deaths from drug overdose are attributable to prescription
drugs, with opioid painkillers accounting for the vast majority of these (71%) [5]. However, the
problem of misuse/abuse is worldwide [610].
The bitter irony is that there are no convincing data that oxycodone (the opiate base for
drugs such as Percocet and Oxycontin) has any value in treating patients with neuropathic
pain, including that associated with diabetic neuropathy, postherpetic neuralgia, or other
neuropathic pain conditions [11]. Similarly, there is no compelling evidence of efcacy for
the use of buprenorphine (a thebaine derivative opioid that acts as an agonist at m-opioid
and ORL-1 receptors, whereas it is an antagonist at k- and d-opioid receptors) in treating
neuropathic pain [12].

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Trends
Neuropathic pain results from injury to
the central and/or peripheral nervous
system. Such injury can result in neuronal excitability, the basis of which
includes altered expression, trafcking,
and functioning of ion channels
expressed by primary sensory neurons,
including CaV, HCN, and NaV channels.
Extensive pharmacological data indicate
that it is possible to synthesize novel
chemical entities (NCEs) that selectively
inhibit channel function within each
channel family. In preclinical tests, NCEs
that selectively target CaV, HCN, and
NaV channels have all demonstrated efcacy in various models of neuropathic
pain; replicating those results in humans
has been difcult, but NaV blockers (at
least) show promise.
Improvements in animal and human
testing methodologies are needed to
identify and develop safe and effective
NCEs as antihyperalgesics.

1
Department of Anesthesiology, Weill
Cornell Medical College, New York,
NY, USA
2
Department of Medicine, Weill Cornell
Medical College, New York, NY, USA

*Correspondence:
pag2014@med.cornell.edu
(P.A. Goldstein).

http://dx.doi.org/10.1016/j.tips.2016.05.002
2016 Elsevier Ltd. All rights reserved.

TIPS 1334 No. of Pages 21

Neuropathic pain syndromes represent a heterogeneous collection of conditions that can be


broadly grouped into the following categories: (i) central nervous system (CNS) lesions, (ii)
complex neuropathic disorders, (iii) peripheral nervous system (PNS) focal and multifocal lesions,
and (iv) PNS system generalized polyneuropathies [13]. Within each group, multiple distinct
conditions exist, and the underlying pathways leading to the common phenotypic feature (pain)
are often variable [1416]. Consequently, to cover the entire spectrum of potential neuropathic
pain treatments is beyond the scope of this review. However, commonalities do exist; one such
common pathway is characterized by neuronal hyperexcitability and spontaneous activity [17];
such behavior can arise from several distinct processes, including altered expression, trafcking,
and functioning of ion channels expressed by primary sensory neurons [15]. In turn, the resulting
hyperexcitability may underlie the sensory abnormalities associated with neuropathic pain
(allodynia, hyperalgesia, spontaneous pain). As will be discussed later, changes in ion channel
expression and function at the level of the primary afferent can be seen in distinct animal models
of neuropathic pain, including that induced by peripheral nerve injury, diabetes, and exposure to
chemotherapeutics, all of which are common etiologies of neuropathic pain in the human
population [13]. While many channels contribute to the electrophysiological properties of
neurons (Figure 1B), there is strong preclinical evidence implicating the involvement of three
voltage-gated ion channel families in particularCaV, HCN, and NaVall of which are fundamentally involved in regulating neuronal excitability. Consequently, ion channel-driven hyperexcitability in sensory neurons presents the unifying theme for this review, and we will rst examine the
preclinical data connecting CaV, HCN, and NaV channels to neuropathic pain and then the
human data where it exists.

Potential Therapeutic Targets


CaV Channels
Voltage-gated Ca2+ channels (CaV) are broadly classied into three closely related groups based
on genes coding the obligatory pore-forming / subunit: CaV1, CaV2, and CaV3 [18,19] (Figure 2).
Three genes comprise the CaV3 subfamily (Figure 2A), and these channels give rise to the lowvoltage activated (LVA) T-type current. T-type Ca2+ currents are present in dorsal root ganglia
(DRG) neurons [2023], the basis of which is CaV3.2 and CaV3.3, at least in small- and mediumsized sensory neurons [2426], including those that are IB4 positive [27,28], a carbohydrate
marker for non-peptidergic nociceptors. This expression pattern suggests that CaV3 channels
might contribute to nociceptive sensory processing [29] and, as such, might also hold the
potential for therapeutic targeting.
CaV3 channels were rst shown to contribute to neuropathic pain by Slobodan Todorovic and
colleagues who demonstrated that mibefradil (a T-type CaV channel antagonist [18,30]) could
block the thermal hyperalgesia produced by intradermal injection of either dithiothreitol or
L-cysteine, reducing agents that augment heterologously expressed CaV3.2 channel and T-type
currents in DRG neurons [31]. Subsequent work by Bourinet et al. showed that antisense targeting
of CaV3.2 induced a signicant decrease in the expression of CaV3.2 mRNA and protein expression as well as a large reduction in CaV3.2-dependent T-type currents in nociceptive DRG neurons.
In normal and neuropathic (chronic constriction injury model; CCI) rats, the CaV3.2, but not CaV3.1
or CaV3.3, antisense treatment resulted in major antinociceptive, antihyperalgesic, and antiallodynic effects, suggesting that CaV3.2 plays an important function in sensory transduction in acute
and neuropathic pain states [32]. In the Bennet CCI model, it was shown that T-type current density
increases in response to chronic constriction injury of the sciatic nerve [33], and this observation
was conrmed by Yue and colleagues, who observed an increase in T-current density and
amplitude following spinal nerve ligation; these changes were accompanied by increases in mRNA
levels for both CaV3.2 and CaV3.3 (note that a similar upregulation in CaV3.2, but not CaV3.1 or
CaV3.3, mRNA occurs in a carrageenaninammatory model [34]) as well as a neuropathic
phenotype that could be relieved by mibefradil [35] (and see also [36]).

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(A)

Cortex

Thalamus

Amygdala

Brainstem

Primary
aerent
Dorsal root
ganglion
Spinal cord

(B)

Somatodendric

Distal
dendrites
Excitatory inputs
(EPSPs)

Axonal

Soma
Proximal
Acon potenals
dendrites
Kv4.2
Kv3 throughout dendrite
Kv2.1

Axon
terminals
Nav
Kv1
Cav

Kv1
JXPs

HCN
AIS

HCN

Cav

Inhibitory inputs
(IPSPs)

Nav
KCNQ

Nodes of ranvier
Nav, KCNQ, Kv3.1b

Figure 1. Schematic Displaying General Sites of Origin of Neuropathic Pain. (A) Neuropathic pain arises from injury
to neurons in the peripheral and/or central nervous system. Amplication of signals arising in primary afferents can result
from several distinct processes, including altered expression and trafcking of receptors and ion channels as well as from
changes in ion channel threshold and kinetics. Such changes can lead to increased spontaneous activity (i.e., action
potential ring) and/or cellular responsiveness to sensory stimuli. (B) General localization of voltage-gated ion channels in a
model neuron. In general, NaV channels are found in the axon initial segment (AIS), nodes of Ranvier, and presynaptic
terminals. KV1 is found at the juxtaparanodes (JXPs) in adult myelinated axons and presynaptic terminals. The KV channel
KCNQ is found at the AIS and nodes of Ranvier, and KV3.1b is also found at the nodes of Ranvier. Excitatory and inhibitory
inputs (EPSPs and IPSPsexcitatory and inhibitory postsynaptic potentials; yellow and blue presynaptic nerve terminals,
respectively) from the somatodendritic region spread passively to the AIS, the site of action potential (AP) generation; APs
propagate to the presynaptic axon terminals to activate voltage-gated Ca2+ (CaV2.2) channels, thereby increasing
intracellular Ca2+ levels that trigger vesicle fusion and exocytosis. Hyperpolarization-activated cyclic nucleotide-gated
(HCN) channels have a gradient distribution that increases in density from the soma to the distal dendrites (dark blue
shading); the variable and highly localized distribution of HCN channels allows them to participate in setting resting
membrane potential, input resistance, and dendritic integration of synaptic signals. KV2.1 is found in clusters on the soma
and proximal dendrites (light yellow ovals). KV3 is found throughout the dendrite, while KV4.2 is located more prominently on
distal dendrites (light blue shading). Dendritic CaV channels increase in density toward the proximal dendrites and the soma.
(Image and legend adapted, with permission, from [228].).

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(A)

(C)

40

20

60

80

P/Q
N
R

Cav3.1 (CACNA1G)
Cav3.2 (CACNA1H)
Cav3.3 (CACNA1I)

T
T
T

Extracellular

30 

LVA

Cav2.1 (CACNA1A)
Cav2.2 (CACNA1B)
Cav2.3 (CACNA1E)

100 

170 

HVA

Cav1.1 (CACNA1S)
Cav1.2 (CACNA1C)
Cav1.3 (CACNA1D)
Cav1.4 (CACNA1F)

Current
L
L
L
L

Intracellular

100

Amino acid sequence identy (%)

(B)

(D)

EC loop I

1
Domain: I

II

III

IV

+H N
3

CO2

VSD II

+H N
3

Outside

+H

CO2

CO2

+H

Inside

+H

CO2

VSD I

Pore domain
+ VSD IV

VSD III

Figure 2. CaV Channel Family Tree and Structural Architecture. (A) Sequence similarity of voltage-gated calcium channel /1 subunits. Phylogenetic
representation of the primary sequences of CaV channels. Only the membrane-spanning segments and pore loops (350 amino acids) are compared. Gene names
are shown to the right of the protein name along with the original current descriptors. L-, P/Q-, N-, and R-type currents are classied as high voltage activated (HVA),
whereas T-type currents are low voltage activated (LVA). (B) Subunit structure of CaV channels. Predicted / helices are depicted as cylinders. The lengths of lines
correspond approximately to the lengths of the polypeptide segments represented. (C) Overall architecture of the rabbit CaV1.1 complex. The overall electron microscopy
(EM) density map (left) and structure (right) of the rabbit CaV1.1 complex. The density corresponding to the b subunit was generated from the 6.1 map at a contour level
of 0.0135, and the other regions were from the 4.2 map at a contour level of 0.0435. (D) Ribbon representation (side view) of the CaV3.1 homology structure model with
the ab initio extracellular loop (EC loop 1) connecting IS5IP shown on top (gray, surface representation). Domains IIV (S1S6) are labeled as shown. Extracellular
cysteines are shown as spheres. (A, B, and legends) Modied, with permission, from [18]. (C and legend) Modied, with permission, from [229]. (D and legend) Modied,
with permission, from [230].

In addition to nerve injury-induced neuropathic pain, CaV3 channels contribute to painful diabetic
neuropathy (PDN) [37]. Yusaf et al. demonstrated that streptozocin (STZ)-induced diabetes
increased CaV3.1 mRNA expression in medium- to large-sized DRG neurons and CaV3.3 mRNA
expression in small- to medium-sized cells [38], while Cao et al. demonstrated that STZ
treatment increased CaV3.2 mRNA levels [39]. Consistent with those observations, electrophysiological studies found that STZ treatment increased T-current density and peak amplitude
[27,40], and the pharmacological prole of those currents strongly suggested that the observed
effects resulted from an increase in CaV3.2 channel expression [27]. Using a similar approach as
Bourinet et al., Messinger and colleagues used the STZ diabetic rat model to study the effects of
antisense oligonucleotide suppression of CaV3.2 expression. They found that downregulation
of CaV3.2 channel expression had a profound antihyperalgesic effect in diabetic rats that was

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accompanied by a signicant decrease in T-type Ca2+[9_TD$IF] currents in DRG neurons while glycemic
control with daily insulin injections reversed the observed effects both in vivo and in vitro. These
observations lend further credence to the idea that CaV3.2 channels contribute to the cellular
hyperexcitability underlying the development of PDN [41].
Leptin-decient (ob/ob) mice are genetically predisposed to developing diabetes and PDN
[42,43]. In vitro, small DRG neurons from ob/ob mice demonstrate a developmentally transient
increase in T-type Ca2+ current density, which is accompanied by persistent thermal and
mechanical hypersensitivity in vivo [44]; the hypersensitivity was relieved by the neuroactive
steroid and selective T-channel antagonist, (3b,5/,17b)-17-hydroxyestrane-3-carbonitrile
(ECN) [45]; mice lacking CaV3.2 did not develop STZ-induced thermal or mechanical hypersensitivity and ECN had no effect on paw withdrawal latency [44].
Thus, there is compelling evidence indicating that CaV3 antagonists might have therapeutic
value as antihyperalgesics for the relief of PDN and/or neuropathic pain following nerve injury.
Recent work with distinct molecular classes of CaV3 channel blockers has shown preclinical
promise (Table 1). Other approaches to suppressing T-type currents, and relieving neuropathic pain, include deglycosylation of CaV3.2 channels with neuraminidase [46] and inhibition of CaV3.2 activity by enhancing ubiquitination due to inhibition of the deubiquitinating
enzyme, USP5 [47].

Table 1. Recent Preclinical Trials with CaV3 Channel Blockers


Name

Compound

Species

Model

Ref.

TTA-P2

3,5-Dichloro-N-[1-(2,2dimethyltetrahydro-pyran-4ylmethyl)-4-uoro-piperidin-4ylmethyl]-benzamide

Mouse

Inammatory painintradermal
formalin
Neuropathic streptozocininduced diabetesthermal
sensitivity

[231]

TTA-A2

2-(4-Cyclopropylphenyl)-N((1R)-1-{5-[(2,2,2-triuoroethyl)
oxo]-pyridin-2-yl}ethyl)
acetamide

Mouse

Acute nociceptionmechanical
and thermal sensitivity
Neuropathic painbutyrate
enema-induced colonic
hypersensitivity

[232]

Compound 9

N-Alkylated carbazole-3carboxylic derivative

Mouse

Neuropathic painpartial
sciatic nerve injurymechanical
hypersensitivity
Inammatory painintraplantar
formalin

[233]

ABT-639

4-Chloro-2-uro-N-(2uorophenyl)-5-[(8aR)hexahydropyrrolo[1,2-a]
pyrazin-2(1H)-ylcarbonyl]
benzenesulfonamide

Rat

Neuropathic painspinal nerve


ligation, sciatic nerve chronic
constriction injury, and
vincristine-induced
Monoiodoacetic acid-induced
knee joint pain model
Capsaicin secondary
hypersensitivity

[49]

Compound 7b

2-(4-(3,4-Dichlorophenyl)-5,6dihydropyridin-1(2H)-yl)-N-((3isopropylisoxazol-5yl)methyl)
ethanamine

Rat

Neuropathic painspinal nerve


ligationmechanical and
thermal hypersensitivity

[234]

Compound 10b

2-(4-(3,4-Dichlorophenyl)
piperidin-1-yl)-N-((3isopropylisoxazol-5-yl)methyl)
ethanamine

Rat

Neuropathic painspinal nerve


ligationmechanical and
thermal hypersensitivity

[234]

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But what is true in rodent (or other animal) models is not necessarily true in humans [48]. Thus,
orally administered ABT-639 (which was shown to produce dose-dependent analgesia in a rat
model of knee joint pain and antihyperalgesia in peripheral nerve injury, chemotherapy- and
capsaicin-induced rat neuropathic pain models [49]) failed to suppress C-ber activity [50] or to
improve self-reported pain scores in prospective, randomized, double-blind controlled trials
of subjects with PDN [50,51]. The failure of ABT-639 to demonstrate any meaningful efcacy led
the authors to state that the data [do] not support T-type calcium channel CaV3.2 as a potential
target for treating diabetic neuropathic pain [51]. Based on positive results in a human
experimental pain model [52] obtained with a less selective and more CNS-penetrant T-type
Ca2+[10_TD$IF] blocker (Z944) [53], however, the authors nal conclusion was that targeting of T-type
calcium channel blockers for the treatment of chronic pain should not be discarded without
further investigation.
So where does this apparent contradiction leave us? Is this an issue of potency, selectivity, site of
action, or something else? In the study by Ziegler el al. [51], 100 mg of ABT-639 was orally
administered to patients every 12 h, resulting in weekly mean plasma levels ranging from 1313 to
2932 ng/ml (2.96.4 mM), which were in the range of the Cmax and Ctrough levels previously
reported in humans on the same dose schedule [54]. More important, the reported plasma
concentrations were commensurate with the IC50 values for T-type currents in rat DRG neurons
(8 mM) and heterologously expressed human CaV3.2 channels (2 mM) [49]. Thus, it is unlikely that
insufcient drug plasma levels were the source of the negative results, and whether a more
potent CaV3.2 blocker would have demonstrated efcacy is unclear. An important limitation of
the study, however, was that it was only 6 weeks in duration, which may have been too short to
detect efcacy. If preclinical efcacy was solely a function of ABT-639-mediated suppression
of CaV3.2-driven hyperexcitability in DRG neurons, then comparable efcacy should have been
observed in humans as steady-state plasma levels are observed by day 5 of a twice daily dose
regimen [54]. But as noted earlier, pain, including neuropathic pain, has an important supraspinal, affective component. Thus, inhibition of CaV channels on central neurons might contribute to the antihyperalgesic effect reported for Z944, and such central efcacy certainly would not
preclude an effect on CaV channels present in peripheral neurons. But what about something
else? Changes in connectivity between the default mode and salience (which includes the
prefrontal cortex) networks have been demonstrated in subjects with numerous chronic pain
states [5557]. The maladaptive plasticity seen in chronic pain can be thought of as one type
of learned behavior [58], which is consolidated over time, implying that its erasure, or reversal,
will also require time; how much time is unknown, but if it is proportional to the time required to
learn the behavior, then short-term assessment, as in the study by Ziegler et al., will fail to detect
drug efcacy in the face of long-term acquisition and consolidation. Thus, additional long-term
studies are needed [1_TD$IF]with [12_TD$IF]ABT-639, and other CaV3.2 antagonists, before abandoning them as
potential antihyperalgesics.
The study by Ziegler and colleagues used multiple, validated, pain assessment tools, including
the Neuropathic Pain Symptom Inventory total intensity score, which included questions that
explore both spontaneous and evoked pain symptoms. However, the study could not demonstrate human in vivo target engagement as there are no validated molecular biomarkers for pain,
and it did not utilize functional magnetic resonance imaging (fMRI), which may have been able to
detect early changes in brain circuits involved in pain perception. fMRI has been used to correlate
the effects of subject expectation on the analgesic efcacy of a m-opiate receptor agonist
(remifentanil) with changes in activity in the underlying neural circuitry; based on those results, the
argument was made that an individual's expectation of a drug's effect critically inuences its
therapeutic efcacy and that regulatory brain mechanisms differ as a function of expectancy. . . it
may be necessary to integrate patients beliefs and expectations into drug treatment regimens
alongside traditional considerations in order to optimize treatment outcomes [59]. Whether that

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argument supports Ziegler et al.s nal contention that we should not discard T-type calcium
channel blockers for the treatment of chronic pain without further investigation [51] remains to
be seen. Demonstration of an effect on an imaging signal may be difcult to reconcile with a
patient's report of no relief from a given intervention since pain is by denition a subjective
experience. That caveat notwithstanding, imaging studies are likely to be incorporated into future
clinical trials barring the advent of other validated biomarkers [6062].
It is worth briey mentioning CaV2.2 channels as therapeutic targets for the treatment of
neuropathic pain. mRNA coding for CaV2.2 is present in DRG neurons [63] as is the corresponding high-voltage activated N-type current [6466]. In vitro, CaV2.2-generated currents
are potently inhibited by the Conus snail toxin -conotoxinGVIA [6769], and in acutely
prepared rat spinal cord slices, block of CaV2.2 reduced Ad ber-evoked transmitter release
(presumably due to inhibition of Ca2+ inux into the axon terminal), as well as the magnitude of
depolarization-evoked Ca2+ transients in Lamina I neurons [70]. In vivo, inhibition of CaV2.2
channels by -conotoxinGVIA [71] or synthetic conotoxin derivatives SNX111, SNX159, or
SNX239 [72] was shown to relieve mechanical and cold allodynia in rat models of neuropathic
pain. These, and other results, led to the development, and introduction into clinical practice, of
the synthetic CaV2.2 channel blocker ziconotide (Prialt; Elan Pharmaceuticals, San Diego, CA,
USA) for the treatment of severe chronic pain not relieved by systemic analgesics, adjunctive
therapies, or intrathecal morphine [73]; although effective, ziconotide has limited widespread use
due to the requirement for intrathecal administration coupled with serious neurological and
psychiatric related adverse events. Despite the poor riskbenet prole, efforts have continued
at developing novel chemical entities (NCEs) that target CaV2.2 on the assumption that the
adverse side effects are drug, rather than class, specic. One strategy has been to develop orally
active tetrahydroisoquinoline derivatives that are CaV2.2 selective; such agents do not inhibit
cytochrome P450 activity and have shown antihyperalgesic efcacy in rat spinal nerve ligation
models without overt CNS toxicity, making them plausible candidates for further development
[74,75]. In a break from the highly selective, one target tradition, attempts to develop multitarget
bifunctional molecules with agonist activity for m-opiate receptors and blocking activity for
CaV2.2 have been undertaken [76]; in vivo, such molecules are weakly analgesic, and the
analgesia appears to result from m-opiate receptor activation rather than CaV2.2 inhibition. Given
that multiple pathways contribute to neuropathic pain, the development of bifunctional ligands
certainly has appeal; given the signicant risks associated with opiate analgesics, however,
designing a drug that includes m-opiate receptor agonist activity may not be desirable.
HCN Channels
Hyperpolarization-activated cyclic nucleotide (HCN)-gated ion channels are members of the KV
superfamily that are weakly selective for K+ over Na+ and are activated by membrane hyperpolarization, not depolarization [77,78]. Four mammalian genes have been identied, each
coding for a single channel isoform (HCN14; Figure 3), which are the basis of the neuronal
pacemaker current, Ih [7983]. HCN channels assemble as homotetramers and heterotetramers
with only the HCN2/HCN3 combination disfavored [8488].
Originally described as a current, If, responsible for the phase 4 spontaneous depolarization that
precedes the cardiac action potential [89], Ih (synonymous with If) is now known to regulate
rhythmic and subthreshold excitability throughout the PNS and CNS by controlling the resting
membrane potential, input resistance, and synaptic integration [78,90,91]. HCN14 channels
are variably expressed throughout the nervous system [92,93]. In large and medium diameter
(Ab) cutaneous primary sensory neurons, HCN1 and HCN2 expression dominates while HCN3
is low and HCN4 minimal; in smaller cells (Ad, C), HCN1 is robust in subsets of C-ber
nociceptors (both IB4+ and IB4) and noxious cold-sensing neurons [9496]. Importantly, with
regard to drug development, HCN4 is the predominant HCN isoform expressed in the human

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(A)

Pore domain

(C)

mHCN2 (mHcn2)
mHCN4 (mHcn4)
mHCN1 (mHcn1)
mHCN3 (mHcn3)

HVIH
SPIH
VSD I

25
20
15
10
5
0
Amino acid sequence dierences (%)

VSD III
C-linker
+ CNBD

(B)

(D)

K /Na

G
Y

Outside

4
2 1

6
Inside

S4-S5linker

HN

A
C-linker
C
CNBD

cA
M
P

D
E-F

C
COOH

Figure 3. HCN Channel Family Tree and Structural Architecture. (A) Phylogenetic tree deduced from sequence
analysis for the amino acid sequences for murine (m)HCN14, SPIH (Strongylocentrotus purpuratus), and HVIH (Heliothis
virescens) proteins. (B) HCN channels are tetramers (inset). One monomer is composed of six transmembrane segments
including the voltage sensor (S4) and the pore region formed of S5 and S6 and the intervening loop. The pore region
contains the selectivity lter carrying the GYG motif. The carboxy terminal channel domain is composed of the C-linker and
the cyclic nucleotide-binding domain (CNBD). The C-linker consists of six /-helices, designated A0 to F0 . The CNBD follows
the C-linker domain and consists of four /-helices (AC and P) with a b-roll (incorporating the P-helix, P not shown in the
cartoon) between the A- and B-helices (thick gray line). Human mutations involved in Ih channelopathies are indicated as
gray circles. (C and D) Homology model of human HCN1 based on X-ray crystal structures of K+ channels and the HCN2
cytoplasmic domain, built using the program Modeller [235]. The structural templates were PDB 2R9R [236] (VSDs, green),
3BEH [237] (pore, blue), and 1Q5O [238] (C-linkerCNBD, grey). The modeled S4S5 linker is highlighted (red). Side (C) and
top (D) views were generated in PyMOL. (A and legend) Modied, with permission, from [239]. (B and legend) Modied, with
permission, from [77].

heart [97,98], especially in the sinoatrial node, where the HCN4/HCN1 mRNA ratio is 7 [99] (but
see [100]). HCN2 is present but there is little or no HCN1 or HCN3 [77,78,101]. The expression
prole suggests that therapeutic targeting of Ih in the PNS may be best achieved with antagonists selective for HCN1 and/or HCN3 [101].
Animal models of peripheral nerve injury demonstrate hyperalgesia and allodynia [102,103].
Injured DRG neurons show increased spontaneous rhythmic electrical activity [104106], and
this increased activity is Ih-dependent [107109]. In peripheral nerve injury models of neuropathic pain, there is an increase in HCN expression in sensory neurons that is more pronounced
for HCN1 than for HCN2 [109], and HCN subunit trafcking is altered [108110], with accumulation of channel protein in the axon rather than the soma [109]. Increased expression is
accompanied by increased Ih amplitude [108,111113] and sensory cell hyperexcitability

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TIPS 1334 No. of Pages 21

[108,112114], both of which are inhibited by superfusion with the HCN channel blocker
ZD7288 [107109,111,115]. Spontaneous activity in the sciatic nerve ipsilateral, but not
contralateral, to the injury is Ih-dependent [109], and low-dose intraperitoneal administration
of ZD7288 alleviates mechanical allodynia [108,115,116]. Those results strongly suggest that
HCN channels expressed on sensory neurons are the primary site of action for ZD7288 but
variability in efcacy (possibly due to drug pharmacokinetics) following proximal (perineural) or
distal (intraplantar) injection precludes exact anatomic localization of the site of analgesic activity
of ZD7288 [108,109,116,117].
Increases in HCN expression in sensory neurons are not limited to those seen following direct
nerve injury. In the STZ diabetic rat model, HCN1 and HCN3 channel proteins were overexpressed in A-ber nodose ganglion neurons, while HCN2 and HCN3 channel proteins were
overexpressed in C-ber neurons, and the increase in channel expression was accompanied by
a corresponding increase in Ih in both cell types [118]. Similarly, exposure to the antineoplastics
oxaliplatin [119] or paclitaxel [120], both of which are associated with chemotherapy-induced
neuropathic pain [121,122], increased HCN1 mRNA expression and paclitaxel increased
excitability of lumbar DRG neurons [120].
Ivabradine is a selective HCN channel pore blocker that blocks all HCN isoforms and
native pacemaker currents in the low micromolar range (in vitro, the IC50 of ivabradine for
heterologously expressed HCN channels is 1 to 2.5 mM [123,124], which closely corresponds to the IC50 of 2.8 mM for native If in sinoatrial node cells [125]); it is clinically approved
throughout Europe and Asia (as Procoralan, Coralan, Corlentor, or Coraxan; Servier,
Suresnes, France) and the USA (as Corlanor; Amgen, Thousand Oaks, CA, USA) as a pure
bradycardic agent for the treatment of chronic angina pectoris and heart failure. In agreement
with the molecular, cellular, and behavioral data, ivabradine has been shown to be antihyperalgesic in multiple animal models (inammatory, nerve injury, chemotherapy induced)
of neuropathic pain [119,120,126]. At present, two trials are listed in the EU Clinical Trials
Register databaseii aimed at studying the effect of ivabradine on pain, one in healthy human
volunteers (EudraCT number: 2012-005627-32), and one in individuals with neuropathic
pain (EudraCT number: 2011-003933-32). As the dose required to produce antihyperalgesia
is essentially the same as that required to produce bradycardia (at least in rodents [126]), it
will be interesting to see if drug efcacy is limited by an unacceptable cardiovascular side
effect prole.
With regard to interpretation of the above clinical trials, it is worth commenting on the potency
with which ivabradine works clinically versus its effects on HCN channels. The initial dose
recommendation for ivabradine is 5 mg oral (p.o.) twice daily (BID). In human subjects, the
plasma concentration (Cmax) following a 5 or 10 mg p.o. dose is 9.7 and 16.3 ng/ml,
respectively, and the terminal half-life (t1/2) is 1.9 h [127,128]. In response to a 20 mg p.o.
BID dose, the human plasma concentration is 85  46 ng/ml at the end of a 5 day dose regimen,
and is essentially identical to the plasma concentration obtained with a single 20 mg oral
dose (83  31 ng/ml), suggesting that drug accumulation does not occur in response to
BID dose [129]. The bradycardic effect is dose-dependent [129,130] and can be detected
at dose regimens as low as 2.5 mg p.o. BID [130]. Effect onset is also rapid (occurs following
a single dose) [129] and reaches sustained efcacy at the end of 1 month [131]. That such a dose
regimen is associated with a physiologically detectable and relevant bradycardia is somewhat
surprising as the corresponding molar concentrations for the reported plasma levels are 19,
32, and 167 nM, concentrations that should elicit minimal inhibition of heterologously expressed
HCN channels and native If. If similarly modest suppression of Ih is sufcient to dampen
excitability in DRG neurons, signicant pain relief might be obtainable without the need to
completely suppress HCN function.

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TIPS 1334 No. of Pages 21

Our demonstration that the alkylphenols 2,6-di-tert-butyl-phenol (2,6-DTBP) and 2,6-di-isopropylphenol (propofol) impair HCN1 gating (in contrast to ivabradine, which is a pore blocker)
and ameliorate mechanical allodynia and thermal hyperalgesia [132] is consistent with alkylphenol-mediated suppression of excitability of HCN1/2 rich Ab mechanoreceptors and/or Ad or
C-type nociceptors. As alkylphenol-mediated inhibition of HCN1HCN2 heteromers is comparable to inhibition of HCN1 homomers [133], pharmacological targeting of sensory neuron Ih
channels containing the HCN1 isoform, alone or in combination with HCN2, may provide relief
for a wide range of painful peripheral neuropathies. As HCN4 is insensitive to relevant concentrations of 2,6-DTBP [132], we predict that 2,6-DTBP, or a suitably optimized congener, will
selectively target HCN1 and thus minimize or eliminate drug-induced bradycardia (the primary
cardiac effect of pan-HCN blockers such as ZD7288 and ivabradine [134137]). Furthermore,
unwanted CNS side effects may be avoided by restricting penetration of alkylphenol compounds
across the bloodbrain barrier.
NaV Channels
Voltage-gated sodium (NaV) channels are the basis of the Na+-dependent action potential in
excitable cells; as with CaV and HCN channels, there are multiple obligatory pore-forming
/ subunits (NaV1.11.9), each being a distinct gene product (Figure 4); coexpression with
one of four b subunits results in channels with altered biophysical properties [138]. Three
channelsNaV1.7, NaV1.8, and NaV1.9have garnered the most attention as potential therapeutic targets because gain-of-function mutations in these isoforms have been clearly linked
to neuropathic pain in humans [139142]. Gain-of-function mutations in NaV1.7 have been
identied in patients with inherited erythromelalgia [143147], an autosomal dominant condition characterized by burning pain and erythema of the extremities [148], as well as in small
ber neuropathy (also known as painful neuropathy) [149], which typically consists of
small ber related symptoms, including pain, autonomic dysfunction, loss of pinprick and
thermal sensation, allodynia, or hyperalgesia, but without evidence of large ber dysfunction
(i.e., muscle weakness, loss of light touch, and/or proprioceptive or vibratory sensation,
hyporeexia, or areexia) [150]. There is some evidence that gain-of-function mutations in
NaV1.7 may also contribute to PDN [151], but the mechanistic data supporting such a
contribution are more complicated than just a simple case of increased channel activation
[152]. Gain-of-function mutations in NaV1.8 [153155] and NaV1.9 [156,157] have also been
identied in patients with small ber neuropathy [153156] and familial episodic pain [157],
which is characterized by intense, relapsing pain primarily in the distal lower extremities (but
occasionally in the upper body, mostly in upper extremity joints) appearing late in the day,
diaphoresis, and pain relief with oral anti-inammatory analgesics. The gain-of-function
mutations result in numerous changes in channel gating (e.g., depolarized slow and fast
inactivation, impaired slow inactivation, hyperpolarized activation, enhanced recovery from
fast inactivation, slowed deactivation), the net result being hyperexcitability of DRG neurons
[140]. Interestingly, however, one gain-of-function mutationNaV1.9Leu811Prois associated
with congenital insensitivity to pain in humans, likely due to inactivation of other Na+ and Ca2+
currents contributing to action potential generation in DRG neurons and resultant conduction
blockade [158]; these results highlight the complex interactions underlying electrogenesis in
DRG neurons [159] and serve as a cautionary example against assuming a one-to-one
correlation between channel function and cellular excitability.
If DRG hyperexcitability due to enhanced NaV function is the root cause of select neuropathic
pain states, then it stands to reason that selective NaV antagonists should be antihyperalgesic.
Both synthetic [160164] and naturally occurring [165167] small molecule inhibitors of NaV1.7
have shown promise as antihyperalgesics in animal studies, as has a NaV1.7-specic monoclonal antibody [165]. Despite clearly identied limitations in the preclinical data [168,169], a
number of early stage (Phase I/II) human studies testing NaV1.7 and NaV1.8 blockers as

10

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

TIPS 1334 No. of Pages 21

(A)

(C)
rNav1.1 (SCN1A)
rNav1.2 (SCN2A)
rNav1.3 (SCN3A)
rNav1.7 (SCN9A)
rNav1.4 (SCN4A)
rNav1.6 (SCN8A)
rNav1.5 (SCN5A)
rNav1.8 (SCN10A)
rNav1.9 (SCN11A)

20

25

15

10

Amino acid sequence dierences (%)

(B)

Conducon pore module

(D)
NH3+

Voltage sensor module

+H N
3

Outside
O

CO2

3C

Voltage
sensing
+H N
3

Inside

Inacvaon

CO2

S4-S5 linker

Figure 4. NaV Family Tree and Structural Architecture. (A) Amino acid sequence similarity of rat (r)NaV1.1NaV1.9 / subunits. Gene names are shown to the right of
the protein name. (B) A transmembrane folding diagram of the NaV1.2 channel. Cylinders represent /-helical segments. Bold lines represent the polypeptide chains of
each subunit, with a length approximately proportional to the number of amino acid residues in the brain sodium channel subtypes. The extracellular domains of the b1
and b2 subunits are shown as immunoglobulin-like folds. C, Sites of probable N-linked glycosylation; P in red, sites of demonstrated protein phosphorylation by protein
kinase A (circles) and protein kinase C (diamonds); blue, pore-lining segments; yellow circles, the outer (EEEE) and inner (DEKA) rings of amino residues that form the
tetrodotoxin-binding site and ion selectivity lter; green, S1S4 voltage sensors; h in blue circle, inactivation particle in the inactivation gate loop; blue circles, sites
implicated in forming the inactivation gate receptor. (C) Intramembrane view (left panel) of structural model of NaV1.7 channel transmembrane domains. Domain I, light
blue; Domain II, salmon; Domain III, cyan; Domain IV, lime. Cytosolic view (right panel) of the structural model of NaV1.7 channel transmembrane domains. (D) Side view of
NaVAb channels: voltage-sensing module (green); pore module (purple); S4S5 linker (red). (A and legend) Modied, with permission, from [138]. (B, D, and legend)
Modied, with permission, from [240]. (C and legend) Modied, with permission, from [241].

antihyperalgesics can be identied in publically accessible clinical research databases (Table 2;


[170,171]).
In a Phase II randomized, double-blind, placebo controlled study of 81 subjects with pain
associated with lumbosacral radiculopathy (identier NCT01561027), the NaV1.7 blocker
CNV1014802 was reported in a company (Convergence Pharmaceuticals) press release to
improve Pain IntensityNumerical Rating Scale scores by a small (0.43), but statistically
signicant (P = 0.0265), amount,iii but no other details were provided. A small single-center
study examined the safety, efcacy, tolerability, and pharmacokinetics of topically applied XPF002 (XEN402 8% w/w ointment) in subjects with primary/inherited erythromelalgia (identier
NCT01486446); the small (n = 8; n = 7 in XPF-002 group and n = 1 in placebo group) and
parallel (as opposed to crossover) group study design preclude drawing any denitive conclusions from the posted data on the ClinicalTrials.gov website. A second study using topically
applied XPF-002 in subjects with postherpetic neuralgia has also been completed (identier
NCT01195636); data posted at ClinicalTrials.gov indicate that no effect of study drug as
compared with placebo was observed for the primary endpoint (change in mean daily pain

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

11

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

Name

Compound

Target

Company

Model

Clinical Trial Identiera[6_TD$IF]

Statusb

AZD3161

N-[(3S)-5-(5Methoxypyrazin-2-yl)-3,4dihydro-2H-chromen-3-yl]6-(2,2,2triuoroethoxymethyl)
pyridine-3-carboxamide

NaV1.7

AstraZeneca

UV-C light-induced
sensitization

NCT01240148

Completed
No results posted

CNV1014802
(raxatrigine)

2S,5R-5-[4-[(2Fluorophenyl)methoxy]
phenyl]pyrrolidine-2carboxamide

NaV1.7

Convergence

Trigeminal neuralgia

NCT01540630

Lumbosacral
Radiculopathy

NCT01561027

Completed
No results posted
Completed
See text

4-[2-(3-Amino-1H-pyrazol4-yl)-4-chlorophenoxy]-5chloro-2-uoro-N-4thiazolylbenzenesulfonamide 4methylbenzenesulfonate

NaV1.7

Diabetic peripheral
neuropathy
Inherited erythromelalgia

NCT02215252

(7S)-10 -[[5-(Triuoromethyl)
furan-2-yl]methyl]spiro[6Hfuro[2,3-f][1,3]
benzodioxole-7,30 -indole]20 -one

NaV1.7

Inherited erythromelalgia
Postherpetic neuralgia

NCT01486446

DSP-2230

NaV1.7/1.8

Sumitomo
Dainippon
[8_TD$IF]Pharma

Healthy volunteers with


capsaicin or UV-B lightsensitized skin

ISRCTN80154838

Completed
No results posted

PF-04531083

N-[6-Amino-5-(2-chloro-5methoxyphenyl)-2pyridinyl]-1-methyl-1Hpyrazole-5-carboxamide

NaV1.8

Pzer

Healthy volunteers with UV


light-sensitized skin

NCT01127906

Completed
No results posted

PF-05089771

XEN402
(funapide)

Pzer

Xenon and Teva


Pharmaceuticals

NCT01195636

NCT indicates trials registered at www.clinicaltrials.gov; ISRCT indicates trial registered at www.isrctn.com.
As of 16 March, 2016.

NCT01769274

Completed
No results posted
Completed
No results posted

Completed
see text
Completed
See text

TIPS 1334 No. of Pages 21

12
Table 2. Phase I/II Human Clinical Trials with NaV1.7 or NaV1.8 Channel Blockers

TIPS 1334 No. of Pages 21

score from baseline to week 3, respectively, 0.94 (1.30 to0.58), n = 56, and[14_TD$IF] 0.97 (1.33
to0.61), n = 56). Data for orally administered XEN402 (identier NCT01090622) have been
reported in the peer-reviewed literature; this was from a single-center, multiple-dose, in-patient,
randomized, double-blind, placebo-controlled, crossover trial in subjects (n = 4) with inherited
erythromelalgia, and the authors reported that XEN402 (i) signicantly reduced the ability to
induce pain by heat or exercise, (ii) increased the time to maximal pain induction, and (iii)
signicantly reduced the amount of pain after induction [172]. While by no means conclusive,
those results plausibly demonstrate efcacy and in vivo target engagement in humans, the sine
qua non for proof-of-principle with regard to contemporary drug development.
NaV1.8, too, has received signicant attention with regard to drug development [173]. As with
NaV1.7, both synthetic [174181] and naturally occurring [182184] small molecule NaV1.8selective inhibitors have been described, which possess antihyperalgesic activity in preclinical
models of neuropathic pain. Of those, PF-01247324 [6-amino-N-methyl-5-(2,3,5-trichlorophenyl)pyridine-2-carboxamide] appears particularly promising. PF-01247324 inhibits both
heterologously expressed human NaV1.8 channels and tetrodotoxin-resistant NaV currents
in rat DRG neurons in a state- and frequency-dependent manner, reduces excitability of both
rat and human DRG neurons in vitro, has high (F = 91%) oral bioavailability, and produces
antinociceptive effects in inammatory and peripheral nerve injury neuropathic pain models
[180].
Human trials have been initiated to study the efcacy of NaV1.8 inhibitors as antihyperalgesics,
one with DSP-2230 ([2_TD$IF]Sumitomo[15_TD$IF] Dainippon Pharma, Osaka, Japan) and the other with PF04531083 (Pzer, New York, NY, USA) [170,171]. DSP-2230 has been studied in human control
subjects using capsaicin- and UV-B light-induced cutaneous sensitization but no results have
been reported (identier ISRCTN80154838). Similarly, the study on the effect of PF-04531083
on heat pain in healthy volunteers, also with UV light-sensitized skin, has been completed
(identier NCT01127906) and again no results reported. The structural similarity between PF01247324 and PF-04531083 (both are substituted carboxamide derivatives) suggests that PF04531083 may yet prove to be effective, but one can reasonably infer that the NCT01127906
study results were less than impressive given that the study was closed to subject accrual in
2010 and no further information provided. Additional clinical trials with NaV1.8 blockers are
necessary to determine the efcacy of such antagonism in patients with neuropathic pain
associated with NaV1.8 gain-of-function mutations; if safety and efcacy can be demonstrated
in this patient population, it might then be reasonable to determine whether such agents have
broader indications for use.
As noted earlier, NaV1.9 gain-of-function mutations have been identied in patients with small
ber neuropathy [156] and familial episodic pain [157], and a compelling argument has been
made for NaV1.9 as validated human therapeutic target [142]. No highly selective blocker of
NaV1.9 has been identied thus far [184], and the search for suitable candidates has been
hampered by exceedingly poor heterologous expression in host cells. Using a NaV1.4/1.9
chimeric approach, it has been shown that limited NaV1.9 expression results arise from the
C-terminal structure of the protein [185]. Use of chimeras that preserve the entire NaV1.9
transmembrane domain offers hope for development of inhibitors, although only those that do
not act on the intracellular motifs of the isoform.
Is a drug that is selective for a wild-type channel isoform an a priori condition for clinical efcacy?
The answer is maybe not. Mexiletine [1-methyl-2-(2,6-xylyloxy)ethylamine hydrochloride] is a
Class 1b antiarrhythmic that is structurally related to lidocaine; it blocks multiple NaV-type
channels, including NaV1.2 [186], NaV1.4 [187], NaV1.5 [188], NaV1.7 [189], as well as tetrodotoxin-resistant Na+[13_TD$IF] currents (presumably mediated by NaV1.8 and/or NaV1.9; [138]) in DRG

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TIPS 1334 No. of Pages 21

neurons [186]. Early reports suggested that orally administered mexiletine could possibly
improve neuropathic pain associated with idiopathic sensorimotor polyneuropathy, chemotherapy administration, diabetes [190,191], and erythromelalgia [192,193]. Subsequent reports also
suggested that oral mexiletine could improve pain symptoms in some, but perhaps not all,
patients with NaV1.7-linked erythromelalgia [189,194,195]. A patient with the NaVI848 T variant
reported mild improvement in their pain when treated with acetaminophen plus mexiletine,
whereas a patient with the NaVV1316A variant reported little or no relief with a poly drug regimen
that included mexiletine [189]. Notably, the mexiletine IC50 for NaV1.7I848 T channel currents (at
steady state) is 1.08 mM, whereas the IC50 for wild-type NaV1.7, NaV1.7I136 V, and NaVV1316A
channels is 1.77, 2.03, and 1.73 mM, respectively [189]. Another mutation (Nav1.7V872G) is
also linked to mexiletine-responsive erythromelalgia; this channel has a mexiletine IC50 that is
similar to that of the wild-type channel, but shows more pronounced use-dependent block [194].
More recently, it has been shown that mexiletine impairs NaV1.7 channel gating and decreases
window currents in mutant NaV1.7L58F channels to a greater degree than wild-type channels
[196]; while the NaV1.7L58F mutation is linked to erythromelalgia [197,198], it is unknown
whether mexiletine is clinically effective in that patient population, but the in vitro data suggest
that it may be efcacious. For disorders such as erythromelalgia that exhibit genetic heterogeneity, appropriate genetic screening of patients may be required when considering whether an
NCE has clinical utility or not.

Concluding Remarks
It is evident that the data from preclinical animal models offers, in many instances, compelling
arguments for a particular molecular pathway as being of fundamental importance to the
development, maintenance, and therapeutic target value in neuropathic pain. Based on those
results, human clinical trials are designed, subjects enrolled, data collated and subjected to
multivariate analysis, and report, all too frequently, no signicant effect [199]; the results with the
CaV3.2 blocker ABT-639 are a case in point. This apparent disconnect between the animal and
human data has led to repeated calls for a fundamental rethinking of how we approach the
problem of developing new analgesics for the treatment of neuropathic pain [48,199,200].
Many, but by no means all, of the basic molecular and cellular aspects of acute nociception
appear to be conserved across species [199]. But mice are far from human, however, and pain is
not merely an unpleasant sensory experience, but includes an emotional/affective component
that the most commonly used tests for nociception (mechanical and thermal testing with von
Frey laments and a Hargreaves apparatus, respectively) cannot gauge. In the same vein, relying
on constitutive gene deletion (global or spatially restricted) animal models to conrm whether the
putative target protein is necessary and sufcient for the development and maintenance of
neuropathic pain can lead to false conclusions as to the relative contribution of the protein in
question to the pathological state [201]; exemplifying this point is the observation that neither
NaV1.7 nor NaV1.8, alone or in combination, appears to be required for the development of
neuropathic pain [202], yet selective NaV antagonists are antihyperalgesic in neuropathic pain
models. Similarly, Hcn1 or Hcn2 gene deletion minimizes or abolishes different aspects of
neuropathic pain [95,203,204] with HCN1 clearly contributing to cold allodynia [95,203]. While
many other aspects of neuropathic pain are preserved despite HCN1 deletion, the role of HCN1
in pathological-mediated neuropathic pain may not be equivalent to measuring the initiation/
development of such pain when it is constitutively absent; thus, HCN1 expression is upregulated
in response to nerve injury [109], diabetes [118], and exposure to chemotherapeutics [119,120].
To examine this further, the efcacy of ivabradine and alkylphenol inhibitors of HCN1 need to be
examined in the absence of HCN1.
So perhaps we are designing the wrong paradigms and measuring the wrong outcome(s) in the
current preclinical studies and what we are seeing are in effect false positives. Alternative tests

14

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

Outstanding Questions
What are the molecular targets in
peripheral sensory neurons that are
most relevant to the development
and maintenance of neuropathic pain?
Do the same molecular targets contribute equally to neuropathic pain regardless of the inciting event?
Is the current approach to identifying
putative targets in animals correct, or
should we rst be identifying the targets
in humans with neuropathic pain and
then establishing the mechanisms/
pathways in animals for validation (i.
e., going from bedside to bench
rather than bench to bedside)?
Are the current widely used approaches
to measuring pain in animals[16_TD$IF] [17_TD$IF] mechanical threshold testing with von Frey laments and thermal sensitivity testing
using the Hargreaves method[18_TD$IF] [19_TD$IF] sufcient when assessing neuropathic pain
or should additional testing modalities
be routinely incorporated?
Are the validated survey tools for
assessing human pain in general, and
neuropathic pain in particular, sufcient
to detect antihyperalgesic efcacy when
evaluating NCEs? Can we establish new
biomarkers, be they molecular, composite using multiple measures, or
image (fMRI or PET scan) generated,
that can be used to conrm in vivo target
engagement? How would we reconcile
discrepant results between biomarker
studies and self-report of effect?
In the past, highly selective therapeutics has been the gold standard with
regard to drug development. In the
event that multiple targets contribute
in some combination to a given type
of neuropathic pain (i.e., diabetic painful neuropathy, where both CaV3 and
HCN channels appear to play a contributory role), is a highly selective drug
in fact desirable? Would a combination medication that has active ingredients that target each pathologic
component separately be the more
logical approach? What about a single
drug but one that was less selective?

TIPS 1334 No. of Pages 21

that may offer a better assessment of complex animal behavior, and which may capture the full
neuropathic experience, include place conditioning and avoidance [205207], analgesic selfadministration [208,209], pain-induced rodent facial expression [210,211] (similar to the scale
used to assess pain in human premature neonates [212]), motor activity (wheel running
[213,214] and burrowing [215,216]), and vocalization [217,218]. Identication of a putative
molecular target, and corresponding novel selective therapeutic agent, will likely require testing
using multiple preclinical models to provide a cumulative predictive measure of both the target
and the ligand. As with human clinical trials, there is a need for consistent, uniform reporting of
preclinical in vivo methodologies as well as clearly dened a priori approaches to data analysis
and interpretation, particularly with regard to estimates of number needed to treat (NNT) and
clinical effect size [219].
To paraphrase Shakespeare, the fault, perhaps, is not in our models, but in ourselves. Neuropathic pain can result from both de novo and inherited mutations and, as such, genome wide
association studies (GWASs) may aid in the identication of specic mutations that contribute to
neuropathic pain, such as those associated with inherited erythromelalgia. Identifying relevant
molecular targets using GWAS approaches will likely provide the most meaningful results where
a single, or few, gene(s) is/are involved. These results can then be used to construct appropriate
animal studies in which novel therapeutics can be tested prior to initiating Phase I human studies.
In studies that rely on subjective reports of improvement, placebo effects are frequently high in
human trials of analgesics for the treatment of neuropathic pain, making it difcult to discern true
clinical benet of the active comparator [220]. As recommended by The Initiative on Methods,
Measurement, and Pain Assessment in Clinical Trials (IMMPACT) group, better design and
improved reporting of numerous parameters (including those broadly pertaining to patient
factors, study design, study site, and outcome measures), will enhance the sensitivity of
randomized clinical trials [221]. By way of example, this would include the use of a crossover
study design as it may require a smaller sample size than a parallel-designed study and may have
increased assay sensitivity; other recommendations based on retrospective quantitative analysis
of randomized controlled trials (RCTs) or relevant observational studies were that study duration
be at least 12 weeks and that subjects receive appropriate training as personal expectations
(i.e., bias) can inuence reporting of active treatment and placebo group improvement. Had the
ABT-639 study [51] followed the IMMPACT guidelines, it is possible the authors might have
reached a very different conclusion. Finally, the identication of validated, molecular, biomarkers
for neuropathic pain will facilitate the testing of potential therapeutics in both the preclinical and
clinical setting; unfortunately, such markers do net yet exist although the use of miRNAs holds
promise [222]. In vivo imaging [as with positron emission tomography (PET) and/or fMRI] is an
alternative biomarker methodology [223], which may be able to identify pain phenotypes [224]
and guide analgesic drug development [225,226]. Such imaging biomarkers may also identify
supraspinal circuits that contribute to the cognitive/affective components of neuropathic pain,
which may provide alternative therapeutic targets to those present in sensory neurons [59,227].
So many targets, so many potential novel analgesics. In many instances, data from animal
model studies provide compelling arguments supporting the role of select CaV, HCN, and
NaV channels in neuropathic pain arising from a variety of pathophysiological states. For NaV
channels, human correlates exist conrming the role of these channels in peripheral neuropathic pain and, at present, offer an excellent opportunity for NCE development for specic
neuropathic conditions. CaV2.2 channels also present an exciting avenue for additional
drug development, but eliminating CNS toxicity (as seen with ziconotide) will be crucial in
determining the translational potential of such compounds. Finally, CaV3.2 and HCN (HCN1
in particular) channels present wide open opportunities with regard to drug development;
whether they contribute to a specic neuropathic condition (as seen with NaV1.7 and
erythromelalgia, for example) or are more generally involved in hyperexcitability and/or

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

15

TIPS 1334 No. of Pages 21

spontaneous activity in human primary afferent neurons is unknown. Ultimately, which NCEs,
if any, will transition from bench to bedside remains to be seen; numerous questions must be
addressed to successfully bridge the gap between the apparent promise of preclinical data
and the paucity of such promise in human trials (see Outstanding Questions). And the search
goes on.
Resources
i

www.iasp-pain.org/Taxonomy

ii

www.clinicaltrialsregister.eu

iii

www.convergencepharma.com/userles/le/140923_LSR_FINAL.pdf

References
1.

International Association for the Study of Pain, Subcommittee on


Taxonomy (1986) Classication of chronic pain. Descriptions of
chronic pain syndromes and denitions of pain terms. Pain
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