Acute and Neurophatic Pain

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6.14 Acute and Neuropathic PainP Honore and M F Jarvis, Abbott Laboratories, Abbott Park, IL, USA

& 2007 Elsevier Ltd. All Rights Reserved.

6.14.1 Introduction: Pain States 328

6.14.2 Neuropathophysiology of Pain 328

6.14.3 Experimental Pain Models 331

6.14.3.1 Models of Acute Pain 331

6.14.3.1.1 Acute thermal pain 331

6.14.3.1.2 Acute mechanical pain 332

6.14.3.1.3 Acute chemical pain 332

6.14.3.2 Models of Nociceptive Pain 333

6.14.3.2.1 Adjuvant-induced arthritis 333

6.14.3.2.2 Unilateral inflammation 333

6.14.3.2.3 Models of osteoarthritic pain 333

6.14.3.3 Models of Neuropathic Pain 334

6.14.3.3.1 Direct trauma to nerves 3346.14.3.3.2 Inflammation/neuritis/nerve compression 334

6.14.3.3.3 Diabetes 335

6.14.3.3.4 Chemotherapy-induced neuropathic pain (vincristine/paclitaxel/platine) 335

6.14.4 Clinical Trial Issues 335

6.14.4.1 Translational Medicine in Pain 337

6.14.5 Current Treatments 337

6.14.5.1 Opioids 338

6.14.5.2 Anti-Inflammatory Drugs 338

6.14.5.2.1 Nonsteroidal anti-inflammatory drugs 338

6.14.5.2.2 Cyclooxygenase-2 inhibitors 339

6.14.5.3 Analgesic Adjuvants 3396.14.5.3.1 Amitriptyline 339

6.14.5.3.2 Antiepileptics 339

6.14.5.3.3 Pregabalin 339

6.14.5.3.4 Antidepressants 339

6.14.6 Unmet Medical Needs 340

6.14.7 New Research Areas 340

6.14.7.1 Neuronal Nicotinic Receptor Agonists 340

6.14.7.2 Vanilloid Receptor Modulators 340

6.14.7.3 Excitatory Amino Acid Receptor Antagonists 341

6.14.7.4 Calcium Channel Modulators 342

6.14.7.5 Cannabinoids 342

6.14.7.6 Sodium Channel Modulators 343

6.14.7.7 Purines 344

6.14.7.7.1 P1 receptor agonists 344

6.14.7.7.2 P2 receptor antagonists 344

6.14.7.8 Emerging Pain Targets 345

6.14.8 Conclusions 346

References 346

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6.14.1 Introduction: Pain States

The International Association for the Study of Pain (IASP) defines pain as an unpleasant sensory and emotional

experience associated with actual or potential tissue damage, or described in terms of such damage.1 This definition

clearly indicates that pain is a multidimensional experience. Pain is not homogenous and can be classified temporally as

acute or chronic. Acute or physiological pain is an early warning against potential injury, a vital defense mechanism,

whereas chronic pain does not play any useful role. As such, chronic pain can be very detrimental to the quality of life of

an individual, disrupting sleep and normal living, and degrading health and functional capability.2

Chronic pain is one of the most common complaints for which individuals solicit medical attention. It can affect

general and psychological health (as is evident by the high degree of comorbidity of chronic pain and emotional

disorders such as depression and anxiety), and can also have deleterious socioeconomic consequences since it is often

associated with the loss of work time and an increased use of healthcare resources. Published estimates of the

prevalence of chronic pain typically range from 2% to 45% with 50% of respondents reportedly suffering from chronic

pain.3 Interestingly, there was no significant difference between genders and, as might be expected, the proportion and

the degree of pain significantly increased with age. Furthermore, 3340% of chronic pain patients included in their

study were unhappy with medical examinations, medical tests, and treatments related to their chronic pain state. 4 This

lack of satisfactory pain relief in chronic pain patients was also identified in a 2004 Americans Living with Pain Survey

conducted on behalf of the American Chronic Pain Association.5 In this study, 50% of the chronic pain patients

surveyed felt that their pain was not under control. In the face of this growing unmet medical need is an increasing

awareness of undertreated pain resulting in a more aggressive use of analgesics, especially opioids. As the population ofelderly people continues to increase the demand for therapies to treat arthritis, pain associated with osteoporosis, and

other painful diseases of the aged will also increase. These results clearly illustrate the need for better and more

efficacious pain management medications, programs, and therapies. One of the greatest challenges in creating more

efficacious medications for pain control has been the heterogeneity of the condition itself, including: the causes and

underlying pathologies; the redundancies in pain perception; and the usefulness of current pharmacological therapies.

6.14.2 Neuropathophysiology of Pain

The conceptualization of the neurobiology of pain has undergone continuous refinement with increasing knowledge of

multiple nociceptive targets and pathways.6,7 The psychophysical parameters used to describe nociceptive processing

have thus been refined to differentiate acute withdrawal behaviors in response to dangerous (e.g., sharp or hot stimuli)stimuli in the environment (acute nociception) from increased sensitivity to mildly painful stimuli (hyperalgesia) or

to otherwise innocuous stimuli (allodynia) (Figure 1).8 An increase in stimulus intensity in any sensory modality will

eventually become noxious (Figure 1). Obviously, the sensation of noxious environmental stimuli (acute pain) is

physiologically protective. However, following injury, this psychophysical function shifts such that previous noxious

stimuli are now perceived as exceedingly painful (hyperalgesia). Additionally, tissue injury results in ongoing

or spontaneous pain and the perception that normally nonnoxious stimuli are pain generating (allodynia). It is now

Hyperalgesia

Allodynia

Innocuous Noxious

Stimulus intensity

Pain

sensation

0

100

50 Injury Normal

Low High

Figure 1 Psychophysical representation of hyperalgesia and allodynia sensory sensitivity. Following injury, being tissue or

nerve injury, pain transmission and perception are changed so that a normally painful stimulus is going to be felt more painful

(hyperalgesia) and a normally nonpainful stimulus is going to be felt as painful (allodynia). (Adapted from Cervero, F.; Laird, J. M.,

Pain 1996, 68, 1323.)

328 Acute and Neuropathic Pain


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well appreciated that distinct sensory mechanisms contribute to physiological pain, to pain arising from tissue

damage (inflammatory or nociceptive pain), and to pain arising from injury to the nervous system (neuropathic

pain).9

Nociceptive pain is caused by the ongoing activation of A-d and C-nociceptors in response to a noxious stimulus

(injury, disease, inflammation) (Figure 2). It can be further classified into visceral pain (deep cramping sensation

associated with referred pain), superficial somatic pain (skin; well-localized sharp, pricking, or burning sensation), and

deep somatic pain (muscle, joint capsules, and bone; diffuse dull or aching sensation). Under normal physiological

conditions, there is a close correspondence between pain perception and stimulus intensity, and the sensation of pain is

indicative of real or potential tissue damage. As the nervous system becomes sensitized (responding more strongly than

normal to peripheral stimuli), in addition to spontaneous pain, nociceptive pain is also associated with evoked

hyperalgesic and allodynic conditions.1012 In general, nociceptive pain abates completely upon the resolution of injury

if the disease process is controlled. Because of this, the use of disease-modifying therapies is being emphasized in the

treatment of nociceptive chronic pain as illustrated in the treatment of rheumatoid arthritis not only by

antiinflammatory agents but also by biological therapies such as tumor necrosis factor-a (TNF-a) antagonists.13

Unlike nociceptive pain, neuropathic pain can persist long after the initiating injurious event has been removed and

any damage has healed. This then leads to abnormal processing of sensory information by the nervous system.

Neuropathic pain can be classified as peripheral (painful peripheral mononeuropathy and polyneuropathy) or central

(post stroke, following spinal cord injury) and can originate from nerve injury following a wide array of conditions/

events, e.g., direct trauma to nerves, inflammation/neuritis/nerve compression, diabetes, infections (herpes zoster,

human immunodeficiency virus (HIV)), tumors (nerve compression/infiltration), toxins (chemotherapy), and primary

neurological diseases.14,15 Following nerve injury, changes occur in the central nervous system (CNS) that can persist

indefinitely. Under these conditions of sensitization, pain can occur without a specific stimulus or can be

disproportionate to the stimulus intensity. The sensation of neuropathic pain may also be constant or intermittent and

is felt in many different ways (e.g., allodynia or hyperalgesia associated with mechanical or thermal stimuli but also

spontaneous sensations such as burning, tingling, prickling, shooting, deep aching, and spasm).14,15

Descendinginhibitions

Nociceptors

A fibers A fibers

Brain

Ascendingpain

pathways

Pathophysiological pain

Peripheralsensitization

Nociceptors

Descendinginhibitions

Ascendingpain

pathways

Brain

Centralsensitization

Spinal cord

Sprouting

Normal pain

Spinal cord

Figure 2 Normal and pathophysiological transmission of pain. Under normal conditions, pain stimuli such as noxious heat are

transmitted from the peripheral site (e.g., skin or joints) through the nociceptive primary afferent fibers to the spinal cord and the

brain. Pathophysiological conditions are associated with peripheral and central sensitization. Peripheral sensitization can result

from the sensitization of nociceptors by inflammatory mediators, neurotrophic factors released during tissue damage or by

inflammatory cells. Peripheral sensitization is also associated with intense, repeated, or prolonged action potential generation in

primary sensory afferents that is mediated by altered expression and activity of voltage-gated sodium and calcium channels.

Consequences of peripheral sensitization are a lowering of the activation threshold of nociceptors and an increase in their firing

rate. These changes result in the production of hyperalgesia and allodynia associated with nociceptive chronic pain. Central

sensitization (long-lasting increases in dorsal horn neuron excitability and responsiveness) is associated with spontaneous

dorsal horn neuron activity, responses from neurons that normally only respond to low intensity stimuli (altered neural

connections following sprouting of Ab fibers to superficial laminae) and reduction in central inhibition. Central sensitization is

associated with persistent pain, hyperalgesia, allodynia, and the spread of pain to uninjured tissue. In addition, it reflects a

complex series of changes occurring in the spinal cord that may promote long-lasting increases in dorsal horn neuron

excitability including the involvement of astrocytes and microglia activation.

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Tissue injury results in the release of pronociceptive mediators that sensitize peripheral nerve terminals (peripheral

sensitization), leading to phenotypic alterations of sensory neurons and increased excitability of spinal cord dorsal horn

neurons (central sensitization).7,9 Nerve injury may be associated with abnormal firing of the injured neurons, leading also

to central sensitization and phenotypic changes in spinal cord neurons.16,17 In addition, descending supraspinal systems

modulate nociceptive responses.18 A multitude of receptors, transmitters, second messenger systems, transcription

factors, and other signaling molecules are now appreciated to be involved in pain pathways (Figures 2 and 3).6

As noted above, two mechanisms play a key role in the development and maintenance of chronic pain, namely

peripheral and central sensitization.19 Peripheral sensitization can result from the sensitization of nociceptors by

inflammatory mediators (e.g., prostaglandin E2 (PGE2), serotonin (5HT), bradykinin, epinephrine, adenosine), by

neurotrophic factors released during tissue damage (e.g., nerve growth factor (NGF)) or by inflammatory cells

(proinflammatory cytokines including interleukin-1 (IL1)). Peripheral sensitization is also associated with intense,

repeated, or prolonged action potential generation in primary sensory afferents that is mediated by altered expression

and activity of voltage-gated sodium and calcium channels.6 Consequences of peripheral sensitization are a lowering of

the activation threshold of nociceptors and an increase in their firing rate. These changes result in the production of

hyperalgesia and allodynia associated with nociceptive chronic pain. In addition, peripheral sensitization plays also an

important role in the development and maintenance of central sensitization.6,17

Central sensitization (long-lasting increases in dorsal horn neuron excitability and responsiveness) is associated with

spontaneous dorsal horn neuron activity, responses from neurons that normally only respond to low-intensity stimuli

(altered neural connections following sprouting of Ab fibers to superficial laminae), expansion of dorsal horn neuron

receptive fields, and reduction in central inhibition.2022 Central sensitization is associated with persistent pain,

hyperalgesia, allodynia, and the spread of pain to uninjured tissue, i.e., secondary hyperalgesia due to increased receptor

Dorsal root ganglionprimary afferent cell body

A

SPBDNFGALNPYNav1.3

C

SPCGRP

TRPV1ASICs

Nav1.8

Peripheral terminalprimary afferent fiber

P2X3 Nav1.3

TRPV1

mGLU

TRPs

opioid

ASICs

TNFIL1

Spinal cord

Nav1.8

TRPs

P2X3

opioid NK-1

NMDA

2

GABA-BCCKB

A1

mGLU

CGRP-1

Glia

Central terminalprimary afferent fiber

Post

synaptic

neuron

SP

NE5HT

GABA

ENK

GLU

ACh

CGRP

Descending systemsor

interneurons

Figure 3 Pain transmission sites can be simply divided into two: the peripheral compartment (e.g., skin, muscle, organs) that

encompasses primary afferent fibers and dorsal root ganglions and the central compartment that includes the spinal cord and

brain. A multitude of receptors, transmitters, second messenger systems, transcription factors, and other signaling molecules

located all along pain transmission pathways are now appreciated to be involved in pain signaling. Tissue injury results in the

release of pronociceptive mediators that activate and sensitize peripheral nerve terminals (peripheral sensitization) through

various receptors/channels, leading to phenotypic alterations of sensory neurons with changes of receptor expression in DRG

cell bodies and changes in neurotransmitter contents and increased excitability of spinal cord dorsal horn neurons (central

sensitization) due to a variety of changes in receptor expression patterns and neurotransmitter release. In addition, interneurons

and descending supraspinal systems modulate nociceptive responses either through excitatory or inhibitory effects. 5HT

(serotonin), A1 (adenosine 1), a2 (alpha 2 adrenergic), ACH (acetylcholine), ASICs (acid sensing ion channels), BDNF (brain

derived nerve growth factor), CCKB (cholecystokinin), CGRP (calcitonin gene related peptide), ENK (enkephalines), GABA, GAL

(galanin), GLU (glutamate), mGlu (metabotropic glutamate receptors), Nav1.3 (sodium channels), Nav1.8 (sodium channels),

NE (norepinephrine), NK-1 (neurokinin-1 receptor), NMDA (N-methyl-D-aspartate receptor), NPY (neuropeptide Y), opioid, P2X3

(ATP receptor), SP (substance P), TRP (transient receptor potential), and TRPV1 (vanilloid receptor 1).



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field of spinal neurons. In addition, it reflects a complex series of changes occurring in the spinal cord that may promote

long-lasting increases in dorsal horn neuron excitability. This process is also know as wind-up in that the response of

sensitized dorsal horn neurons is exaggerated relative to the normal situation.2022 While both peripheral and central

sensitization play a role in nociceptive chronic pain, central sensitization clearly plays a key role in neuropathic pain.

Thus, central sensitization also explains the observation that established pain is more difficult to suppress than acute

pain because of the maladaptive changes that have taken place in the CNS.6,17 Interestingly, not only neurons, but also

glia, e.g. astrocytes and microglia, as well as infiltrating mast cells are involved in the generation and maintenance of

central sensitization.23,24

In addition to the activation of pronociceptive inflammatory and/or neurotrophic messengers noted above, the

sensitization of the nervous system in response to chronic pain involves the alteration and/or activation of many

neurotransmitter systems that have been extensively reviewed elsewhere.6,15 Chronic pain is mediated by altered

neuronal excitability involving activation of sodium and calcium channels in both peripheral and spinal neurons.

Additionally, there is enhanced glutamatergic activity and a concomitant decrease in GABAergic inhibitory

neuromodulation at the level of the dorsal spinal horn.6,15 This altered neurochemical profile contributes to the

heightened state of neuronal excitability (e.g., wind-up) and can be viewed as a shift in the balance of excitatory and

inhibitory systems that also incorporates activation of intracellular signaling cascades (e.g., ras-mitogen-activated protein

kinase (MAPK) pathway) and recruitment of neurotrophic neuropeptides including substance P, neuropeptide Y,

and brain-derived neurotrophic factor (BDNF). These changes in spinal neuron neurochemistry are also accompanied

by upregulation of specific excitatory amino acid receptors (e.g., a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid

(AMPA) and N-methyl-D-aspartate (NMDA)), as well as increased calcium and potassium ion channel activity. Taken

together, chronic pain is associated with a large variety of deranged patterns of neurotransmission at multiple levels of

the neuroaxis with considerable target and pathway redundancy. Thus, in the absence of ongoing injury, chronic pain can

be viewed as a disease in itself. The enhanced appreciation of the many neurochemical and neurophysiological

alterations in neuronal function associated with chronic pain has led to the development of both new preclinical models

of pain and a variety of potentially useful therapeutic interventions.

6.14.3 Experimental Pain Models

To facilitate the study of pain transmission and the characterization of novel analgesic compounds, an array of

experimental animal pain models has been developed mainly in rodents, reflecting all types of pain, from acute to

chronic, somatic to visceral, and nociceptive to neuropathic and cancer-related pain. Depending on the model, painmeasurements can encompass spontaneous pain behaviors as well as pain evoked by various sensory modalities. It is

important to note that in rodents, measuring spontaneous pain is very difficult and is generally limited to the

observation of quantifiable nocifensive (pain-escape) behaviors such as hind paw lifting or altered grooming. However,

experimental measures of evoked pain are well characterized and are analogous to clinical diagnostic methods. In the

following overview, acute pain refers to pain that lasts from seconds to a day while chronic pain typically refers to

experimental pain manipulations that persist for at least several days. In addition, this section will focus on the most-

widely used preclinical pain models.

The majority of these animal models of pain were originally developed in rats. Except as noted below, essentially all

have also been successfully carried out using various mouse strains including gene-disrupted (knockout) mice.25

However, significant differences in the basal nociceptive sensitivity and analgesic response have been noted for

different mouse strains serving to further complicate the interpretation of the knockout phenotype. 26

6.14.3.1 Models of Acute Pain

Animal models of acute pain allow the evaluation of the effects of potential analgesics on pain sensation/transmission in

an otherwise normal animal. In addition, the same tests may be used to measure stimulus-evoked pain in animals with

chronic inflammation or nerve injury. Usually, these tests rely on an escape behavior/withdrawal reflex or vocalization as

an index of pain. The animals have control over the duration of the pain, that is, their behavioral response leads to

termination of the noxious stimulus.

6.14.3.1.1 Acute thermal pain

Models have been developed to interrogate acute thermal pain sensitivity, using various means of applying a noxious

heat stimulus to the paw or the tail of rodents. These models have been widely used in the characterization of opioid

analgesics. Usually, latency to behavioral response is recorded and a cutoff is set to avoid any tissue damage to the



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animal. The tail-flick test involves the application of a focused heat (usually light) source on the tail until a tail-flick

(rapid removal of the tail) reflex occurs. This test has an advantage in that it does not involve repeated assessments of

animal behavior, i.e., animals learning with time when the stimulus is going to be applied and anticipating the test.

The hot plate assay uses a hot plate set at a fixed temperature, usually 5055 1C. Latency to licking, shaking of hind

limbs or fore limbs, in addition to latency to jump can be recorded and statistically analyzed for groups of animals. This

assay can be difficult to standardize since the heat stimulus is not delivered in a controlled fashion. Possible sources of

variability include differential exposure to the heated plat depending on how much weight the animal puts on each limb.

Another approach to assess acute thermal pain is the use of a radiant heat source.27 Using this methodology, the

temperature of the heat source applied to the hind paw increases over time until it reaches a painful threshold. Latency

to hind paw withdraw is recorded and analyzed. In each test session, each animal is tested in three to four sequential

trials at approximately 5 min intervals to avoid sensitization of the response. One of the advantages of this method

versus the tail-flick assay is that both paws can be tested. This important control has proven a useful behavioral

assessment in models of unilateral inflammation or nerve injury, the contralateral paw serving as control for the injured

paw. In addition, in this assay, rats are confined in plastic chambers but not manually restrained as in the tail-flick assay

or in the immersion tests (see below), decreasing the stress level of the test subjects. This method also uses a heated

(30 1C) glass test surface to prevent paw cooling and to minimize sensitization artifacts.

Acute thermal pain can also be evaluated using a fixed temperature (4550 1C) water bath and assessment of latency

to withdraw of a hind limb or tail from the hot water. One of the advantages of this method is that the water bath can be

set at various temperatures and it can be less sensitive to environmental conditions. However, this assay requires

handling of the animals when testing for nociceptive behavior, making this measure highly dependent on experimenter

experience/comfort handling/restraining animals by hand.

This method can also be used to test for reactivity to cold, using a 4 or 10 1C water bath and recording latency to

withdraw as an index of pain. Another method uses a cold plate cooled by cold water circulating under it. Latency to

nociceptive behavior or duration of guarding behavior can be recorded. As for the hot plate assay, the cold plate test has

the advantage of not requiring animal restraint. However, depending on the position of the animal paw on the plate (or

just above it), the cold stimulation can be very variable. Another widely used method is application of a drop of cold

acetone on the plantar skin of animals resting on an elevated mesh floor. Acetone produces a distinct cooling sensation

as it evaporates. Normal rats will not respond to this stimulus or with a very small response (in amplitude and duration)

while nerve-injured rats will almost always respond with an exaggerated response.

6.14.3.1.2 Acute mechanical painModels have been developed to interrogate acute mechanical pain/sensitivity, using various means of stimulating the

paw or the tail of a mouse or a rat. A common method for the assessment of acute mechanical pain is determination of

withdrawal thresholds to paw/tail pressure using the Randall Selitto test.28 This apparatus allows for the application of a

steadily increasing pressure to the dorsal surface of the hind paw/tail of a rat via a dome-shaped plastic tip. The

threshold (in g) for either paw/tail withdrawal or vocalization is recorded. Usually, two or three measurements are

conducted on each paw or tail. This apparatus was designed originally for measuring mechanical sensitivity of inflamed

paws and its use in normal noninflamed paws can produce great variability in the response, depending on the location of

the stylus (soft tissue between the metatarsal/bone/joint). It is worth noting that training helps generate a more stable

response with this assay.

Another approach for assessment of acute mechanical pain is to use a pinprick, applying painful pressure to the

plantar surface of the hind paw. This is similar to the pricking pain test done during the neurological examination in

patients. The behavior can be measured by the duration of paw lifting following the pinprick application or recorded as

a frequency of withdrawal (percentage of response to the pinprick in 10 trials).

Finally, mechanical hypersensitivity can also be tested with von Frey monofilaments. These are a series of hairs/

nylon monofilaments of various thicknesses that exert various degrees of force when applied to the planter surface of

the hind paw. Responses can be quantified as percentage response or duration of response to a given monofilament

force applied several times, or mechanical threshold can be determined using the updown method. 29

6.14.3.1.3 Acute chemical pain

Usually, when studying acute chemical pain, behaviors such as flinching, biting, or licking the injected paw are

recorded at various time points following the injection of a chemical irritant (capsaicin, formalin, PGE2, mustard oil,

ab-methylene ATP). The duration of the nociceptive behavior as well as the number of behaviors can be quantified and

analyzed. Two models are mostly used to study acute chemical pain: nocifensive behaviors following injection of



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capsaicin or formalin into the hind paw.30 Doses of capsaicin can vary from 1 to 10 mg per 10 mL injected to the dorsal

surface of the rat hind paw. The injection of capsaicin is immediately followed by an intense period of nocifensive

behaviors that are usually recorded for 5 min following capsaicin injection. Following formalin injection (usually 5% per

50mL) into the dorsal or sometimes plantar surface of the rat hind paw, a biphasic behavioral response can be observed.

Phase I of the formalin response is defined as the period of time immediately following injection of formalin until

10 min after the formalin injection and corresponds to acute thermal pain by direct activation of nociceptors by

formalin. Following a quiet period of little or no nocifensive behavior, the second phase of the formalin response can be

observed (2060 min post formalin injection) that corresponds to a more persistent inflammatory state.

6.14.3.2 Models of Nociceptive Pain

Models of nociceptive pain are defined as models of pain following tissue injury induced by trauma, surgery,

inflammation, and cancer. As stated above, spontaneous pain in these models is difficult to measure. However, evoked

pain behaviors have been well characterized and can be induced by the methods described above in the acute pain

section. The focus of this section will be on models of nociceptive pain, mimicking as closely as possible rheumatoid

arthritis and osteoarthritis clinical conditions since they have been the most studied and widely used. Models of

postoperative pain or cancer pain will not be described, as they are recent and still under validation.

6.14.3.2.1 Adjuvant-induced arthritis

Experimental arthritis is generated by an intravenous injection of complete Freunds adjuvant (CFA) at the base of the

tail. The development of the joint inflammation is progressive and dramatic, leading to a multijoint arthritis with

dramatic swelling and permanent joint tissue destruction.31 In this model, it is clear that the animals are in chronic

pain, all their joints are swollen, they have decreased appetite, they limp, and have lower threshold for limb withdrawal

or vocalization to paw pressure/joint manipulation. This model is rarely used today as the polyarthritic rat has significant

systemic disease with abnormal hunchback posture and piloerection.

6.14.3.2.2 Unilateral inflammation

To further study inflammatory pain, various models have been developed to induce a localized inflammatory reaction by

injecting various substances, e.g., formalin, carrageenan, or CFA into the paw or the joint. Following the initial injection,

pain can be measured minutes to days later, at the site of inflammation or away from the primary site of injury. Usually,the inflamed paw/joint becomes very sensitive to both thermal and mechanical stimuli while the contralateral paw

remains normal. Sometimes, secondary mechanical hypersensitivity can also develop on the contralateral side as

observed 2 weeks following carrageenan injection into the knee joint when testing on the contralateral paw. These

models of more localized inflammation/inflammatory pain have been widely used in pain research to test the effects of

potential analgesic compounds but also in electrophysiological and gene expression studies to determine the plastic

changes that initiate/maintain chronic inflammatory pain.

6.14.3.2.3 Models of osteoarthritic pain

More recently, models have been developed to mimic osteoarthritic (OA) pain observed in the clinic. Contrary to

rheumatoid arthritis (RA) and the models of inflammatory pain, OA in the clinic and in animal models is not associated

with a large amount of inflammation. In addition, to mimic more closely the clinical situation, pain evaluation in OApain models relies on functional measures such as weight bearing or grip force of the affected limb rather than

evaluation of withdrawal latencies to thermal or mechanical stimuli. Two models have been widely used, intraarticular

administration of sodium monoiodoacetate (MIA) into the knee and partial meniscectomy. 32 Contrary to what is

observed in the polyarthritic rat, no changes in body weight were observed over a 4-week period after either iodoacetate

injection or partial medial meniscectomy. In addition, the general health of the animals is good with no signs of

spontaneous nociceptive behavior, impaired locomotion, or distress. Furthermore, both iodoacetate injection and partial

medial meniscectomy in the knee joint of the rat induced histological changes and pain-related behaviors characteristic

of clinical OA. Although the behavioral changes and histology both worsened over time, the majority of the pain

responses were apparent within one week of surgery or iodoacetate injection. It is important to note that the pain

behaviors are less pronounced in the surgery model than in the MIA model and that these findings agree with the

clinical situation. Indeed, magnetic resonance imaging (MRI) studies have shown that although meniscal lesions in

humans are common, they are also rarely associated with pain.



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6.14.3.3 Models of Neuropathic Pain

6.14.3.3.1 Direct trauma to nerves

To mimic nerve injury observed in the clinic, a number of different animals models have been developed. One of the most

studied models is the L5L6 spinal nerve ligation (SNL, Chung model) (Figure 4) model.33 In this model, following

sterilization procedures, a 1.5 cm incision is made dorsal to the lumbosacral plexus, the paraspinal muscles are separated from

the spinous processes, the L5 and L6 spinal nerves are isolated, and tightly ligated with 30 silk thread. Usually the animals

are allowed to recover from surgery for 7 days before being tested for mechanical allodynia using von Frey monofilaments(updown method or percentage response to 10 applications of innocuous or noxious von Frey monofilament). While the

spinal nerve injured rats also develop cold allodynia and thermal hyperalgesia, they have a greater degree of mechanical

allodynia and most pharmacological studies with these animals have involved mechanical allodynia endpoints.

Another widely used model of direct nerve injury is a partial nerve ligation model (PNL) (Figure 4).33 The sciatic

nerve is exposed unilaterally, just distal to the descendence of the posterior biceps semitendinosus nerve from the

sciatic. The dorsal 1/31/2 of the nerve thickness is then tightly ligated with an 80 silk suture. Following injury, these

animals develop guarding behavior of the injured hind limb suggesting the possibility of spontaneous pain. In addition,

the animals develop mechanical allodynia as well as thermal hyperalgesia and bilateral mechanical hyperalgesia.

6.14.3.3.2 Inflammation/neuritis/nerve compression

Neuropathic pain can also result from inflammation around peripheral nerves and peripheral nerve compression. Two

preclinical models have been developed to attempt to mimic this phenomenon. The first model is the chronic

constriction injury (CCI) (Bennett model) (Figure 4) of the sciatic nerve model.33 In this model, a 1.5 cm incision is

made 0.5 cm below the pelvis. The biceps femoris and the gluteus superficialis are separated and the sciatic nerve

exposed, isolated, and four loose ligatures (50 chromic catgut) with 1 mm spacing are placed around it. CCI animals

develop mechanical allodynia, cold allodynia, and thermal hyperalgesia. When compared to the SNL injured animals,

CCI animals do develop thermal hyperalgesia and cold allodynia to a greater extent.

The second model, developed more recently, is the SIN model or zymosan-induced sciatic inflammatory neuritis.34

In this model, a chronic indwelling perisciatic catheter is used to inject zymosan around the sciatic nerve. After aseptic

exposure of the sciatic nerve at midthigh level, the gelfoam is threaded around the nerve so as to minimize nerve

displacement. Suturing and insertion of a sterile dummy injection tube during implantation maintained catheter

patency and ensured replicable drug delivery close to the nerve. After anchoring to the muscle, the external end is

tunneled subcutaneously to exit 1 cm rostral to the tail base. After removal of the dummy injector, the external end ofthe silastic tube is protected. Usually, catheter placement can be verified at sacrifice by visual inspection. The catheter

Sural

PNL

CCISciatic

SNL

Peroneal

Tibial

DRG

L4

L4L5

L6 Spinal cord

Figure 4 Animal models of neuropathic pain. This schematic illustrates the three main rodent models of neuropathic pain

associated with direct nerve injury; the L5L6 spinal nerve ligation (SNL) model (Chung model), the chronic constriction injury

(CCI) of the sciatic nerve model (Bennett model), and the partial nerve ligation (PNL) model (Seltzer model).



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is used for a single injection 45 days after surgery conducted in freely moving rats. In this model, perisciatic zymosan

injection induces unilateral mechanical allodynia at low dose and bilateral mechanical allodynia at high dose.

Interestingly, the same high dose injected into gelfoam in neighboring muscles does not induce mechanical allodynia,

suggesting that immune activation must occur in close proximity to peripheral nerves to create allodynia and that

zymosan spread to systemic circulation cannot explain allodynia created by perisciatic zymosan. Interestingly, no

thermal hyperalgesia is observed in this model.

6.14.3.3.3 Diabetes

Another major cause of neuropathic pain in the clinic is neuropathic pain observed in diabetic patients. In rodents, this

is mimicked by streptozotocin (STZ) injection to induce diabetes and subsequent neuropathic pain symptoms. 33

Usually, diabetes is induced by a single injection of STZ (75 mg kg1 intraperitoneal). Diabetes is confirmed by testing

for blood glucose levels. Not all animals show signs of neuropathic pain immediately following STZ administration.

Generally it takes usually between 4 and 8 weeks to observed neuropathic pain symptoms, mostly mechanical allodynia

assessment with von Frey monofilaments, in a group of streptozotocin-treated rats.

6.14.3.3.4 Chemotherapy-induced neuropathic pain (vincristine/paclitaxel/platine)

The last type of neuropathic pain models are chemotherapy-induced neuropathic pain models. Cancer-related pain is

a significant clinical problem that will likely increase in its extent as the average lifespan continues to rise and

cancer therapies continue to improve. The two main sources of cancer-related pain are that from the malignancy itselfand from the treatments utilized to alleviate the cancer (surgery, radiation, and chemotherapy). Peripheral neuro-

pathy and subsequent neuropathic pain related to chemotherapeutic treatment can be dose limiting, and the pain is

often resistant to standard analgesics. To date, no one drug or drug class is considered to be both a safe and effective

analgesic in the treatment of chemotherapy-induced pain, and three preclinical models of chemotherapy-induced

neuropathic pain have been recently developed to further our understanding of the pathophysiology of such

neuropathic pain states. Chemotherapy-induced neuropathic pain can be induced by the injection of either vincristine,

platine, or paclitaxel.33 Depending on the experimental protocol, they can be injected as a bolus, for several days

or weeks or as a continuous intravenous infusion using osmotic pump. Interestingly, as observed in the clinic,

thermal hyperalgesia is not observed in these animals. However, both mechanical allodynia and cold allodynia are

observed.

The differential efficacy of analgesic medications for different types of pain that is seen in the clinic is also observed

in animal pain models. For example, while opioid analgesics like morphine (Figure 5) are potent and efficacious in allanimal pain models, anti-inflammatory agents such as ibuprofen and celecoxib (Figure 5) are most potent and effective

in animal models associated with inflammation, and anti-epileptics like lamotrigine and gabapentin ( Figure 6) are most

potent and efficacious in animal models of neuropathic pain (Table 1). As preclinical models of the various forms of pain

appear to have selective and differential predictive validity for efficacy in the clinical setting, they should be useful in

determining if new chemical entities (NCEs) with a novel molecular mechanism have the promise to be broad-

spectrum analgesics.

6.14.4 Clinical Trial Issues

Traditionally, the assessment of novel analgesics has been based on methods and models based on the clinical utility of

opioid analgesics.14

Many of the endpoints measured involve the use of self-report methodologies including theclassical visual analog scale (VAS) with which patients rate their pain from a score of 0 (no noticeable pain) to 10 (worst

pain imaginable). For the specific assessment of neuropathic pain, clinical studies have used tools like the McGill Pain

Questionnaire, Neuropathic Pain Scale, and the Neuropathic Pain Symptoms Inventory. While the use of many of these

analgesic endpoints has been validated in the clinical setting, the use of specific combinations of these scales may be

useful to enhance the sensitivity of clinical outcomes for new analgesic compounds.

Clinical trial designs often employ parallel placebo-controlled and randomized withdrawal types of experimental

manipulations.14,35 The majority of these designs have been well validated using opioids; however, the relative utility in

assessing novel analgesics that target specific aspects of chronic pain (e.g., neuropathic allodynia) await further evaluation

(e.g., non-NSAID (nonsteroidal anti-inflammatory drug) mediated analgesia in the third molar extraction model).

A number of nociceptive tests have also been used in experimental clinical trials including acute heat sensitivity,

topical and intradermal capsaicin, heat/capsaicin combinations, and quantitative sensory testing using both mechanical

and thermal stimuli.36,37

Some of these tests can also be coupled to other functional readouts such as functional MRI



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(fMRI) and nerve conduction velocity assessments to further enhance measurements of drug action.38 For example, in

assessing the acute antinociceptive effects of remifentanyl, the apparent sensitivity of fMRI analysis of human cortical

oxygen utilization was significantly greater than patients subjective ratings using traditional VAS scales. The use of

fMRI coupled with experimental pain models like the capsaicin-evoked secondary hyperalgesia model may provide a

reliable early assessment of novel analgesic efficacy. Many of these techniques have received clinical validation using

opioids and are now being used to characterize mechanistically novel therapeutics.

NH2 CO2H NH2 CO2H

CH3H3C

N

NH2O NCH3

CH3

N

N N

Cl Cl

NH2

NH2

CH3NH

S

O

Gabapentin Carbamazepine Amitriptyline

Lamotrigine Duloxetine

Pregabalin

Figure 6 Analgesic adjuvant agents.

Morphine Oxycodone Fentanyl Tramadol

O

NCH3

H

HO

HO

O

NCH3

OH

HO

O

N

N

H3C

O CH2N(CH3)2

HO

H

CH3O

NN

F

F

F

H3C

SO2NH2

O

H3CO2S

O

Celecoxib Rofecoxib

H3C

CH3COOH

CH3N

COOH

CH3

CH3OCl

O

Ibuprofen Indomethacin

O CH3

O

COOH

Aspirin

Figure 5 Opioid analgesics, nonsteroidal anti-inflammatory analgesics, and cyclooxygenase-2 (COX-2) inhibitors.



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6.14.4.1 Translational Medicine in Pain

A major issue in all aspects of drug discovery is the extent of predictivity of the preclinical animal models used to

characterize NCEs to the clinical situation. Of all areas of research, based on validation with opioids and nonsteroidal

anti-inflammatory drugs (NSAIDs), pain models were always considered highly robust. However, the recent failure of

multiple NCEs targeted for the neurokinin-1 (NK-1) or substance P receptor has raised serious concerns about the

translation of preclinical analgesic data to analgesic efficacy in patients.39,40 Structurally diverse NK-1 antagonists from

several companies were evaluated clinically for acute pain (third molar extraction, migraine, or diabetic neuropathy).40

While these NCEs attenuated nociceptive responses sensitized by either inflammation or nerve damage, they

exhibited little effect on baseline (acute) nociception in preclinical models of pain. However, these same NCEs failed

to demonstrate significant analgesic efficacy in early clinical trials. This situation has led to the re-evaluation of both

the predictability of the preclinical animal models as well as the utility of clinical trial designs that were customized to

assess analgesic by opiate and/or NSAID mechanisms. Is should also be noted that with the exception of the formalinmodel described above, animal pain models largely depend on stimulus-evoked pain behaviors as experimental

endpoints. Consequently, assessments of ongoing nociception that are the foundation of most clinical studies are not

modeled very well preclinically. It remains unclear whether the disconnect between the preclinical and clinical data for

NK-1 antagonists reflects species-dependent roles of substance P in chronic pain or an imprecision in relating the

nociceptive state of experimental animals to that of humans. Clearly, additional research is needed to adequately

resolve these translational research issues for NK-1 antagonists. This issue highlights the practical need for the

development of accurate and cost-effective translational medicine approaches to assess analgesic efficacy such as the

coupling of fMRI studies with pain modeling in clinical studies described above.38

6.14.5 Current Treatments

Currently available analgesic agents can be broadly categorized as nonopioid analgesics (acetaminophen and NSAIDs),opioid analgesics (morphine and fentanyl), and adjuvant analgesics or coanalgesics (e.g., antiepileptics, anesthetics, and

antidepressants). Nonopioid analgesics are mostly used to relieve mild to moderate nociceptive pain, adjuvant

analgesics are used to relieve neuropathic pain, and opioid analgesics are used to treat severe pain of all origins,

depending on the dose prescribed. Based on a number needed to treat analysis, a variety of opioid medications

including fentanyl and oxycodone produce equivalent analgesic efficacy in neuropathic pain as compared to gabapentin,

tramadol, and analgesic adjuvants like tricyclic antidepressants and anticonvulsants.41 The primary adverse event

associated with chronic opioid therapy is decreased gastrointestinal (GI) motility. Respiratory depression and opioid

dependence, which are routinely cited as a major issue in the use of opioids for pain therapy, are significantly less

prevalent in chronic pain patients. This finding has led to ethical concerns related to patients not being given sufficient

pain medication due to legal issues with drug scheduling.35,42

While currently available analgesics have therapeutic utility in different pain states, all suffer from drawbacks in

clinical use. The opioids can produce tolerance and dependence, constipation, respiratory depression, and sedation.

Table 1 Effects of clinically used analgesics in preclinical models of acute, nociceptive, and neuropathic pain; relative

analgesic efficacy of clinically useful analgesics in experimental pain models in the absence of psychomotor side effects. Data

derived from both in house and literature values (see text)

Acute pain Nociceptive pain Neuropathic pain

T C F Car CFA OA RA SNL CCI Vinc

Morphine 2 2 3 4 5 4 5 2 3 3

Ibuprofen 0 1 2 5 5 2 NT 2 3 0

Celecoxib 0 1 2 5 5 5 1 2 3 0

Gabapentin 0 1 3 2 3 2 NT 5 5 3

Lamotrigine 2 2 3 2 3 2 NT 5 4 3

Scale: 0, no effect; 1, o20% effect; 2, 2040% effect; 3, 4060% effect; 4, 6080% effect; 5, 80100% effect, NT, Not

tested. Abbreviations: T, tail flick; C, abdominal constriction; F, formalin; Car, carrageenan; CFA, complete Freunds

adjuvant; OA, osteoarthritis; RA, rheumatoid arthritis; SNL, spinal nerve ligation; CCI, chronic constriction injury; Vinc,

vincristine.



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The NSAIDs are associated with GI side effects and increased bleeding time, and do not effectively ameliorate severe

pain. It has been estimated conservatively that 16 500 NSAID-related deaths occur among patients with rheumatoid

arthritis or osteoarthritis every year in the USA.43 The development of selective COX-2 inhibitors (e.g., celecoxib and

rofecoxib (Figure 5)) offered NSAID-like analgesia with the potential for diminished GI side effects. However, in light

of the withdrawal of rofecoxib from the market, the cardiovascular safety of this class of analgesics is questionable. 44

Adjuvant agents, e.g., gabapentin, lamotrigine, and amitryptiline, whose mechanism of action may be primarily

mediated via a nonselective block of sodium channels, are associated with CNS and cardiovascular side effects.

Currently available analgesics also have limited utility in the treatment of neuropathic pain. The anticonvulsant

adjuvant gabapentin (Figure 6), which also has a poorly defined mechanism of action, has demonstrated clinical utility

in the treatment of some forms of neuropathic pain.45 Thus, there is a significant unmet medical need for safer and

more effective analgesic agents.

6.14.5.1 Opioids

Opioid analgesics produce their effects by binding and activating the opioid receptor subtypes (e.g., m, d, k receptors)

in the CNS.6 The cloning and characterization of the major opioid receptor subtypes (m, OP1; d, OP2; and k, OP3) has

stimulated significant basic and clinical research to discover new opioids with improved target selectivity, safety, and

efficacy. Moreover, each of these opioid subtypes has been further subdivided into putative subtypes, and an orphan

member of this family, ORL-1 (OP4), has also been described.

46

Given the anatomical distribution of the opioid receptors in the CNS, opioid analgesics can block pain transmission

from the periphery to the spinal cord by blocking neurotransmitter release from the primary afferent fibers and by

directly decreasing activation of postsynaptic dorsal horn neurons. The broad spectrum analgesic efficacy of the opioids,

like morphine, fentanyl, and oxycodone (Figure 5), coupled with the fact that these agents do not show analgesic

ceiling effects makes opioid compounds the mainstay in the control of moderate to severe pain. 46 The analgesic actions

of opioid drugs are mediated at multiple sites of action including primary sensory afferent neurons, the dorsal horn of

the spinal cord, and sites within the brain such as the brainstem and midbrain.

This multitude of opioid interactions also contributes to the side effects associated with opioid analgesic therapy

including dependence, tolerance, immunosuppression, respiratory depression, and constipation.41 Opioid dose titration

can be achieved to manage some nociceptive conditions; however, this strategy does not provide full efficacy in all

chronic pain syndromes such as cancer and neuropathic pain.35,41 In addition, acting at higher brain centers, opioid

analgesics can decrease pain transmission from the spinal cord to the brain, alter the limbic system, and increasedescending inhibitory pathways to modulate pain transmission at the spinal level. While nociceptive pain is generally

more responsive to opioid analgesics than neuropathic pain, nearly all types of pain respond to the right dose of opioid

analgesics. Opioid analgesics are usually recommended to treat moderate to severe pain that does not respond to

nonopioid analgesics alone and are often prescribed in combination with nonopioid analgesics. They do play a key role

in the treatment of acute pain (postoperative pain), breakthrough pain, cancer pain, and some types of chronic

noncancer pain.35 However, the use of opioid analgesics is associated with side effects such as sedation, confusion,

respiratory depression, constipation, nausea, and vomiting that can limit their utility. More recently, a number of

controlled release or extended release formulations of opioids have been developed in an effort both to enhance the

analgesic coverage of these medications and to reduce the severity of the adverse events.

The discovery of the opioid receptors in 1973 and their subsequent cloning has conceptually provided the tools

necessary to develop receptor subtype selective NCEs that may improved efficacy and/or reduced side effect liabi-

lities that would make them more useful analgesic agents. However, since 1975, more than $3 billion has beenspent on research to find such improved NCEs but none has lived up to its preclinical promise in the clinical

situation.

6.14.5.2 Anti-Inflammatory Drugs

6.14.5.2.1 Nonsteroidal anti-inflammatory drugs

The primary mechanism of action of NSAIDs is the blockade of prostaglandin synthesis via inhibition of the cyclo-

oxygenase enzymes (COX-1 and COX-2). NSAIDs like aspirin, diclofenac, and ketorolac (Figure 5) have anti-

inflammatory, antipyretic, and analgesic effects, and their anti-inflammatory effects can indirectly relieve nociceptive

pain by reducing inflammation and tissue swelling, although potency across these effects can be compound dependent.

These characteristics make them particularly efficacious is nociceptive pain conditions associated with peripheral

inflammation.



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6.14.5.2.2 Cyclooxygenase-2 inhibitors

Recently, selective inhibitors of COX-2 (the COX enzyme isoform inducible by inflammation), e.g., celecoxib and

rofecoxib (Figure 5), have demonstrated similar efficacy to classical NSAIDs with reportedly fewer GI side effects at

therapeutic doses.47 However, this is controversial and these agents have also recently been associated with a

significant increase in the risk of cardiovascular disease that has brought the entire class under intense scrutiny with

rofecoxib having been withdrawn from the market.44,48 As stated above, NSAIDs relieve mild to moderate pain

associated with trauma, surgery, cancer, and arthritis. They are especially effective for certain types of somatic pain such

as muscle and joint pain, sprains, bone/dental pain (tooth extraction), inflammatory pain, osteoarthritic pain, and in

combination with disease-modifying therapies in rheumatoid arthritic pain.49

6.14.5.3 Analgesic Adjuvants

Adjuvant analgesics have a primary indication other than pain but have demonstrated analgesic effects in particular

pain conditions. Antiepileptic drugs such as gabapentin, amitriptyline, carbamazepine, and lamotrigine (Figure 6) are

one type of adjuvant analgesic and are used primarily in the treatment of neuropathic pain. 45 While it is hypothesized

that their analgesic effects are due to their ability to reduce membrane excitability and tone down a hyperexcitable

nervous system, their exact mechanism(s) of action remains unclear and not all antiepileptic drugs are good analgesics.

As a class, they are most efficacious at treating peripheral neuropathic pain, postherpetic neuralgia, trigeminal

neuralgia, and diabetic neuropathic pain.50

6.14.5.3.1 Amitriptyline

Amitriptyline is a tricyclic compound that has been approved for treatment of major affective disorders (e.g.,

depression) since the 1950s.51 The antidepressant actions of amitriptyline generally are associated with blockade of the

uptake of serotonin and norepinephrine in the CNS; however, amitriptyline possesses a multiplicity of other distinct

pharmacological activities, e.g., antagonist actions at histamine, muscarinic, a1-adrenergic, and serotonin receptors at

namomolar concentrations and at a number of ion channels (e.g., sodium, calcium, and potassium) at micromolar

concentrations.52,53 Amitriptyline also has micromolar affinity for blocking the uptake of the antinociceptive and anti-

inflammatory purine adenosine.52 In addition, amitriptyline has peripheral anti-inflammatory/analgesic actions in

several in vivo models that are associated with acute, local delivery of low concentrations of amitriptyline. Many of

these pharmacological activities are likely to contribute to its analgesic and anti-inflammatory actions.

6.14.5.3.2 Antiepileptics

Recent data suggest that newer antiepileptic drugs such as gabapentin or lamotrigine (Figure 6) are better alternatives

to older agents of this class. Lamotrigine has been shown to be effective in patients with trigeminal neuralgia, complex

regional pain syndrome, and neuropathic pain associated with multiple sclerosis and HIV infection.54,55 In addition, it

has been shown that gabapentin provides pain relief in diabetic neuropathic pain conditions and postherpetic neuralgia;

it also has a more favorable side effect profile compared to other neuropathic pain agents.55 Open-label studies suggest

that gabapentin also may be useful in the management of trigeminal neuralgia, central pain, phantom limb pain, and

neuropathy associated with HIV infection. Common side effects of antiepileptic drugs as a class include sedation,

mental clouding, dizziness, nausea, or unsteadiness, and patients need to start at low doses and go through a slow

titration in order to diminish the risk of side effects.

6.14.5.3.3 PregabalinPregabalin (Figure 6), an alkylated analog of GABA, is a more potent anticonvulsant than gabapentin and has shown

enhanced analgesic potency as well.56 Pregabalin is clinically effective in pain associated with diabetic neuropathy and

postherpetic neuralgia. The enhanced potency of pregabalin relative to gabapentin appears to be related to

pharmacokinetic properties rather the mechanism of action. In this regard, the analgesic mechanism(s) for these

anticonvulsant compounds has not been definitively determined. Recent data indicate that these GABA analogs bind

the a2d subunit of voltage-gated calcium channels with high affinity.57 However, it should be noted that binding to the

a2d subunit is not likely to fully account for the analgesic properties of gabapentin or pregabalin.58

6.14.5.3.4 Antidepressants

In addition to antiepileptic drugs, tricyclic antidepressant drugs like amitriptyline are also used as adjunct analgesics in

the treatment of neuropathic pain.14,45 While other antidepressant medications such as selective serotonin reuptake

inhibitors (SSRIs) have not proven to be particularly efficacious in treating neuropathic pain, some of the newer



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combination serotoninnorepinephrine reuptake inhibitors such as duloxetine and venlafaxine significantly attenuate

neuropathic pain. Indeed, duloxetine (Figure 6) is the newest approved medications for the treatment of the pain

associated with diabetic neuropathy.59

Interestingly, in parallel to their preferred efficacy in the clinic, clinically used analgesics also have a differential

analgesic profile in preclinical pain models (see Table 1). While opioid analgesics like morphine are potent and efficacious

in all animal models of pain, including acute pain, pain associated with inflammation in which they are particularly potent,

and pain associated with neuropathy, NSAIDs such as ibuprofen and celecoxib are most potent and effective in animal

models associated with inflammation, with near to no efficacy in models of acute pain and neuropathic pain, and

antiepileptics like lamotrigine and gabapentin are most potent and efficacious in animal models of neuropathic pain.

6.14.6 Unmet Medical Needs

Due to the relatively poor efficacy to tolerability ratio for opioid analgesics in treating neuropathic pain, this area

represents a major unmet medical need. While gabapentin and other adjuvant analgesics have been reported to be

clinically effective in treating neuropathic pain, the efficacy rates are relatively small and are often accompanied by side

effects of sufficient magnitude to limit compliance.7 This situation is not surprising in that essentially all clinically used

analgesic adjuvants were originally developed to treat other indications. Their clinical utility in treating pain has been

based largely on clinical serendipity.

In the case of inflammatory or nociceptive pain, opioids provide significant analgesic efficacy; however, long-termuse of these analgesics is limited by both opioid-mediated side effects including constipation, and regulatory concern of

opioid dependence and abuse liability. NSAIDs also provide moderate pain relief in these pain states, but are associated

with GI disturbances. The COX-2 inhibitors represent an analgesic advance due to their enhanced GI safety profile;

however, the long-term cardiovascular safety of these agents is controversial.

6.14.7 New Research Areas

The identification clinically useful analgesic targeting nonopioid mechanisms has been challenging as demonstrated by

the failure of several novel tachykinin NK-1 receptor antagonists in clinical trials.39 Other novel analgesic agents have

either not been advanced or are used in only limited conditions due to mechanism related toxicities. For example, the

analgesic efficacy of ziconotide, a selective neuronal calcium channel (N-type, Cav2.2) blocker (discussed in more detailbelow) is limited to intrathecal administration in order to minimize severe cardiovascular adverse effects. Additionally,

the development of several classes of adenosine kinase inhibitors as analgesics was halted due to the occurrence of

vascular microhemorrhages in brain.60 Despite these difficulties in translating advances in pain neurobiology into clinical

useful analgesics, a number of novel analgesic mechanisms and compounds have been identified and validated in

preclinical models. Some of these are described below.

6.14.7.1 Neuronal Nicotinic Receptor Agonists

Activation of neuronal nicotinic receptors (NNRs) represents a novel approach to pain management supported by the

observation that epibatidine (isolated from Epipedobates tricolor) had significantly greater analgesic potency than

morphine in assays of acute thermal pain.61,62 While the mechanism of action of epibatidine was unknown, it was

subsequently found to be a picomolar agonists at NNRs. NNR agonists with higher affinity for the a4b2 subunit (thepredominant subtype in the CNS) relative to the a1b1dg nicotinic acetylcholine receptor subunit (located at the

neuromuscular junction) had analgesic efficacy with a larger therapeutic window from severe side effects than did

epibatidine.6367 These observations facilitated the discovery of ABT-594 (Figure 7), an a4b2-preferring NNR agonist

that was synthesized independently of the identification of the mechanism of action of epibatidine. ABT-594 has

broad-spectrum analgesic activity in both acute (hot plate, tail flick, formalin) inflammatory (CFA), and neuropathic

pain models. Importantly, ABT-594 showed less potential for analgesic tolerance than morphine in animal models and

did not produce pharmacologic dependence.6365

6.14.7.2 Vanilloid Receptor Modulators

The analgesic actions of topically applied capsaicin, the active ingredient in hot chillies, has been known for many years;

however, the clinical utility of vanilloid-derived analgesics has been limited by the initial burning sensation these



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compounds elicit. The cloning and characterization of the capsaicin-sensitive vanilloid receptor (TRPV1) has greatly

enhanced understanding of the mechanism by which vanilloids, acid, and heat may alter nociceptor sensitivity.68 TRPV1

receptors are one member for a larger family of transient receptor potential (TRP) proteins, several member of which

have been associated with sensory function including cold (TRPA1, TRPM8) and mechanical (TRPV3, TRPV4)

sensitivity.68 These findings have also led to the discovery of several distinct classes of TRPV1 antagonists (Figure 8).69,70

These compounds potently and selectively block capsaicin activation of TRPV1 in a competitive manner. Interestingly,

these antagonists also effectively block TRPV1 activation by acid and heat, indicating that these agents may exert channel

modulating activity as compared to direct channel activation. These latter effects may be species dependent with some

capsaicin-competitive antagonists being ineffective in blocking acid activation of rat TRPV1 receptors. TRPV1

antagonists have demonstrated analgesic efficacy in animal models of inflammatory pain, thus illustrating their potential

to be clinically effective analgesics.

6.14.7.3 Excitatory Amino Acid Receptor Antagonists

The excitatory amino acid (EAA) glutamate functions as a primary excitatory neurotransmitter in the CNS, and

activation of EAA-specific ionotropic and metabotropic receptor superfamilies in the spinal cord underlies the process

of central sensitization involved in chronic pain.16 Activation of the heteromultimeric NMDA receptors (NR1/NR2B/

NR2D subunits) expressed in spinal cord contributes to the expression of tactile and thermal hyperalgesia. A number

of competitive and noncompetitive NMDA receptor antagonists including (7)-CPP, MK-801, ketamine,

and dextromethorphan (Figure 9) block hyperalgesia in animal models and attenuate the process of central

sensitization.71,72 A problematic issue associated with NMDA receptor antagonists is their psychotomimetic effects

that include both dysphoria and cognitive impairment. This has led to the search for ligands that are selective for

specific NMDA channel subunits (e.g., NR2B) that may alter nociceptive processing with an improved therapeutic

window relative to previous NMDA antagonists. CP-101,606 (Figure 9), a selective NR2B antagonist, effectivelyreduced pain in low back pain and spinal cord injury patients.73 Adverse events associated with CP-101,606 treatment

were dizziness, and hypoesthesia, but were tolerated by the patients.

Memantine (Figure 9) is a low-affinity (NR1/NR2B IC50820 nM) noncompetitive NMDA antagonist that has

analgesic efficacy in humans. In early clinical studies, memantine attenuated ongoing neuropathic pain symptoms in

both diabetic and postherpetic neuralgia patients that memantine failed in Phase III pain indication trials.74 In addition

to these agents, other NMDA antagonists are being investigated preclinically as potential analgesic. For example,

MRZ-2/579 (Figure 9), a low-affinity noncompetitive NMDA antagonist, attenuates carrageenan-induced thermal

hyperalgesia at doses that do not affect sensory-motor function.75 GV 196771A (Figure 9) modulates NMDA receptor

function by blocking the glycine binding site of the NMDA receptor complex. Like memantine and dextromethorphan,

GV 196771A produces antihyperalgesia in animal models at doses that do not elicit CNS side effects.76Additionally, GV

196771A is only weakly active in cerebral stroke models suggesting differences in the physiological substrates of

nociception and neuroprotection. NMDA receptor antagonists can provide opioid sparing effects and may prevent the

ABT-594

NO

HN

Cl

Figure 7 ABT-594, a neuronal nicotinic receptor (NNR) agonist.

AMG 9810

O

OHN

O

OHN

O

Cl

N

NH

HN

O

F

FF

SB-366791 A-425619

Figure 8 Novel nonvanilloid TRPV1 receptor antagonists.



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tolerance related to prolonged opioid use. As noted above, late stage clinical trials of a dextromethorphan/morphine

combination (MorphiDex) are ongoing. Antagonists of the kainic acid subtype of the glutamate receptor include LY

293558 (Figure 9) which was active in acute migraine.77

6.14.7.4 Calcium Channel Modulators

Modulation of N-type (Cav2.2) calcium channels has been shown to provide an avenue for development of novel

analgesics, as exemplified by ziconotide, a 25 amino acid polycationic peptide originally isolated from the venom of a

cone snail. Its delivery is limited to the epidural and intrathecal routes, as systemic administration has led to a risk

for orthostatic hypotension.78 Ziconotide is approved for treating intractable cancer pain and chronic neuropathic

pain. The clinical efficacy of ziconotide provides important validation of this novel analgesic mechanism. In addition

to N-type calcium channels, T-type channels (Cav3.1 and Cav3.2) have also been implicated in nociceptive processing.

T-type calcium channels are expressed on dorsal root ganglion neurons and intrathecal antisense treatment targeting

the Cav3.2 subtype of T-type calcium channels effectively blocked all low-voltage calcium currents in dorsal root

ganglion neurons and significantly attenuated both acute and inflammatory pain.79

6.14.7.5 Cannabinoids

Marijuana (cannabis) has been used to relieve pain for centuries.80 However, clinical evaluation of the major active

cannabinoid, D9-tetrahydrocannabinol (D9-THC), has produced equivocal results in chronic cancer pain patients.

Furthermore, the analgesic actions of D9-THC could not be clearly separated from the other well-described

psychotropic actions ofD9-THC. Investigation of the pharmacological actions of the cannabinoids has been greatly

aided by the recent discovery of specific cannabinoid receptor subtypes (CB1 and CB2), elucidation of their signaltransduction pathways, and the identification of putative endogenous ligands (e.g., anandamide).80 High densities of

CB1 receptors are found in the CNS, while CB2 receptors are localized primarily to immune cells and peripheral nerve

terminals. These advances in cannabinoid pharmacology suggest the possibility of identifying receptor subtype

selective ligands.

Cannabimimetics have been shown to produce antinociception in animal pain models via spinal and supraspinal

actions on CB1 receptors, and by peripheral actions at CB2 receptors on sensory afferents and, indirectly, on immune

cells. Recent compounds in preclinical development include agonists with improved oral bioavailability and/or

enhanced receptor subtypes selectivity. CT-3 (Figure 10) is an orally active and nonselective analog of THC that dose-

dependently reduces acute nociception in the rat. Recently, CT-3 has been tested in the clinic in a phase II trial

in chronic neuropathic pain patients and the results suggested that CT-3 could be useful in treating this condition.80

O-1057 (Figure 10) is a potent and moderately CB1 receptor selective analog of CT-3 that has improved water

solubility and acute antinociceptive actions.67

HU-308 (Figure 10) is a novel, highly CB2 receptor selective agonist

CH3

CH3

H2N

Memantine

HN

O

OH

N

O

Cl

Cl

NH

H

H

COOHNN

NNH

GV 196771A LY 293558

NH3+

H3C

H3CH3C

CH3CH3

MRZ-2/579

N

OH

HO

OH

CP-101,606

O

NCH3

H

CH3O

Dextromethorphan

Figure 9 Antagonists for excitatory amino acid (glutamate) receptors.



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(Ki CB1410 mM, CB223 nM) that has antinociceptive effects in the persistent phase of the mouse formalin test, but

was inactive in the acute phase of the formalin test.81 While no effects of HU-308 were observed on motor function,

antinociceptive doses of the compound also reduced GI motility and blood pressure.82

More recently, the potential analgesic profile of CB2 agonists has been extensively characterized in in vivo pain

models in rodents. PRS-211375 (Figure 10) has been shown to be CB2 selective (CB2 Ki9 nM; CB1 Ki300 nM)

and a full agonist in adenylyl cyclase and GTPgS binding assays with similar potencies as observed in binding assays.

PRS-211375 has good CNS penetration and has shown efficacy in various animal models including formalin,

neuropathic pain models, acute thermal pain, visceral pain, and CFA-induced arthritis.83 PRS-211375 recently

completed phase I clinical trial. AM-1241 (Figure 10) is another selective CB2 agonist used in preclinical models toidentify the potential analgesic profile of CB2 agonists. This compound has been shown to decrease acute,

inflammatory, and neuropathic pain, its effects mediated through the release of b-endorphin and effects on opioid

receptors.84 GW405833 (Figure 10), another CB2 agonist, has also been shown to produce analgesia in animal models

of chronic pain including postoperative pain. However, contrary to the results obtained with AM-1241, the analgesic

effects of GW405833 are not mediated through activity at opioid receptors.85

6.14.7.6 Sodium Channel Modulators

The activation of voltage-gated sodium channels is necessary for the generation of neuronal action potentials. A feature

common to the local anesthetics and most analgesic adjuvants (e.g., carbamazepine, lamotrigine, and amitriptyline) is

their ability to block sodium channels and this property may underlie the clinical utility of these agents in reducingpain. However, all of these agents possess other pharmacologically relevant activities that results in limits their

analgesic effectiveness in the clinic. Notably, these include CNS sedation and/or untoward cardiovascular effects.

The cloning and characterization of several sensory nerve-specific sodium channel subtypes has raised interest in

the possibility of developing subtype-specific inhibitors which might overcome the cardiovascular and proconvulsant

liabilities of nonselective agents. The voltage-gated sodium channel gene family consists of multiple members, termed

Nav1.1 through Nav1.9.86 At least six of these channels are found in the peripheral nervous system.87 Structurally, the

family has a high overall degree of similarity (around 50% identity), with subfamilies being very closely related (up to

90% identity). Susceptibility to blockade by natural toxins, particularly tetrodotoxin (TTX), has been typically used to

classify sodium channel currents. Two TTX-resistant channels are present in the periphery: Nav1.8 (also called PN3 or

SNS) and Nav1.9 (also called NaN). Nav1.8 is likely to be the more important sodium channel in regulating nociceptive

signaling since in vivo antisense experiments targeting Nav1.9 did not reduce chronic neuropathic pain.88 Nav1.8

immunoreactivity is increased in the carrageenan inflammatory pain model, and increased proximal to the site of nerve

OR

O

CO2H

R = H

R =O

N+O

H

OCH3

H3CO

CH2OH

CT-3

O-1057

HU-308

N

OI

NO2N

CH3

AM-1241

O

O

OH

O

OO

PRS-211375

N

O

N

O

Cl Cl

OGW405833

Steriochemistry

Figure 10 Agonists for cannabinoid receptors.



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injury in rats and humans. Antisense oligonucleotides against Nav1.8 prevented thermal hyperalgesia or mechanical

allodynia from developing in animal models of neuropathic pain, and were also effective at reducing prostaglandin-

induced hyperalgesia. Nav1.8 knockout mice demonstrated a diminished response to noxious mechanical stimuli and

delayed inflammatory hyperalgesia. While the PN3/SNS subtype has been a major focus of research, other sodium

channels may also be appealing targets for pharmaceutical intervention of pain.89,90 These data indicate that the

development of antagonist that are selective for specific sodium channel subunits, like Nav1.8 may provide novel

analgesic agents. The feasibility of this approach is likely to be difficult based on the poor selectivity of currently

available sodium channel blockers, however, two NCEs have been recently described that show increased potency for

tetrodotoxin-resistant sodium channels as compared to the typical analgesic adjuvants. Ralfinamide (NW-1029)

(Figure 11) inhibits TTX-resistant currents in rat dorsal root ganglion neurons with an IC50 value of 10mM and dose-

dependently reduced allodynia in neuropathic pain models.91 CDA-54 (Figure 11) is a potent (IC50 B200 nM)

nonselective sodium channel blocker that reduces pain in inflammation pain models.92

6.14.7.7 Purines

6.14.7.7.1 P1 receptor agonists

The systemic or spinal administration of adenosine (ADO) or ADO A1 receptor selective agonists has been shown to

provide clinically effective analgesia.93 However, analgesic efficacy of systemically administered ADO in neuropathic

pain patients has been variable.94

In contrast, intrathecal injection of ADO or an A1 receptor agonist appears toconsistently reduce pathological pain in neuropathic subjects or in healthy volunteers given intradermal capsaicin to

induce central sensitization.9597 Despite these promising results, their profound effects on cardiovascular function

have hampered the development of ADO receptors agonists as analgesics.

Inhibition of the primary metabolic enzyme, adenosine kinase (AK), which regulates ADO availability enhances the

beneficial actions of adenosine at sites of tissue injury or trauma and AK inhibitors are potent antinociceptive and anti-

inflammatory agents with an potentially improved therapeutic window over direct acting ADO receptor agonists.98

ABT-702 (Figure 12), a non-nucleoside AK inhibitor, has both analgesic (e.g., effective in acute nociception) and

antihyperalgesic activity (e.g., effective in blocking both hyperalgesia and allodynia).98 The analgesic actions of ABT-

702 are blocked by selectivity ADO A1 receptor subtype antagonists and the locus of analgesic activity is in the spinal

cord.98ABT-702 also shows significantly greater separation between its antihyperalgesic activity and its effects on CNS

or cardiovascular function as compared to direct-acting ADO receptor agonists.

6.14.7.7.2 P2 receptor antagonists

The cloning and characterization of the family of ATP-sensitive ligand-gated ion channels (P2X receptors) provided

mechanistic insights for the role of ATP as an extracellular signaling molecule.99 The P2X3 channel is localized

O

F HN

NH2O

Ralfinamide CDA-54

O

HNN

OH2NO2S OCF3

Figure 11 Novel voltage-gated sodium channel blockers.

N

N N

NH2

N

Br

N

OABT-702

Figure 12 ABT-702, a nonnucleoside adenosine kinase (AK) inhibitor.



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primarily to sensory neurons, suggesting a role in pain transmission. The P2X3 messenger RNA (mRNA) occurs only in

the trigeminal, dorsal root, and nodose ganglia, and the receptor is selectively expressed in sensory C-fiber neurons that

project to the periphery and spinal cord, and which are predominantly nociceptors. In addition, P2X3 receptors located

presynaptically at the central terminals of primary afferent neurons may have a facilitatory role to enhance

neurotransmission, leading to a further increase in pain sensation.100

A role of ATP in pain transmission is consistent with the observed induction of pain by ATP upon application to

human skin, and with reports that intradermal and intrathecal application of ATP and ATP analogs (e.g., ab-methylene

ATP (abmetATP)) into the rat hind paw evokes acute nociceptive behavioral responses. Transgenic disruption of P2X3receptors in rodents via knockout, antisense, or short interfering RNA (siRNA) manipulations leads to decreased

nociceptive sensitivity.101,102 A-317491 (Figure 13), a potent and selective antagonist of homomeric P2X3 and

heteromeric P2X2/3 receptors that when given systemically and intrathecally dose-dependently reduces nociception in

animal models of inflammatory and neuropathic pain indicating that blockade of spinal P2X3 receptors may provide

broad-spectrum analgesic effects in chronic pain states.100

P2Y2 receptors signal through protein kinase C (PKC) and, in turn, modulates the activation of TRPV1 receptors.103

Intrathecal antisense studies have also shown that P2X4 receptors acting via a spinal neuralmicroglial interaction alter

nociceptive processing in neuropathic rodents.104 More recently, P2X7 receptor knockout mice have been characterized

and show reduced inflammation and neuropathic pain as compared to wild-type mice.105

P2X7 receptors are notlocalized on small-diameter neurons in the periphery, but are found on gial cells in the dorsal root ganglion.106 Blockade

of P2X7 receptors leads to inhibition of IL-1b release from macrophages and glial cells, while antagonists like A-740003

are active in pain models.110 In addition, P2X7 receptors mediate the ability of ATP to stimulate glutamate release from

glial cells, thus providing an additional mechanism for ATP-mediated fast neurotransmission.107

6.14.7.8 Emerging Pain Targets

As indicated above, a developing concept in pain research is the appreciation that neuroimmune interactions

participate in nociceptive signaling in chronic pain states.25 This has greatly added to the potential list of candidate

mechanisms that may offer an avenue for analgesic intervention. In addition to traditional mechanisms associated with

neuronneuron communication, cytokines, chemokines, and inflammatory acute phase proteins are now known to

contribute to nociceptive signaling. This collection of potential analgesic mechanisms is further complemented byfindings that extracellular acid may also play a neuromodulatory role in the transmission of pain signals. Members of the

TRP channel family are sensitive to both endogenous lipids as well as acidic pH.68,69 The activation of specific acid-

sensing ion channels (ASICs) also contributes to the encoding of noxious stimulation.108 The development of selective

antagonists of ASIC3 antagonist A-317567 (Figure 14) provided the demonstration that this acid-sensing channel

contributes to both inflammatory hyperalgesia and pain associated with skin incision.108

As noted above, neurotrophic factors play important roles in the remodeling of the peripheral and central nervous

systems in response to pain. Specifically, nerve growth factor (NGF) is a neurotrophin that is an important survival

factor for sensory neurons.109 However, NGF also has pronociceptive actions, producing pain and enhancing

hyperalgesia in both experimental animals and in human clinical studies. The recent development of small-molecule

antagonists like ALE0540 and PD90780 (Figure 15) as well as anti-NGF antibodies has enabled studies demonstrating

that these agents effectively reduce chronic arthritic pain, skin incision pain, and tactile allodynia following peripheral

nerve injury.109

A-317491

N

O

HO

O

OHHO O

O

O

N

N N

N

N

N

OOMe

OMe

A-740003

Figure 13 A-317491 and A-740003, selective P2X3 and P2X7 ligand-gated ion channel antagonists, respectively.



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6.14.8 Conclusions

The adverse physiological, psychological, and economic effects of inadequate pain management have become

increasingly recognized in recent years. This has been accompanied by a growing awareness on the part of patients and

caregivers that pain need not be tolerated, and an increased emphasis amongst physicians on the proactive treatment of

pain. The unmet need for new analgesics remains substantial. Recent advances in the neurobiology of pain, together

with the development of new preclinical and clinical pain paradigms, have revealed new opportunities for the

development of analgesics, and raised the exciting possibility of entirely novel classes of analgesics in the future.

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