2. Review of literature

Page 4

Immediate response to tissue damage / injury navigates the immune system of human body

for the healing process where acute inflammation is the key response. Resolution of the

inflammation requires the eradication of primary inflammatory cell and their mediators.

Unresolved inflammation resulting due to imbalance between tissue damage and tissue repair,

tends to progress towards chronic inflammatory disorder7. Inflammatory condition becomes

debilitating due to persistent pain and the possible progression towards functio laesa (loss of

functioning). Pain sensation is an integral component of human reflexes, which influences

human response to injurious stimuli. Though regarded a protective mechanism, the

perception of pain completely depends on the individual’s threshold to endure this sensation,

which in turn determines the degree of unpleasantness felt by the human body. Apart from

physiological factors modulating pain sensation, environmental factors also sway pain

perception and response 8.

In pathophysiological term, pain may be defined as ‘sensory consequence of neuronal activity

triggered by noxious stimuli, inflammation or damage to specific nociceptive pathways in the

nervous system9. Clinically, several processes and their secondary consequences (such as

nociception, pain perception, suffering and pain behavior) have been identified, exhibiting

association with pain8, 10

. However, the definition of nociception as ‘the detection of noxious

stimuli and the subsequent transmission of encoded information to the brain’, does not

simplify the intricacies involved in the pain pathway.

Painful and tissue-damaging stimuli are detected by the free nerve terminals of the small- and

medium-diameter primary afferent sensory neurons, whose cell bodies are located in the

dorsal root ganglia (DRG) and trigeminal ganglia11

. Most of the signaling cascades that

sensitize the DRG neurons, during persistent inflammatory or neuropathic pain, have been

identified 11

.

2. Review of literature

Page 5

Figure 2.1: The classification of major pain syndromes 12

.

As depicted in Figure 2.1, the pain syndrome has been classified majorly as nociceptive,

inflammatory and neuropathic pain 12

. When the nociceptor detects the noxious stimuli, it

elicits nociceptive pain for example pain after touching a hot object / pain after an intense

pinch (Figure 2.1). Type of pain which occurs in the absence of noxious stimulus is called as

allodynia, and type of pain with an exaggerated response to a noxious stimulus called as

hyperalgesia. The inflammatory condition after tissue injury sensitizes nociceptors by

decreasing their threshold potential which contributes to allodynia / hyperalgesia at an

inflamed site. This pain hypersensitivity is a distinctive component of inflammatory pain. In

the third class of pain which follows after peripheral nerve damage, nociceptors begin to fire

ectopically and contribute to the spontaneous element of neuropathic pain.

In the purview of the present work, inflammatory pain that arises from tissue injury,

involving interplay of several chemical mediators, has been extensively discussed in this

review. As the term inflammatory pain can be associated with a variety of clinical conditions,

2. Review of literature

Page 6

the focus in this review has been primarily on the type of pain arising from microbial

infection and arthritic conditions, thereby affecting the routine activities of patients.

Inflammatory pain develops when the sensitivity of the nociceptive system increases after the

tissue integrity is disrupted by trauma, heat, infection, toxins, inadequate immune responses,

tumors or other insults. After years of extensive research in molecular genetics and

applications of cellular physiology, it is somewhat clear that inflammatory pain signaling

mechanisms involve a series of key mediators cross-talking with each other, thus allowing

propagation of the pain cascade.

The initiation of the pain mechanism occurs at the tissue site (cutaneous or deep somatic

tissues) innervated by primary afferent neurons. Nerve endings of these neurons are equipped

with various receptors, called nociceptors, which bind specifically to the molecules present or

released at the tissue site in response to the intensity, localization and timing of the initiating

stimuli. Nociceptors are thinly myelinated Aδ and unmyelinated C fibres whose sensory

endings are so-called “free nerve endings” because they are not equipped with corpuscular

end organs 13

. Most of the nociceptors are polymodal, responding to noxious mechanical

stimuli (painful pressure, squeezing or cutting the tissue), to noxious thermal stimuli (heat or

cold), and to chemical stimuli 13

. These nociceptors are involved in the transduction of

painful stimuli into action potentials 14

. Through the signaling network established around

and within the primary afferent sensory neurons, information is relayed via synapses to the

second-order neurons in the dorsal horn of the spinal cord and further to the supra-spinal

structures, thalamus and brain stem 8.

2.1. The quintessential elements of inflammatory response

Following tissue injury several inflammatory and pain mediators such as ions (K+, H

+),

bradykinin, prostaglandin E2 (PGE2), prostacyclins, purines (ATP), amines [histamine, 5-

2. Review of literature

Page 7

hydroxytryptamine (5-HT)], and nitric oxide are released from damaged cells. At the site of

injury, immune cells especially tissue macrophages are recruited, which release mediators

such as cytokines (tumor necrosis factor-α & interleukins), chemokines (IL-8) and growth

factors (neurotrophic growth factors) 15

.

Figure 2.2: Peripheral mediators of inflammation and their target receptors / ion

channels 15

.

Histamine, a known inflammatory mediator is released from mast cells in response to other

mediators such as substance P (SP) and calcitonin gene-related peptide (CGRP), which are

released by primary afferent sensory fibers. Blood cells release cytokines, complement

factors C3a and C5a, serotonin, platelet-activating factor, neutrophil chemotactic factor,

fibrinopeptides, leukotrienes. Pain response mediators for example, bradykinin and plasmin

derived from plasma; serotonin, acetylcholine and ATP are released by injured platelets and

endothelial cells; PGE2 is produced by the enzymes COX-I and COX-II in damaged cells.

These mediators all together cause vasodilation and thereby increased vascular permeability.

2. Review of literature

Page 8

This finally results into plasma extravasation followed by neutrophils and monocytes

migration to the injury site16

.

Thus, the released inflammatory mediators directly bind to the cell surface nociceptors

including receptor tyrosine kinases (RTK), acid-sensitive ion channels (ASIC), transient

receptor potential (TRP) channels, two-pore potassium channels (K2P), and G protein-

coupled receptors (GPCR) (Figure 2.2).

Table 2.1: Major receptors involved in transduction, conduction and transmission of

pain response13, 15

Receptor/Ion channels Type Distribution Responds

to

Function

Transient receptor potential

(TRP) channel/Vanilloid

receptors (VR)

TRPV1/VR-1

Ligand gated

non-selective

cation

channel

Small to

medium size

neurons in

the

trigeminal

ganglia (TG)

and dorsal

root ganglia

(DRG)

Small

diameter

afferent

neurons;

CNS

Capsaicin,

thermal

stimuli

(43°C), H+,

anandamide,

12-(S)-

HPETE

Pain

sensitization

Acid sensing receptors (ASIC)

ASIC-3

Ion channels Throughout

the nervous

system

Dorsal root

ganglion

cells

Low pH -Produce

sustained

sodium

current

-Produce a

rapidly

inactivating

current

Purinergic receptors (P2X)

P2X3

Ionotropic

ligand gated

ion channels

Small

diameter

neurons

AMP, ADP

and ATP

-Fast

synaptic

transmission

Sodium channels

Toxin tetrodotoxin sensitive (TTX-S)

Toxin tetrodotoxin

resistant

Large

diameter

neurons

2. Review of literature

Page 9

(TTX-R)

SNS/PN3 channel

SNS/NaN

Small

diameter

nociceptor

neurons

Calcium channels Voltage

gated

-Involved in

transmitter

release from

the

presynaptic

terminals in

the dorsal

horn

-Prolonged

excitatory

states of the

neuronal

membrane

Tyrosine kinase receptors

TrkA

Nociceptive

sensory

neurones

N-methyl-d-aspartic acid

(NMDA) receptors

Postsynaptic

neurones

2.2. Secondary messengers downstream to receptor/ion channel activation or mediator

release

2.2.1. Cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA)

cAMP, was the first cellular second messenger discovered and also the first one implicated in

pain and nociceptor sensitization. A state of continuously elevated cAMP levels not only

determines the onset but also the duration of hyperalgesia. Researchers have reported robust

sensitization toward physical stimuli (hyperalgesia) and nociceptor sensitization with

intradermal injection of membrane permeable cAMP analogs 17

or the adenylyl cyclase

activator forskolin 18

. Additionally, cAMP also evokes transmitter release and modulates

voltage and ligand-gated ion channels crucial in pain19

.

cAMP signaling is widely held to be synonymous with the activity of its binding partner,

protein kinase A (PKA). Pharmacological along with genetic inhibition of PKA, results in a

reduction of inflammatory mediator-induced hyperalgesic behavior20

, in reduced nociceptor

2. Review of literature

Page 10

discharge 21

and in attenuated stimulus-induced peptide release as reported22

. Ion channels

particularly tetrodotoxin-resistant sodium channel, Nav1.8 (TTX-R INa) and transient receptor

potential cation channel subfamily V member 1 (TRPV1) express PKA phosphorylation sites

and any mutation in these sites results in ablation of channel modulation by PKA19

.

It has recently become clear that cAMP can also activate molecules other than PKA such as

calcium channels23

and the guanosine diphosphate / guanosine triphosphate (GDP/GTP)

exchange factor Epac 24

. Therefore, some of the established data on cAMP signaling

pathways in nociceptor sensitization must also take these additional targets into

consideration25

. While cAMP/PKA signaling is important for inflammatory hyperalgesia, it is

also clear that other second messenger pathways play critical roles in this process.

2.2.2. Protein kinase C (PKC)

PKC plays an important role in nociceptor activation as well as sensitization. Among the six

PKC isoforms, detected in doral root ganglion (DRG) neurons, only PKCε, a member of the

calcium-independent novel PKCs, has been shown to be activated by the inflammatory

mediators such as bradykinin, epinephrine, carrageenan, tumor necrosis factor alpha (TNF-

), and the protease-activated receptor (PAR2)26

. Although, in a PKCε knockout mouse, the

basal threshold to mechanical as well as thermal stimulation was unchanged, sensitization in

response to inflammatory mediator treatment reduced as observed by scientists.27

Sensitization of the nociceptor specific TTX-R INa was found dependent on PKCε activity27

,

and enhanced activity of another ion channel important in inflammatory pain, TRPV1, was

shown to require direct phosphorylation of TRPV1 by PKCε 28

.

2.2.3. Mitogen-Activated Protein Kinases (MAPK)

Mitogen-activated protein kinases (MAPKs) have also recently been implicated in nociceptor

sensitization associated with inflammation and peripheral neuropathy. In nociceptors,

2. Review of literature

Page 11

extracellular signal-regulated kinases (ERK) is activated by nerve growth factor (NGF)29, 30

,

capsaicin, electrical stimulation31

, Freund’s adjuvant32

and nerve transection32

. MAPK p38 is

activated in response to peripheral inflammation33

and activation of TRPV1 leads to p38-

dependent hyperalgesia34

. There is a body of evidence supporting p38 activation in spinal

cord and DRG neurons following inflammation, axotomy, and spinal nerve ligation,

contributing to neuropathic pain35, 36

. In the spinal nerve ligation model of painful peripheral

neuropathy, TNF- was found central to p38 phosphorylation and mechanical hyperalgesia37-

39. Receptor activation and retrograde transport of locally produced NGF also resulted in p38

activation leading to increased expression of TRPV133

. Other members of the MAPK family,

c-Jun amino-terminal kinase 1 (JNK) and ERK5, are also implicated in nociception19

.

2.2.4. Nitric Oxide (NO)

The second messenger nitric oxide (NO), other than the kinases, also contributes to induction

of pain and sensitization. The NO-producing enzyme, nitric oxide synthase (NOS), is found

in small- and medium-diameter, nociceptive DRG neurons. The nitric oxide synthase (NOS)

expression is increased in DRG neurons by noxious irritants as well as nerve injury40

.

Exposure to inflammatory mediators increases the production of cyclic guanosine

monophosphate (cGMP), the downstream effector of NO. In turn, inhibition of NOS

suppresses activity in dorsal roots originating from sciatic neuromas41

and reduces thermal

hyperalgesia established by chronic constriction injury or hind paw inflammation42

. In

cultured DRG neurons, prostaglandin E2 (PGE2)-induced increase of TTX-R INa was partially

suppressed by NOS inhibitors43

.

2.2.5 Bradykinin and its signaling pathway

Bradykinin (BK) is considered a primary mediator of pain and inflammation, exhibiting dual

role as potent and direct activator of nociceptors, and as inducer of prolonged inflammatory

2. Review of literature

Page 12

hyperalgesia. Subsequent to tissue damage, kallikreins, a subgroup of serine proteases, act on

kininogen precursors, present in blood stream, to yield BK. Bradykinin is rapidly inactivated

by proteolytic enzymes yielding des-[Arg9] bradykinin, a major active metabolite. However,

before inactivation, BK initiates an inflammatory positive feedback cycle, stimulating the

release of prostaglandins and cytokines, which in turn sensitize the inflamed tissue to BK44

.

This way, the magnitude of pain sensation, triggered by BK, amplifies several times.

The actions of BK are mediated through the activation of two types of G-protein coupled

receptors, BK1 and BK2. The BK1 receptor is expressed as a result of tissue damage and

inflammatory signals such as nerve growth factor (NGF) and the cytokines, tumor necrosis

factor (TNF ) and interleukin-1β/6 (IL-I β/6), whereas the BK2 receptor is constitutively

expressed45

.

Figure 2.3: Downstream signaling of bradykinin pathway46

Patients with rheumatoid arthritis, have an elevated level of circulating bradykinin thus

confirming the involvement of bradykinin in inflammatory pathophysiology. Bradykinin has

2. Review of literature

Page 13

also been observed to mediate bone resorption in chronic inflammatory conditions such as

periodontitis, osteomyelitis and rheumatoid arthritis44

.

2.2.6. Prostaglandins and their signaling pathway

Prostaglandins (PGs), another important mediator of pain and fever, are generally considered

to be sensitizing agents and they directly activate the nociceptors only in some case. PGs are

synthesized by the constitutive enzyme, cyclo-oxygenase-1 (COX-1). An isoform enzyme,

COX-2, is induced in peripheral tissues by inflammatory stimuli, cytokines and growth

factors.

Prostaglandins bind to specific G-protein coupled receptors and stimulate second messengers

such as protein kinase A (PKA) and PKC, thus mediating an increase in intracellular Ca2+

.

When PG act on EP2 receptor linked to Gs protein, it causes the activation of adenylyl

cyclase and PKA. PKA directly phosphorylates the TTX-R sodium channel Nav1.8 and

increases the magnitude of the peak currents. A resultant hyperpolarising shift in the voltage

dependence of activation is observed that leads to an increase in sensory neuron excitability45

.

2. Review of literature

Page 14

Figure 2.4: Downstream signaling of arachidonate pathway46

Prostaglandins, particularly PGE2 contribute to the sensitization of nociceptors and

mechanoreceptors in inflamed tissue as well as in inflamed joints47

. In addition to peripheral

sensitizing effects, PGE2 plays an important role in pain signaling also. PGE2 released in the

dorsal horns of the spinal cord, following nociceptive stimulation elicits multiple actions,

which include

(i) facilitation of neuropeptide release from central nociceptor terminals48, 49

(ii) spinal dis-inhibition by suppressing glycine receptor mediated inhibitory currents50, 51

(iii)increase excitability of dorsal horn neurons by depolarizing postsynaptic membranes52

2.2.7. Pro-inflammatory cytokines

Cytokines [tumor necrosis factor alpha (TNF- ), interleukin (IL)-1, and IL-6], are long

peptides, released by the immune cells (activated lymphocytes & macrophages). They may

act directly on nociceptors, leading to spontaneous pain or act indirectly by stimulating the

2. Review of literature

Page 15

release of prostaglandins through activation of the arachidonic acid pathway in the

phospholipid layer of membranes of damaged cells. Cytokines play role in both acute and

chronic phases of inflammation. During acute phases, cytokines appear to induce

sensitization via receptor-associated kinases and phosphorylation of ion channels, whereas in

chronic inflammation transcriptional up-regulation of receptors and secondary signaling

become important8, 53

.

Interleukin-1β and TNF-α, the first cytokines to be formed after tissue damage or infection,

affect directly specific receptors on sensorial neurons, leading to the “cascade” synthesis of

other effectors, such as other cytokines, chemokines, prostanoids, neurotrophins, nitric oxide,

kinins, lipids, adenosine triphosphate (ATP), and members of the complement pathway54

. In

central nervous system, these elements in turn cause glial cell proliferation and hypertrophy,

releasing relevant proinflammatory cytokines, TNF-α, IL-1β, and IL-6, forming a complex

network of independent activation54, 55

. The exclusive role of TNF- α is pictorially depicted

below.

2. Review of literature

Page 16

Figure 2.5: TNF-α mediated pain sensitization56

Cytokines are pleiotropic, which means they have a broad range of redundant, frequently

overlapping functions. To elaborate, different types of cells secrete the same cytokine, and a

single cytokine can affect several types of cells. Cytokine stimulates its target-cells to

produce more cytokines, establishing a cascade in their own formation. These extracellular

signaling proteins bind to their specific receptors, activating intracellular messengers that

regulate gene transcription. Therefore, cytokines influence the activity, differentiation,

proliferation, and survival of immune cells, in addition to regulating the production and

activity of other cytokines, influencing the inflammatory response 54

.

2. Review of literature

Page 17

2.2.8. Other important mediators of inflammation and pain

2.2.8.1. Adenosine triphosphate (ATP) signaling

ATP can elicit pain upon infusion into the skin via ATP-gated ion channels of the purinergic

receptors (P2X) family. P2X3 is expressed exclusively in small diameter nociceptive sensory

neurons, whereas P2X4 is expressed on microglia cells in dorsal horn. After nerve injury,

ATP acts on P2X4 stimulating the release of brain-derived neurotrophic factor (BDNF)57

.

BDNF acts on neurons to reduce the expression of an anion transporter resulting increased

intracellular Cl−

concentration. This disrupts normal inhibition of pain signaling as the

inhibitory transmitter -amino butyric acid (GABA) behaves as an excitatory transmitter

rather than an inhibitory one.

2.2.8.2. Neurotrophic growth factor

Neurotrophic growth factor (NGF) contributes significantly to neuron sensitivity modulation

during inflammation. NGF production is increased by inflammatory mediators particularly

IL-1β and TNF-58

. In human subjects, NGF produces cutaneous hyperalgesia at the

injection site and widespread deep pain persisting for several days. These sensitizing effects

are believed to be mediated partially by the direct action on nociceptors and partially via

mediators released by NGF- activated mast and other inflammatory cells8. NGF is secreted by

Schwann cells, mast cells, and macrophages. NGF interacts with mast cells and causes

degranulation and subsequent release of mediators such as 5-HT, histamine, and NGF itself.

NGF plays a major role in modifying the activity of sensory neurons via modulation of gene

expression through binding to its receptor neurotrophic tyrosine kinase receptor type 1

(TrkA) expressed in nociceptive sensory neurons. NGF up-regulates expression of voltage-

gated sodium channels, TRP channels, substance P, calcitonin-gene-related peptide (CGRP),

and bradykinin receptors45

. NGF also sensitizes nociceptor responses through post-

2. Review of literature

Page 18

translational modifications. For example, sensitization of TRPV1 by NGF is partly due to

phosphorylation by extracellular signal regulated kinase (ERK) activated by phosphatidyl

inositide-3 kinase (PI3K)59

.

In inflamed tissues, NGF promotes macrophage proliferation, degranulation, and release of

inflammatory mediators including NGF itself generating a self-activating cycle. Nerve

growth factor has both peripheral and central action in the nervous system by genetic

alteration and post-translational receptor and ion channels (such as TRPV1, PKA, PKC,

MAPK, and tetrodotoxin-resistant sodium channels) regulation, inducing thermal and

mechanical hyperalgesia. Nerve growth factor can also cause peripheral sensitization through

the activation of 5-lipoxygenase, which converts arachidonic acid in leukotrienes that cause

nociceptive afferents to become excitable to thermal and mechanical stimuli54

.

2.2.8.3. Serotonin (5-hydroxytryptamine, 5-HT) signaling

5-HT is released from platelets and acts on 14 distinct receptor types. Of these receptors, 5-

HT3 receptor is not linked to a G-protein mediated second messenger pathway. Instead, it

forms a ligand gated ion channel and causes Na+ influx leading to neuronal excitation.

Another receptor 5-HT1B receptor, upon ligand binding, inhibits adenylyl cyclase via Gi

and/or Go, regulating the release of 5-HT itself and several other neurotransmitters. 5-HT1B

receptor is present in the sensory neurons, spinal cord and many central nervous system

regions. 5-HT receptor antagonists are known to have antinociceptive effects within the

dorsal root ganglion and peripherally, whereas intra-thecal administration of 5-HT1B receptor

antagonists blocks 5 HT-induced analgesia and produces a moderate hyperalgesic response

(Giordano & Dyche, 1989).

2. Review of literature

Page 19

2.2.8.4. Glutamatergic signaling

Glutamate plays a key role in transmission of excitatory information from primary afferent

sensory neurons to dorsal horn neurons at the synapse in the spinal cord 60

. Excitation of the

nociceptive sensory neurons by tissue damage and nerve injury evokes a continuous release

of glutamate from central terminals of noxious sensory neurons. The released glutamate acts

on postsynaptic glutamate receptors such as N-methyl-d-aspartic acid (NMDA) receptors,

leading to central sensitization. Glutamate receptors also localize on central terminals of

primary afferent sensory neurons, where they are involved in controlling neurotransmitter

release.

The scaffolding protein Homer1 binds metabotropic glutamate receptors and inositol 1,4,5-

triphosphate (IP3) receptors in the postsynaptic compartment, forming an efficient signaling

complex that generates IP3 and releases Ca2+

from intracellular pools. Homer1a, an activity

dependent splice variant of Homer1, lacks the ability to link glutamate receptors to IP3

receptors and competitively disassembles synaptic glutamatergic signaling complexes.

Homer1a is rapidly induced in spinal neurons after peripheral injury in a NMDA receptor-

dependent manner, hence evokes a feedback mechanism to reduce glutamate-induced release

of Ca2+

from intracellular pools61

. Preventing upregulation of Homer1a using shRNAs in

mice exacerbates inflammatory pain, and heterotopic expression of Homer1a in specific

spinal segments reduces inflammatory hyperalgesia. Thus, Homer1 is involved in pain

plasticity.

2.3. Peripheral sensitization to inflammatory mediators

Inflammatory mediators modulate the response of primary afferent neurons to subsequent

stimuli, a mechanism termed as peripheral sensitization. The peripheral terminals of

nociceptors express receptors for the inflammatory mediators. Two important receptors are

2. Review of literature

Page 20

the transient receptor potential cation channel subtype V1 (TRPV1) and voltage-gated

sodium channels (e.g. Nav1.8 or 1.9), which detect noxious stimuli and transduce them into

electrical energy. Binding of the mediators to their receptors result in lowering of the

activation threshold of the TRPV1 and the Nav ion channels, by two proposed mechanisms,

(i) by inducing phosphorylation events at regulatory amino acid residues (ii) by increasing

expression levels. This increase in the sensitivity of the peripheral terminals of nociceptors in

inflamed tissue is termed peripheral sensitization and contributes to inflammatory pain

hypersensitivity or hyperalgesia8.

2.4. Central sensitization to inflammatory mediators

In addition to creating the peripheral “inflammatory exudate”, tissue injury also stimulates the

release of neurotransmitters (e.g. glutamate, substance P) from the central terminals of

nociceptors and augments the production of prostaglandin E2 (PGE2) and pro-inflammatory

cytokines (e.g. IL-1β, Il-6 and TNF- ) in the spinal cord. This causes additional excitation

and dis-inhibition of dorsal horn neurons and generates abnormal responses to sensory signals

from the periphery62, 63

. The pain spreads to regions beyond the site of tissue damage and

innocuous tactile stimulation now is processed to cause a painful sensation. This mechanism

underlying pain hypersensitivity is termed central sensitization64

. The processes underlying

sensitization, invoked by injury, reflect the plasticity of the nociceptive system. They are

usually reversible within hours to days following adequate responses of the nociceptive

system (e.g. in postoperative pain). Chronic inflammatory disease may condition persistent

modification of the architecture of the nociceptive system, which may lead to long lasting

changes in its responsiveness. These mechanisms contribute to chronic pain.

2. Review of literature

Page 21

2.5. The signal sequence and interplay

Cascade of signaling starts when bradykinin stimulates synthesis/release of prostaglandins.

Prostaglandins together with bradykinin sensitize the primary afferent nociceptive nerve

fibers. This sensitization occurs in response to stimulation by ATP, acetylcholine, serotonin,

mechanical and thermal stimuli16, 65

. Among prostaglandins, PGE2 results in direct peripheral

sensitization by acting on EP1 and EP4 receptors. This causes activation of TRPV1, TTX-R

Na+, T-type calcium channel, purinergic P2X3 receptors channels and inhibition of voltage-

gated potassium currents62

. Also, PGE2 indirectly sensitizes sensory neuron to bradykinin.

Substance P and CGRP released by the primary afferent sensory fibers contribute to the pain

response by triggering the release of histamine from mast cells, which, in turn, excites the

peripheral afferent sensory fibers16, 65

. Spinal CGRP receptors also appear to be involved in

the generation and maintenance of capsaicin-induced inflammatory pain66

. An intensified

sensation of pain occurs when primary afferent sensory fibers of nociceptive neurons are

sensitized by the excessively released mediators16, 67

.

Proinflammatory cytokines provoke inflammatory hyperalgesia by the vicious network of

mechanism. Importantly, TNF-α, IL-1β and IL-6 through bradykinin receptor modulation

stimulates the synthesis and release of CGRP and substance-P directly or through increasing

the sensitivity of TRPV-1 on DRG neurons. In damaged tissue and spinal cord, these

cytokines further induce the expression of COX-1 enzyme, leading to elevated level of

PGE254

. Thus, these pro-inflammatory cytokines have the propensity to induce the expression

of other mediators of inflammation and vice versa forming a vicious nociception cycle.

2. Review of literature

Page 22

Figure 2.6: Interplay between proinflammatory cytokines, substance P, CGRP, COX-

1/2 and PGE254

.

1.5. Transcription regulation of inflammatory mediators

At the nuclear level, transcription of proinflammatory mediator is regulated by the family of

transcription factors. For example rheumatoid arthritis, an autoimmune disease with chronic

inflammatory and persistent pain, has been investigated extensively to understand the role of

transcription factor(s) for over expression of pro-inflammatory mediators and thus in disease

progression. The major members of the transcription family which are involved

pathogenically are activator protein-1 (AP-1), nuclear factor-kappaB (NF-κB), signal

transducer and activator of transcription protein (STAT4), nuclear factor of activated T-cells

(NFAT), Forkhead (Foxo3a), Interferon (type 1)-related (non STAT) IRF-1,

CCAAT/enhancer-binding protein (C/EBP) (Figure 2.7) 68

.

2. Review of literature

Page 23

Figure 2.7: Trancription signaling in inflammatory arthritis68

.

2.5. Therapeutic interventions developed so far

An understanding of the intricacies involved in the pain and inflammation pathway, has

directed efforts towards developing target centric drugs. Of the many channels and receptors

expressed in nociceptive sensory neurons, so far quite a few have been identified, expanding

the avenues of knowledge about the intracellular transduction mechanisms activated in the

process. Identifying such selective molecules and pathways as therapeutic targets for the

treatment of pain, has been a challenging and arduous task. As many of these targets are

involved in a variety of physiological functions, it has proved difficult to develop analgesics

without undesirable side effects. However, research has been progressive to discover target

specific drug candidates, with minimal side effects on other physiological systems.

Exciting progress is being made in discovering the mechanisms operating in sensory

pathways of pain. Consequently, multiple potentially useful targets for novel analgesics have

been identified. Clinical treatment of pain, however, is still largely confined to opioids and

non-steroidal anti-inflammatory drugs (NSAIDs). COX-2 selective NSAIDs (cox-ibs) may be

2. Review of literature

Page 24

a clinical advance in terms of gastro-intestinal toxicity but not in terms of other side effects or

efficacy. Taking into consideration the partial effectiveness of currently available treatment

measures and the increasing number of elderly population, a rising prevalence of age-related

painful conditions like arthritis is expected and therefore successful pain treatment is sought

for. To bridge the gap between the advancing understanding of the neurobiology of pain and

the lack of progress in clinical pain therapy a greater effort is required to develop new

analgesics and to change the empirical pain treatment to a mechanism based and

individualized approach to pain management. This section outlines some advances in the

pharmacology of pain with a focus on inflammatory pain in arthritis.

As observed in arthritic pain, at a local level, mediators released from synovium, bone or

other tissues induce the sensitization of articular pain receptors69

. In chronic conditions such

as osteoarthritis (OA) or rheumatoid arthritis (RA), neural sensitization is not just confined to

the periphery. Clinically, this presents enhanced pain perception at the site of injury, along

with the development of pain and tenderness in normal tissues. Spinal nociceptive processing

in arthritic patients is under the influence of descending inhibitory controls and inputs from

other somatic structures70

. Both previous pain episodes and genetic factors are likely to

influence activity. Pain mechanisms in OA are still unclear, however, sensitization of

nociceptors present in synovium/capsule/ subchondral bone held responsible for the

propagation and maintenance of pain. Synovial free nerve endings are reported in pain

mediation1. An increased expression of pain mediators, such as substance P (SP) and

calcitonin gene related peptide (CGRP), was observed in the subchondral bone in knee joints,

hip joint capsule and soft tissues in patient with painful OA71, 72

.

The involvement of multiple mediators provides an opportunity for the intervention,

commonly used therapeutic strategies (acupuncture, transcutaneous electrical nerve

stimulation etc.) and pharmacological agents such as NSAIDs and the weaker opioid drugs

2. Review of literature

Page 25

which exert their effect on these mediators69

. Treatment with systemic or topical therapies

designed to reduce inflammatory mediators might be expected to have a beneficial effect,

which is in accord with clinical experience73

.

2. Review of literature

Page 26

Table 2.2: Currently available therapy for inflammation and pain

Drugs/drug candidates Function

-Non-steroidal anti-inflammatory drugs8 Inhibit COX enzymes and reduce formation of prostaglandins

-Nitric oxide releasing derivatives of NSAIDs8

-Nitro aspirin

Greater anti-nociceptive and anti-inflammatory effects

No damage to gastro-intestinal tract74

-EP receptor antagonists8 Block binding of PGE2 in animal models

-IP receptor antagonist8 Block binding of PGI2 in animal models

-Opioid agonists8

(Loperamide)

-Effective in experimental arthritis.

-Blocks spontaneous firing of fibres innervating inflamed skin

-Anti-nociception in inflammatory conditions

-Cannabinoids8

(Anandamide)

-Blocks nociception

-Activation of CB1 receptor is negatively coupled to adenylate

cyclase and blocks excitability and activation of primary afferents75

-Activation of CB2 receptor produces antinociceptive effects via

inhibition of immune cell function76

-P2X receptor antagonists77

-Preclinical efficacy in rodent models of neuropathic pain

-AZD9056, a P2X7 antagonist, which is into phase II clinical trials

for rheumatoid arthritis.

2. Review of literature

Page 27

-B-type natriuretic peptide (BNP)78

-Expressed in CGRP containing small neurons and IB4

(isolectin B4)-positive neurons

-Natriuretic peptide receptor-A (NPR-A)- BNP receptor

(present in CGRP-containing neurons)

-BNP negatively regulates nociceptive transmission through

presynaptic receptor NPR-A

-BNP was found to reduce the firing frequency of small DRG

neurons in the presence of glutamate by opening large-conductance

Ca2+

activated K channels (BKCa channels).

-Intrathecal injection of BNP has exhibited inhibitory effects on

formalin-induced flinching behavior and CFA-induced thermal

hyperalgesia in rats.

-Blockade of BNP signaling by BNP antibodies or cGMP-dependent

protein kinase (PKG) inhibitor KT5823 impaired the recovery from

CFA-induced thermal hyperalgesia.

CGRP receptor antagonist79

BIBN4096BS

-Effective in rat pain model of inflammation, induced by injection of

complete Freund adjuvant (CFA)

-Reduces inflammatory pain and spinal neuronal activity

-In vivo electrophysiological experiments suggest a predominantly

peripheral site of action

-BIBN4096BS inhibits vasodilatation, thus reducing infiltration of

inflammatory cells to the site of injury.

2. Review of literature

Page 28

2.6. Drawbacks associated with the available treatments

Nonsteroidal anti-inflammatory drugs (NSAIDs) are chemically diverse class of drugs which

are the mainstay for the treatment of inflammatory disease. These pharmaceutical agents

constitute one of the most widely used class of drugs, with more 70 million prescriptions and

more than 30 billion over-the counter tablets sold annually in the US alone. Currently

available NSAIDs intervene by the inhibition of cyclooxygenase (COX) and, consequently,

prevent the conversion of arachidonic acid into prostaglandins. The main limitations of

NSAIDs are on account of their side-effects, which include gastrointestinal ulcerogenicity

and bronchospasm, both being attributed to their nonselective inhibition of COX.

Gastrointestinal complications are strongly associated with the use of conventional NSAIDs

and are recognized as the most prevalent and severe cause of drug toxicity in the USA 80

.

Millions of patients use NSAIDs for the relief of various types of arthritis pain, stiffness, and

related symptoms. 16500 patients die due to NSAIDs induced gastric intolerance, which is

the third major cause of death among the disease associated mortality80

. This is attributed to

nonselective inhibition of COX isoenzymes and subsequent diminished PGE2 resulting in

mucosal damage to corpus area of stomach. So the requirement of balance between risk and

efficacy, selective NSAIDs such as cyclo-oxygenase (COX)-2 inhibitors were first introduced

with the purpose of providing symptomatic pain relief along with lesser gastrointestinal risk.

Later cardiovascular risk emerged with selective COX-2 inhibitor therapy attributed towards

disturbed vasoconstriction-vasorelaxation and platelet aggregation. Inhibition of endothelial

COX-2 by the cox-ibs directs the physiological shift towards elevation in TxA2 generation via

COX-1. This results in increased platelet aggregation and potent vasoconstriction leading to

cardiovascular complications.

2. Review of literature

Page 29

Another group of drugs available in the market, targeting specific cytokine network for the

treatment of chronic inflammatory condition such as RA are the biologics. For example TNF-

α antagonist (infliximab, adalimumab), IL-6 antagonist (tocilizumab), IL-1 antagonist

(anakinra). These drugs are very expensive. There is also the possibility of individual

showing hypersensitivity reaction to these biologics is in question. Thus, there is an unmet

demand of small molecules having diverse activity against inflammation and associated pain,

which can ameliorate the disease progression with minimum or no risk to patient (s).

2.7. Design and discovery of thiazolidin-4-ones for the treatment of inflammation and

pain

Thiazolidin-4-one derivatives were chosen for anti-inflammatory and analgesic screening.

The basis for selecting these compounds hails from the scientific history reporting their

potential as anti-inflammatory agents. In 1999, Goel et al reported anti-inflammatory action

of a molecule from the series of 2-Substituted -3-(4-bromo-2carboxyphenyl)-5-methyl-4-

thiazolidinones81

. This molecule exhibited less ulcerogenic properties as well. Vagdevi et al.,

(2006) synthesized nine thiazolidinone derivatives of naphtho [2,1-b]furan. Out of nine

compounds, one was reported to exhibit anti-inflammatory activity 82

. Kumar et al., (2007)

synthesized eight 2-(4’-oxo-2’-phenyl-thiazolidin-3’-yl-aminomethyl)-3[4”-(p-chlorophenyl)-

thiazol-2’’-yl]-6-bromoquinazolin-4-ones 83

. They screened all the compounds for anti-

inflammatory and analgesic activities, out of which one compound exhibited potential anti-

inflammatory and analgesic activity.

Taranalli et al., (2008) synthesized eight thiazolidin-4-one compounds and three spiro

derivatives of thiazolidin-4-one compounds. All the compounds showed significant anti-

inflammatory, analgesic and antipyretic activities. Out of the eight thaizolidin-4-one

compounds, five showed significant inhibition of COX-2 activity without inhibiting the

COX-1 activity 84

.

2. Review of literature

Page 30

In our laboratory, Kalia et al., (2007) synthesized N-[2-(3, 5-Di-tert-butyl-4-hydroxyphenyl)-

4-oxo-thiazolidin-3yl]-nicotinamide and reported anti-inflammatory activity in three

experimental models of inflammation5. This motivated us to explore more for the activity of

these derivatives against inflammation and associated pain disorder and to understand their

cellular and molecular mechanism of action.

2. Review of literature

Page 31

Table 2.3: Thiazolidin-4-ones (TZOs) basic ring structure and list of TZOs

2-(4-chlorophenoxy)-N-(2-(aryl)-4-oxothiazolidin-3-yl) acetamide

S. No. Compound

code -Ar (Aryl) R1 R2 R3 IUPAC name

1. Fural-

TGA O

2-furyl

H H H

2-(4-chlorophenoxy)-N-(2-(furan-

2-yl)-4-oxothiazolidin-3-yl)

acetamide

2. PCB-TGA

Cl

4-chlorophenyl

H H H

2-(4-chlorophenoxy)-N-(2-(4-

chlorophenyl)-4-

oxothiazolidin-3-yl) acetamide

S

N

O

ArNH CO C

R1

R2

O Cl

R3

2. Review of literature

Page 32

3. BHT-TGA

OH

3,5-diisobutyl 4-

hydroxybenzyl

H H H

2-(4-chlorophenoxy)-N-(2-(3,5-di-

tert-butyl-4-hydroxyphenyl)-4-

oxothiazolidin-3-yl)acetamide

4. ONB-TGA

O2N

2-nitrophenyl

H H H

2-(4-chlorophenoxy)-N-(2-(2-

nitrophenyl)-4-oxothiazolidin-3-

yl)acetamide

5. ANI-TGA

OCH3

4-anisolyl

H H H

2-(4-chlorophenoxy)-N-(2-(4-

methoxyphenyl)-4-oxothiazolidin-

3-yl) acetamide

6. CLOANI-

TGA

OCH3

4-anisolyl

CH3 CH3 H

2-(4-chlorophenoxy)-N-(2-(4-

methoxyphenyl)-4-oxothiazolidin-

3-yl)-2-methylpropanamide

2. Review of literature

Page 33

7. PBB-TGA

Br

4-bromophenyl

H H H

N-(2-(4-bromophenyl)-4-

oxothiazolidin-3-yl)-2-(4-

chlorophenoxy)acetamide

8. Dichloro-

TGA Cl

Cl

3,4-dichlorophenyl

H H H

2-(4-chlorophenoxy)-N-(2-(3,4-

dichlorophenyl)-4-oxothiazolidin-

3-yl) acetamide

9. PY-TLA N

2-pyridyl

H H

CH3

2-(4-chlorophenoxy)-N-(5-methyl-

4-oxo-2-(pyridin-2-yl)thiazolidin-

3-yl) acetamide

10. ANI-TLA

OCH3

4-anisolyl

H H

CH3

2-(4-chlorophenoxy)-N-(2-(4-

methoxyphenyl)-5-methyl-4-

oxothiazolidin-3-yl) acetamide

2. Review of literature

Page 34

2.8. Design and discovery of heterocyclic homoprostanoids for the treatment of

inflammation and pain

Prostanoids are the product of cyclooxygenase biosynthetic pathways and constitute a family

of lipid mediators of widely diverse structures and biological actions. Besides their known

pro-inflammatory role, several studies have revealed the anti-inflammatory effects of various

prostanoids and established their role in the resolution of inflammation along with

immunosuppressant action. For instance, PGE2 inhibits the humoral arm of immunity by

preventing the differentiation of B lymphocytes. Also, PGE2 decreases the release of

lysosomal enzymes and reactive oxygen species from neutrophils and histamine from mast

cells. Thus, the concept of developing derivatives of heterocyclic homoprostanoid appealed

the researchers and considerable work has been carried out on these molecules so far. A

pooled review of existing literature on pain modulatory action of heterocyclic

homoprostanoid derivatives is as follows.

Way back in the year 1969, Fried et al., synthesized several derivatives of 7–oxaprostanoic

acid, which were also screened for their biological activities, of which 7–oxa–13 prostanoic

acid exhibited PG–antagonism like effect on isolated tissues85

. Then in 1975, Bender et al,

synthesized a series of novel heterocyclic homoprostanoids86

, among which two molecules

exhibited potency 100 times greater than natural PGE1 and PGE2. Derivatives like cis–

epoxide of oleic acid and (8γ– trans)–10–thioxo–9, 11–dithia–1a–homoprostan–1-oic acid

have been reported to inhibit PGs–synthetase, with the potency twice that of phenylbutazone

and nine times of aspirin.

In 1992, a team of two scientists synthesized a series of novel heterocyclic homoprostanoids

incorporated with 1,2,5–oxadiazole, 2–oxoimidazole, 2–thioxoimidazole,1,4–

dihydropyrazine,1,4–diazepine and quinoxaline ring system. In the series, two molecules,

2. Review of literature

Page 35

with 2- thioxoimidazole and 2–oxoimidazole ring. These were found to possess anti–

inflammatory and analgesic activity.

In previous work from our laboratory6, a series of 19 heterocyclic homoprostanoids with 2-

thioxoimidazole, 2–oxoimidazole and quinoxaline ring were synthesized and screened for

their anti-inflammatory potential in vitro and in vivo. Results from the above work revealed

the anti-oxidant and anti-inflammatory activity of heterocyclic homoprostanoids with

quinoxaline ring. However, the study had its own limitations due to the absence of suitable

molecular approach, which could have suggested the possible mechanism of action. Also, the

potential of these derivatives against inflammation associated pain was not confirmed.

Thus, present work aimed at screening heterocyclic homoprostanoid analogues (Table 2.4)

for anti-inflammatory and analgesic activity in acute and chronic models inflammation and

associated pain with finding out the possible mechanism of action.

Table 2.4: List of synthetic heterocyclic homoprostanoids (HHPs)6

S. No.

Compound

coding

Chemical Structure IUPAC Name

1.

1

8-(5–octyl–2–thioxo–2H–

imidazol–4–yl)octanoic acid

2.

1a

8-(5–octyl–2–thioxo–2H–

imidazol–4–yl)octanoic acid

hydrazide

3.

1b

2-(nitrooxy)ethyl 8-(5-octyl-2-

thioxo-2H-imi dazol-4-yl)

octanoate

2. Review of literature

Page 36

Table 2.4: List of synthetic heterocyclic homoprostanoids (HHPs)6 continued…

4.

1c

8-(5–octyl–2–thioxo–2H–

imidazol–4–yl)-N-(4-oxo-2-

phenylthiazolidin-3-

yl)octanamide

5.

2b

2-(nitrooxy) ethyl 8-(5-octyl-2-

oxo-2H-imidazol-4-yl) octanoate

6.

2c

8-(5–octyl–2–oxo–2H–imidazol–

4–yl)-N-(4-oxo-2-

phenylthiazolidin-3-

yl)octanamide

7.

3

8-(3–octylquinoxalin-2-

yl)octanoic acid

8.

3a

8-(7-methyl-3–octylquinoxalin–

2–yl)octanoic acid

9.

3b

8-(7-chloro-3–octylquinoxalin–2–

yl)octanoic acid

10.

3c

8-(7-nitro-3–octylquinoxalin–2–

yl)octanoic acid

11.

4

8-(4–octyl-1, 2, 5-oxadiazol–3–

yl)octanoic acid

12.

5

8-(5–octyl-3-thioxo-2, 3-dihydro-

1, 2, 4-triazin-6-yl) octanoic acid

13.

6

8-(3–octyl-5, 7-dioxo-6, 7-

dihydro-5H-1, 4-diazepine-2-yl)

octanoic acid