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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
.
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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
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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
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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.
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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
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(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
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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,
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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
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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
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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
.
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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
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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.
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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
.
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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-
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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).
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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
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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.
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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.
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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
.
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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
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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
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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
.
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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
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