Thesis

1. INTRODUCTION 2014
Shri Vishnu College Of Pharmacy Page 1
Histamine: Histamine (2-[4-emidazolyl]ethylamine) was discovered in 1910 by Dale and
Laidlaw and identified as a mediator of anaphylactic reactions in 1932 (Ricardo Criado et
al., 2010).Histamine is a hydrophilic molecule comprising an imidazole ring and an amino
group connected by two methylene groups. The pharmacologically active fom at all
histamine receptors is the monocaionic Nγ-H tautomer. The three classes of histamine
receptors (H1,H2,H3) can be activated differently by analogs of histamine. Thus, 2-
methylhistamine preferentially elicits responses mediatted by H1 receptors, whereas 4(5)-
methylhistamine has a preferential effect on H2 receptors (Black et al.,1972). A chiral
analog of histamine with restricted conformational freedom,(R)-α-methylhistamine, is the
preferred agonist at H3 receptor sites (Arrang et al., 1987).
Distribution and Biosynthesis of Histamine:
Distribution: Histamine is a widely, if unevenly, distributed throughout the animal
kingdom and is present in many venoms, bacteria and plants. Almost all mammalian tissues
contain histamine in amounts ranging from less than 1µg/g to more than 100µg/g.
Concentrations in plasma and other body fluids generally are very low, but human
cerebrospinal fluid contains significant amounts. The mast cell is the predominant storage
site for histamine in most tissues; the concentration of histamine is particularly high in
tissues that contain large numbers of mast cells, such as skin, the mucosa of the bronchial
tree, and the intestinal mucosa.
Synthesis, Storage, and Metabolism: Histamine, in the amounts normally ingested or
formed by bacteria in the gastrointesttinal tract, is rapidly metabolized and eliminated in the
urine. Every mammalian tissue that contains histtamine is capable of synthesizing it from
histidine by virtue of its content of L-histidine decarboxylase. The chief site of histamine
storage in most tissues is the mast cell; in the blood, it is the basophil. These cells synthesize
histamine and store it in secretory granules. At the secretory granule pH of ~5.5, histamine
positively charged and ionically complexed with negative charged acidic groups on other
secretory granule constituents, priarily proteases and heparin or chondroitin sulfate
proteoglcans (Serafin and Austen,1987). The turnover rate of histamine in secretory is slow,
and when tissues rich in mast cell are depleted of their stores of histamine, it may take
weeks before concentrattions of the autacoid return to normal levels. Non mast cell sites of
histamine formation or storage include cells of the epidermis, cells in the gastric mucosa,

neurons within the central nervous system and cells in regenerating or rapidly growing
tissues. Turnover is rapid at these sites, since the histamine is continously releaseed rather
than stored.
Fig. No 1.0: Histamine biosynthesis and metabolism.
There are two major paths of histamine metabolism in human beings. The more important
of these involves ring methylation to form N-methylhistamine. This is catalyzed by
histamine-N-methyltransferase, which is widely distributed. Most of the N-methylhistamine
formed is then converted by monoamine oxidase (MAO) to N-methylimidazoleacettic acid.
This reaction can be blocked by MAO inhibitors.
Alternatively, histamine undergoes oxidative deamination catalyzed mainly by the
nonspecific enzyme diamine oxidase,yielding imidazoleacetic acid, which is then converted
to imidazoleacetic acid riboside. These metabolites have little or no activity and are excreted
in the urine. One important aspect regarding these metabolites, however, is that it has been
shown that measurement of N-methylhistamine in urine affords a more reliable index of
endogenous histamine producttion than does measurement of histamine, because it
circumvents the problem of artifactually elevated levels of some genitourinary tract bacteria

to decarboxylate histidine. In addition, the metabolism of histamne appears to be altered in
patients with mastocytosis such that measurement of histamine metabolite has been shown
to be a more sensitive diagnostic indicator of the disease than is measurement of histamine
(Keyzer et al.,1983).
Functions of endogenous histamine: Histamine is one of the preformed mediators stored
in the mast cell, its release as a resultt of the interactin of antigen with IgE antibodies on the
mastt cell surface plays a central role in immediate hypersensitivity and allergic responses.
The actions of histamine on bronchial smooth muscle and blood vessels account in part for
the symptoms of the allergic response. Histamine has a major role in the regulattion of
gastric acid secretion.
Role in allergic responses: The principal target cells of immediate hypersensitivity
reactions are mast cells and basophils. As part of the allergic response to an antigen,
reaginic antibodies (IgE) are generated and bind to the surface of mast cells and basophils
via high-affinity Fc receptors that are specific for IgE. This receptor, FcɛRI, consists ofα,β
and two γ chains, all of which have been moleclarly characterized. The IgE molecules
function as receptors for antigens, and FcɛRI, interact with signal transduction systems in
the membrane of sensitized cells. Upon exposure antigen bridges the IgE molecules and
causes activation of tyrosine kinases and subsequent phosphorylation of multiple protein
substrates within seconds after contact with antigen. Prominent among the newly
phosphorylated proteins are the β and γ subunits of the FcɛRI itself and phospholipase Cγ1
and Cγ2. Subsequently, inositol phosphorylated proteins are metabolized, with a result
being the release of Ca2+ from intracellular stores, thereby raising free cytosolic Ca2+ levels.
These events trigger the extrusion of the contents of secretory granules by exocytosis. The
secretory behavior of mast cells and basophils is similar to that of various endocrine and
exocrine glands and conforms to a general pattern of stimulus-secretion coupling in which a
secretagogue-induced rise in the intracellular concentration of Ca2+ serves to initiate
exocytosis. The mechanism by which the rise in Ca2+ leads to fusion of the secretory
granule with the plasma membrane is not fully elucidated,but is likely to involve activation
of Ca2+ /Calmodulin-dependent protein kinases and protein kinase C.
Release of Other Autacoids: The release of histamine provides only a partial explanation
for all of the biological effects that ensue from immediate hypersensitivity reactions. This is

because a broad spectrum of other inflammatory mediators is released upon mast cell
activation.
In addition to acticvation of phospholipase C and the hydrolysis of inositol
phospholipids, stimulation of IgE receptors also activates phospholipase A2, leading to the
production of a host of mediators including platlet-activating factor (PAF) and metabolites
of arachidonic acid.Leukotriene D4, which is generated in this way, is a potent contractor of
the smooth muscle of the bronchial tree. Kinins also are generated during some allergic
responses. Thus, mast cell secretes a variety of inflammatory compounds in addition to
histamine, and each contributes to varying extents to the major symptoms of the allergic
response: constriction of the bronchi, decrease in blood pressure, increased capillary
permeability and edema formation.
Histamine release by Drugs, Peptides, Venoms and Other Agents: Many compounds
including a large number of therapeutic agents, stimulate the release of histamine from mast
cells directly and without prior sensitization. Responses of this sort are most likely to occur
following intravenous injections of certain categories of substances, particularly those that
are organic bases. Among these bases are amides, amidines, quaternary ammonium
compounds, pyridinium compounds, piperidines, alkaloids and antibiotic bases.
Tubocurarine, succinylcholine, morphine, radiocontrast media and certain carbohydrate
plasma expanders also elicit the response. The phenomenon is one of clinical concern, for it
may account for unexpected anaphylactoid reactions. Vancomycin-induced “red-man
syndrome” involving upper body and facial flushing and hypotension may be mediated, at
least in part if not entirely, through histamine release (Levy et al., 1987).
In addition to therapeutic agents, certain experimental compounds stimulate the release
of histamine as their dominant pharmacological characteristic. The archetype is the
polybasic substance known as compound 48/80. This is a mixture of low molecular weight
polymers of p-methoxy-N-methylphenethylamine, of which the hexamer is mostt active
(Lagunoff et al., 1983).
Basic polypeptides often are effective histamine releasers, and their potency generally
increases with the number of basic groups over a limited range. Polymyxin B is very active;
others include bradykinin and substance P. Since basic polypeptides are released upon tissue
injury or are present in venoms, they constitute pathophsiological sttimuli to secretion for

mast cells and basophils. Anaphylotoxins (C3a and C5a), which are low molecular weight
peptides that are cleaved from the complemet system.
Histamine release by other means: Clinical conditions in which release off histamine
occurs in response to other stimuli include cold urticaria, cholinergic urticaria and solar
urticaria. Some of these involve specific secretory responses of the mast cells and indeed,
cell fixed IgE. However, histamine release also occurs whenever there is nonspecific cell
damage from any cause. The redness and urticaria that follow scratching of the skin is a
familiar example.
Gastric acid secretion: Histamine is a powerful gastric secretagogue and evokes a copious
secretion of acid from parietal cells by acting on H2 receptors.The output of pepsin and
intrinsic factor also is increased. However, the secretion of acid also is evoked by
stimulation of the vagus nerve and by the enteric hormone gastrin. In addition, there appear
to be cells in the gastric mucosa that contain somatostatin, which can inhibit secretion of
acid by parietal cells; the release of somatostatin is inhibited by acetylcholine.
Central nervous system: There is substantial evidence that histamine functions as a
neurotransmitter in the CNS. Histamine, histidine decarboxylase, and enzymes that catalyze
the degradation of histamine are distributed nonuniformly in the CNS and are concentrated
in synaptosomal fractions of brain homogenates. H1 receptors are found throughout the
CNS and are densely concentrated in the hypothalamus.
Histamine acting wakefulness via H1 receptors, explaining the potential for sedation by
classical antihistamines. Histamine acting through H1 receptors inhibits appetite (Ookuma et
al., 1993). Histamine-containing neurons may participate in the regulation of drinking, body
temperature and the secretion of antidiuretic hormone, as well as in the control of blood
pressure and the perception of pain.
Back ground of synthetic compounds:
Any heterocyclic skeleton containing nitrogen atom is the basis of several essential
pharmaceuticals and that of many physiologically active natural products. 2(1H)-
Pyridinone is one such nitrogen containing synthetically designed scaffold presenting
broad spectrum of biological activities.

2(1H)- Pyridinone with the formula C5H4NH(O) is a colourless crystalline solid used in
peptide synthesis and is well known to form hydrogen bonded structures somewhat
related to the base-pairing mechanism found in RNA and DNA. It exists in tautomeric
forms and goes by various synonyms such as 2-pyridones, 2(1H)-pyridone, 1-H-pyridine-2-
one, 1,2-dihydro-2-oxopyridine, 2-pyridinol, 1H-2-pyridone, 2-oxopyridone, 2-
hydroxypyridine (Dorigo P et al., 1993).
N
H
O
1
2
3
4
5
6
1H-Pyridin-2-one
Fig. No. 1.1: Structure of Pyridin-2-one
2-Pyridones constitute an important type of heterocyclic which have shown variety of
biological activities and is found in a variety of interesting compounds. It has received
remarkable attention due to its promising features as a key scaffold and in privileged
building blocks.

2. AIM AND OBJECTIVE 2014
Aim:
To evaluate the H1 antagonistic effect of some novel synthetic compounds on mice.
Objective:
 To study H1 antagonistic effect of fused [1,2,4] triazolo pyridinones (A1, A2, A13)
On isolated goat tracheal chain preparation (In-vitro).
 To study the anti-inflammatory effect of [1,2,4] triazolo pyridinones (A1, A2, A13)
by HRBC membrane stabilization method (In-vitro).
 To perform the acute toxicity studies of fused [1,2,4] triazolo pyridinones (A1, A2,
A13).
 To study the H1 antagonistic effect of fused [1,2,4] triazolo pyridinones (A1, A2,
A13) on swiss mice (In-vivo).

3. LITERATURE REVIEW 2014
LITERATUER REVIEW:
Histamine exerts its effects on target cells in various tissues by binding to its four receptors:
histamine receptor (HR)1, HR2, HR3, and HR4. These receptors belong to the G protein-
coupled receptors family (GPCRs). H1 receptor (HR1) is codified in the human
chromosome 3 and is responsible for many symptoms of allergic diseases, such as pruritus,
rhinorrhea, bronchospasm, and contraction of the intestinal smooth muscle. Activation of
HR1 stimulates the signaling pathways of inositol phospholipid culminating in the
formation of inositol 1,4,5-triphosphate (InsP3) and diacylglycerol (DAG), leading to an
increase in intracellular calcium. Moreover, when HR1 is stimulated, it can activate other
intracellular signaling pathways, such as phospholipase D and phospholipase A. Recently, it
was shown that stimulation of HR1 can activate the nuclear transcription factor KB (NFkB).
Both are involved in the development of allergic diseases. Historically, the potency of
antihistamines was verified through standard pharmacological trials, particularly from
guinea pig ileum or tracheal smooth muscle contraction. In these tissues, the drugs cause a
parallel displacement in the histamine concentration/response.
Table 3.1: Histamine receptors and their cellular mechanism:
Histamine
receptor
Cell and tissue expression Activated intracellular signals G proteins
HR1 Nerve cells, airway and vascular
smooth muscles, endothelial cells,
hepatocytes, epithelial cells,
neutrophils, eosinophils,
monocytes, DC, T and B cells.
Main signaling: enhanced Ca2+
Others: PhLC, PhLD, cGMP,
PhLA, NFκB
Gq/11
HR2 Nerve cells, airway and vascular
smooth muscles, hepatocytes,
chondrocytes, endothelial cells,
epithelial cells, neutrophils,
eosinophils, monocytes DC, T and
B cells.
Main signaling: enhanced cAMP
Others: Adenylate cyclase, c-
Fos, c- Jun, PKC, p70S6K
G±S

HR3 Histaminergic neurons,
eosinophils, DC, monocytes low
expression in peripheral tissues. It
inhibits histamine release and
synthesis.
Main signaling: inhibition of
cAMP
Others: enhanced Ca2+, MAP
Kinase
Gi/o
HR4 High expression on bone marrow
and peripheral hematopoietic
cells, eosinophils, neutrophils,
DC, T cells, basophils, mast cells,
low expression in nerve cells,
hepatocytes peripheral tissues,
spleen, thymus, lung, small
intestine, colon and heart. It
stimulates chemotaxis of
eosinophils and mast cells.
Enhanced Ca2+, inhibition of
cAMP.
Gi/o
Adapted source: Jutel M, et al.
Knowledge of molecular biology advanced dramatically over the last few years, especially
of the GPCRs expression in recombinant cell systems. This has changed our understanding
about the way that antihistamines interact with GPCRs to exert their effects. Classical
models of GPCRs need histamine receptors to be occupied by antagonist agents, which
initiate the activation of signal transduction pathways. However, it has been recently shown
that GPCRs may show spontaneous activation, which does not depend upon the occupation
of the receptor by an antagonist. This is denominated constitutional (physiological) activity
of the receptor, which has led to a reclassification of the drugs that act on GPRCs. (Ligand)
drugs traditionally considered antagonists, are now called inverse agonists, that is,
substances capable of reducing the constitutional activity of GPCRs, or neutral antagonists,
when ligands do not alter the basal activity of these receptors (GPCRs), but interfere with
the binding of their agonists. Since antihistamines can theoretically be both inverse agonists
and neutral antagonists, it is not yet clear whether the term “H1 receptor antagonist” is
accurate. Thus, the adoption of the term “H1 antihistamines” has been suggested.

Figure. No. 3.1: Mechanism of histamine receptors activation:
Adapted source: Leurs R, et al

The observation that the constitutional activity of GPCRs is often associated with mutant
GPCRs has strengthened the interest in this phenomenon as being the mechanism for several
genetic diseases. The functional model of GPCRs is constituted by a dynamic equilibrium
between its inactive (R) and active (R*) conformations. Based on this model, the
spontaneous isomerization of HR, independently of the agonist (histamine), from the
inactive state (R) to the active (R*), shifts the equilibrium towards the state of constitutional
activity of the GPCRs. This isomerization involves conformational alterations of the
receptors, which can be spontaneous or induced by mutations that alter the intramolecular
structure of GPCRs. Agonists preferably bind to histamine receptors in their active state to
increase their stability and force an equilibrium shift to the active state. The degree of the
shift will depend on whether it is a full or partial agonist. Conversely, an inverse agonist
preferably binds to the inactive state of the histamine receptor and moves the equilibrium in
the opposite direction, that is, in the direction of the inactive state (R). The degree of this
equilibrium shift will depend on the nature of the inverse agonist. The neutral agonist does
not differentiate between the active and inactive receptor state. Consequently, it binds to
both the active and inactive states and does not shift the equilibrium between the two states;
however, it interferes with the subsequent binding, both of agonists and inverse agonists.
Identification of the constitutional activity of the H1 receptor has suggested that the inverse
agonist could be the action mechanism of the then-called H1 antagonists and now called H1
antihistamines.
In addition, the constitutional activity of H1 receptors is not restricted to the activation of
phospholipase C (PLC), but it also activates the entire genetic transcription mediated by the
kappa B nuclear factor (NFκB). The constitutional activity of the H1 receptor mediating
NFκB activation was inhibited by all the antihistamines tested by Bakker et al. including
cetirizine, ebastine, epinastine, fexofenadine, loratadine, and mezolastine. This indicates that
all these agents act as inverse agonists.

Fig. No.3.11: H1 receptors and their action on the gene transcription of inflammatory
mediators
Adapted source:Leurs R, et al.
Anti-inflammatory properties of H1 antihistamines: H1 antihistamines had the ability to
inhibit the release of histamine from mast cells, several in-vitro and in-vivo studies have
been conducted to determine whether these drugs have properties, other than the inhibition
of histamine effects, that could contribute to the clinical efficacy of allergic diseases control.
It has been postulated that some of the anti-inflammatory effects of H1 antihistamines
follow their interaction with HRs, whereas others are independent of these receptors.
A possible mechanism of action of the inhibition effect of H1 antihistamines on the
accumulation of inflammatory cells and their activation on tissues is its capacity to suppress
NFκB activation, as described (Bakker et al). The NFκB is an omnipresent transcription
factor which binds to regions that promote many genes that regulate the production of
proinflammatory cytokines and adhesion molecules. The NFκB can be activated by
histamine and TNF α. Low concentrations of cetirizine and azelastine suppressed the
expression of NFkB in a parallel manner with the synthesis of cytokines, IL1β, IL6, IL8,
TNFα and GM CSF. In various clinical studies, cetirizine, azelastine, loratadine, and
levocarbastine reduced ICAM-1 expression. If these important anti-inflammatory effects are
secondary to their interaction with HRs, they will occur for all H1antihistamines clinically

used. Nevertheless, the intensity of these effects will depend upon their antihistaminic
potency and their dose (An Bras Dermatol et al., 2010)
H1 antagonistics on CNS:
First generation H1 antagonistics penetrate the blood–brain barrier readily. Their proclivity
to interfere with neurotransmission by histamine at central nervous system (CNS) H1-
receptors potentially leads to drowsiness, sedation, somnolence, fatigue leading to
impairment of cognitive function, memory and psychomotor performance. In addition, the
H1-antihistaminic effects in the brain are primarily responsible for the potentially life-
threatening toxicity of first-generation H1-antihistamines in overdose.
Fig. No. 3.12: The penetration (red colouring) of (A) diphenhydramine, a first-
generation H1-antihistamine, and (B) bepotastine, a secondgeneration H1-
antihistamine, into human brain shown by positron emission tomography
The adverse effects of these drugs have been widely reported, beginning shortly after they
were introduced six decades ago. A major advance in antihistamine development occurred
in the 1980s with the introduction of second-generation H1-antihistamine, which are
minimally or nonsedating because of their limited penetration of the blood–brain barrier. In
addition, these drugs are highly selective for the histamine H1-receptor and have no
anticholinergic effects

Fig. No. 3.13: A map of histaminergic neurons emanating from the tuberomamillary
nucleus in the brain
There are approximately 64000 histamine-producing neurones, located in the
tuberomamillary nucleus of the human brain. When activated, these neurones stimulate H1-
receptors in all of the major parts of the cerebrum, cerebellum, posterior pituitary and spinal
cord. The actions of histamine on H1-receptors in the brain have been implicated in arousal
in the circadian sleep/wake cycle, reinforcement of learning and memory, fluid balance,
suppression of feeding, control of body temperature, control of cardiovascular system and
mediation of stress-triggered release of ACTH and b-endorphin from the pituitary gland.
Furthermore, as neurotransmitter amines in the brain do not work individually but in a
complex integrated network, the central anticholinergic effects of first-generation H1-
antihistamines may contribute to their unwanted CNS effects. First-generation H1-
antihistamines markedly alter the circadian sleep/wake cycle. The release of histamine
during the day causes arousal, whereas its reduced production at night results in a passive
reduction of the arousal response. It is well established that taking first-generation H1-
antihistamines in doses commonly recommended for the treatment of allergic disorders
frequently leads to daytime somnolence, sedation, drowsiness, fatigue and impaired
concentration and memory (M. K. Church et al., 2010).
Pharmacology of antihistamines:
Although the efficacy of the different H1 antihistamines in the treatment of allergic patients
is similar, even when first and second generation antihistamines are compared, they are very

different in terms of their chemical structure, pharmacology and toxic potential. Therefore,
knowledge about their pharmacokinetic and pharmacodynamic characteristics becomes
relevant to the clinical use of these drugs, especially in very young and old individuals,
pregnant women and patients with co-morbidities.
Absorption:
Most H1 antihistamines have good absorption when administered orally, since most of them
reach effective plasma concentration within three hours after administration. The good
liposolubility of these molecules allows them to easily cross cellular membranes, which
facilitates their bioavailability. In some cases, the concomitant administration of these drugs
with the ingestion of particular food items may alter their plasmatic concentration. This is
explained by the presence of active transporting mechanisms of cellular membranes. The
most well-known mechanisms are P glycoprotein (gP) and organic anions transporting
polypeptides (OATP). These glycoproteins and polypeptides are found in the cellular
membrane and serve as active transporting systems for other molecules, for which they
show affinity. In some cases, these systems act as important elements in the absorption
and/or clearance of a few drugs. In other circumstances, they promote tissue detoxification,
depending on whether these transporting systems are localized in the cellular membranes of
the intestinal epithelium (drug absorption) or in the central nervous system (blood-brain
barrier, BHL) or kidneys (excretion), where they detoxify from drugs.
A few antihistamines behave as substrates of these transporting systems, such as
fexofenadine. However, other drugs, such as desloratadine, do not have their intestinal
absorption influenced by transporting systems. Variations in the bioavailability of a few
antihistamines have been documented. When a few antihistamines, such as fexofenadine,
are ingested with food that serves as a glycoprotein substrate, like grape or orange juice, or
with drugs that also have this property, such as verapamil, cimetidine, and probenecid,
variations in their bioavailability have been documented.
Metabolism and Excretion:
Most H1 antihistamines are metabolized and detoxified in the liver by a group of enzymes
that belong to the cytochrome p450 system (CYP). Only acrivastine, cetirizine,
levocetirizine, fexofenadine, and desloratadine prevent this metabolic passage to a relevant
extent, which makes them more predictable regarding their desirable effects and adverse

reactions. Cetirizine and levocetirizine are eliminated in urine, mainly in their unaltered
form, whereas fexofenadineis eliminated in feces, after biliary excretion, without metabolic
alterations. Other H1 antihistamines are transformed in the liver into active or inactive
metabolites, whose plasmatic concentration depends on the CYP system activity. This
activity is, on its turn, genetically determined; thus, some individuals have a high intrinsic
activity of these pathways, while others show a reduced activity of this enzymatic system,
namely the CYP3A4 or CYP2D6. In addition, the CYP system can be altered in special
metabolic conditions, such as infancy, advanced age, hepatic diseases or by the direct action
of other drugs which accelerate or delay the action of these enzymes in the metabolism of
H1 antihistamines.
Drug interactions decrease the plasmatic concentration of H1 antihistamines and,
consequently, reduce their clinical efficacy, such as when CYP3A4 inductors are
administered; for example, benzodiazepines with H1 antihistamines.
Conversely, we can increase the concentration of H1 antihistamines and their
bioavailability, thus intensifying their adverse reactions, such as when drugs that
competitively inhibit their metabolism by CYP are administered; for instance, with the
concomitant use of macrolides, antifungal drugs, and calcium channel antagonists. In such
cases, the safety margins of H1 antihistamines are minimal, with greater likelihood of
adverse effects since their plasma levels are unpredictable.
Classical or first-generation H1 antihistamines:
Classical antihistamines are lipophilic drugs classified into different groups according to
their chemical structure. All of them are metabolized by CYP in the liver and do not serve
as gP substrates. Although not all metabolic pathways are completely known, most classical
H1 antihistamines are metabolized by CYP2D6, and some of them by CYP3A4. Studies
based on the use of diphenhydramine, as an example of a first-generation H1 antihistamine,
have shown that these drugs are not only CYP2D6 substrates, but also inhibit this pathway
of cytochrome p450. This should be considered when other drugs that use this metabolic
pathway are administered concomitantly, such as metoprolol, tricyclic antidepressants and
tramadol. Moreover, classical H1 antihistamines have several adverse effects due to their
actions in muscarinic (anticholinergiceffect), serotoninergic, and adrenergic receptors. First
generation H1 antihistamines are rapidly absorbed and metabolized, which means that they
should be administered three or four times a day. Due to their lipophilic molecular structure,

they cross the blood-brain barrier, bind easily to the cerebral H1receptors, and thereby
create their main adverse effect: sedation. In addition, they do not behave as P glycoprotein
substrate in the endothelium of the blood-brain vessels.
Second-generation H1 antihistamines:
Metabolic interactions of second-generation H1 antihistamines, such as terfenadine,
astemizol, loratadine, desloratadine, ebastine, fexofenadine, cetirizine, levocetirizine,
mizolastine, epinastine, and rupatadine have been intensively studied, since the first reports
of severe cardiac arrhythmia associated with the use of terfenadine. In general, we can state
that second-generation H1 antihistamines act as a gP substrates. Due to this fact, they have
less sedative effects than first-generation H1 antihistamines, since they are removed from
the CNS by gP. Nonetheless, a few second-generation H1 antihistamines undergo an
important initial metabolization in the liver or intestine, mediated by CYP. The metabolism
of H1 antihistamines via CYP3A4 became relevant due to observations of drug interactions
between terfenadine, erythromycin, and ketoconazole. Later, other CYP3A4 substrates
and/or inhibitors, such as fluoxetine, troleandomycin and zileuton, among other drugs, were
investigated in relation to their interaction with terfenadine, which has its plasmatic levels
increased when co-administered with these drugs.
Table 3.11: Chemical classification of H1 antihistamines:
Alkylamin-
es
Ethanolamines Ethylenediamn
-es
Phenothiazin-
es
Piperazin-
es
Piperidines
Bromophen
iramine
Chlorphenir
amine
Dexchlorph
eniramine
Phenirami-
ne
Dimethind-
ene
Triprolkline
Acrivastine
Carbinoxamine
Clemastine
Dimenhydrinate
Diphenhydramie
Doxylamine
Phenyltoxamine
Antazoline
Pyrilamine
Tripelenamine
Promethazine
Mequitazine
Trimepazine
Buclizine
Cyclizine
Meclizine
Oxatomide
Cetrizine
Aaztadine
Ketotifen
Terfenadine
Fexofenadine

Fig. No. 3.14: Symptoms and signs of the adverse effects of first-generation H1
antihistamines:
H1 antihistamines in urticaria:
Second-generation H1 antihistamines are the only drugs with class 1 evidence and A level
of recommendation by evidence-based medicine (EBM) indicated for the treatment of
chronic urticaria (CU), due to the existence of randomized prospective, doubleblind, and
placebo-controlled studies. H1 antihistamines are first line drugs indicated for the
symptomatic treatment of CU (An Bras Dermatol et al., 2010). Second-generation H1
antihistamines offer moderate to good control in 44-91% of all types of urticaria and in 55%
of CU patients. All H1 antihistamines are more effective in reducing pruritus than in
decreasing the frequency, number or size of urticas (Paulo Ricardo Criado et al 2009).
The Role of Antihistamines in Allergic Rhinitis and Asthma:
H1-antihistamines are widely used in the treatment of allergic and nonallergic disorders. In
patients with allergic rhinitis, second generation H1-antihistamines prevent and relieve the
sneezing, itching, rhinorrhea, and nasal congestion that characterize the early and the late
response to allergen. In the treatment of allergic rhinitis, oral H1- antihistamines are more
efficacious than chromones; but less efficacious than nasal glucocorticoids. In a study done

comparing nasal H1-antihistamine and nasal glucocorticoid in patients with allergic and
nonallergic rhinitis, azelastine was as effective as triamcinolone in improving nasal
symptoms; sleep symptoms and quality of life. H1-antihistamines are not medications of
choice in asthmatic patients.
However, comorbidity of asthma and allergic rhinitis is very high (80%) and they have
clinically relevant antiasthmatic properties by controlling rhinitis. Confirming this
statement, it was shown that antihistamines have reduced asthma symptoms in patients with
seasonal allergic rhinitis when given alone or in combination with an antileukotriene. The
early treatment of atopic child (ETAC) study also showed that the onset of asthma was
prevented by continuous antihistamine treatment. Therefore, H1-antihistamines appear to
provide indirect benefit in patients with concomitant asthma and allergic rhinitis.
Combination of H1-Antihistamine and Cysteinyl Leukotriene Receptor Antagonist
Both antihistamines and antileukotrienes have been found to be useful when used alone in
allergic rhinitis and asthma. Combination of both drugs showed a synergistic effect in
treating seasonal allergic rhinitis. The first combination treatment studies with montelukast
was done with second generation antihistamines such as loratadine and cetirizine, followed
by combinations with third generation antihistamines fexofenadine, desloratadine,
levocetirizine. Then fixed combinations of montelukast-levocetirizine and montelukast-
desloratadine tablets were aiming to increase the quality of life of the patients (Ayse
Baccioglu et al., 2003).
Physiologic Role for Histamine in Lung and Asthma:
One of the first identified actions of histamine was its constrictor actions on airway
smooth muscle. This response was subsequently shown to exacerbate disease when Weiss
reported that administration of histamine to asthmatic patients resulted in breathlessness and
decreased vital capacity. This association was strengthened with the observation that the
bronchoconstrictor response to histamine was enhanced in asthmatic versus normal
individuals and that these responses could be blocked by a prototypical H1R antihistamine
drug.2 Subsequently, development of more selective H1R antagonists allowed for studies to
more clearly define the role of histamine and H1R in bronchoconstriction. In one important
study, inhalation of histamine leading to bronchoconstriction and a decrease in FEV1
(forced expiratory volume in 1 second), was significantly inhibited by oral administration of
all H1R antagonists tested, whose efficacy in the lung correlated well with that observed

against histamine induced skin responses in the same patients. In addition, none of the
antihistamines were able to inhibit methacholine induced bronchoconstriction.
Consequently, this one study demonstrated the selectivity and specificity of the H1R
mediated bronchoconstrictor response to histamine, whilst supporting the theory that the
histamine induced vasodilatory responses in skin and bronchoconstrictor responses in lung
are mediated by the same receptor.
While histamine is incontrovertibly a constrictor of large and small airway smooth
muscle, the direct contractile effect of histamine on airway smooth muscle cells is largely
inferred from in vitro studies on human tissue. There is some debate as to whether, in vivo,
H1R present on sensory nerves may contribute to an indirect contractile response via
stimulation of a vagally mediated parasympathetic reflex. Whilst this may be relevant in
some species, the lack of effect of anticholinergics on histamine-induced
bronchoconstriction in humans would seem to argue against this. In addition, a role for
histamine in maintaining bronchial smooth muscle tone, in the absence of nerve innervation
has also been demonstrated. Constitutive production of histamine and cysteinyl leukotrienes
appeared to impart a resting tone on bronchial smooth muscle that could be significantly
reversed by addition of H1R antagonists and CysLT1 receptor antagonists, respectively.
Provocatively, H1R antagonists dosed to asthma patients can produce immediate
bronchodilatory responses, with one study demonstrating that cetirizine was almost as
potent a bronchodilatory agent as a β- adrenergic agonist. However, as discussed in detail
below, these effects are inconsistent and appear to fade with continuous treatment.
The previously mentioned edematous and vasodilator responses observed in the skin to
histamine could additionally have pathological implications in asthmatic airways via the
causation of mucosal edema and the facilitation of plasma proteins and leukocyte movement
into the affected tissue. These vasodilator effects are mediated by H1R on vascular
endothelium which act to increase paracellular permeability, whilst the movement of cells is
additionally facilitated by the H1R dependent upregulation of adhesion molecules such as
ICAM-1, E-selectin and P-selectin on endothelial and epithelial cells, particularly under
inflammatory conditions. Histamine acting on H1R on endothelium and airway epithelium
may have direct pro-inflammatory effects via the release of cytokines such as IL-6 and
IL-824, and has also been shown to augment the release of IL-16, a potent chemokine for T
helper cells, from airway epithelial cells. Interestingly, histamine has been shown to
similarly induce IL-16 release from CD8
+
T cells in a H4R and H2R dependent fashion.

All of these actions of histamine on airway structural cells may combine to promote an
inflammatory phenotype, however a caveat of all these data is that they have largely been
obtained in vitro and their in vivo significance is unknown. Likewise, a role for histamine in
airway smooth muscle cell proliferation in vitro has also been reported and other studies
have suggested histamine receptors to be involved in the hypersecretory response seen in
asthmatic airways. Whilst H1R may be linked to airway secretion via an effect secondary to
its role in plasma exudation, the H2R has been shown to be the sole histamine receptor
involved in mucus secretion, which is consistent with the expression of H2R in secretory
cells from nasal mucosa.
In summary, the pharmacological effects of histamine on the lung recapitulate many of
the pathophysiological symptoms of asthma and that the potential role of histamine as an
important mediator in asthma is further suggested by its increased presence in the asthmatic
airway. In the rest of this chapter we will examine whether this ‘guilt by association’ is
borne out by the evidence from clinical studies with antihistamines and whether newer
insight into the role of histamine in immune modulation, potentially through receptors not
targeted by current antihistamines, may have implications for the future treatment of
asthma.
Immunological Modulation by Histamine
Many cells of the immune system associated with asthma have been shown to express a
range of histamine receptors, notably H1R, H2R and the newly described H4R. In most
cases these receptors are co-expressed so that the net effect of their activation may vary,
depending on the exact expression profile and the concentration of histamine in the
surrounding milieu, and is further complicated since the receptors may have opposing
function. It is therefore difficult to interpret the significance of the in vitro literature in
which many of these conditions may be manipulated to observe a specific effect and hence
preclinical examination of their role in the whole animal is likely the most relevant predictor
of their role in human disease.
Mast Cells and Basophils
Mast cells are not only the main source of histamine in the lung, but are a source of
cytokines and tissue growth factors that may be important in the inflammatory and
remodeling processes observed in asthma. They themselves may be modulated by histamine
through expression of H1R, H2R and H4R on their surface. While histamine does not

appear to affect degranulation of mast cells, it has been shown to be a potent
chemoattractant for mast cells and has been shown to enhance chemotaxis of mast cell
precursors in response to CXCL12, all via activity at the H4R. In addition, inhaled
histamine was able to increase the number of sub and intra-epithelial mast cells in mouse
airways in an H4R dependent fashion.
Interestingly, localization of mast cells to the bronchial epithelium has been observed in
asthmatics after allergen challenge. There are several reports of H1R antagonists reducing
leukotriene and histamine release from human lung mast cells and basophils , but these
effects are believed to be independent of their antagonism of histamine, as previously
reviewed. Histamine acting at the H2R does appear to have inhibitory effects on mast cells,
decreasing histamine and cytokine release and may have similar actions on basophils. H4R
has also been detected on basophils but its role in their function has yet to be studied.
Fig.No. 3.15: Potential role of histamine in asthma:

Potential role of histamine in asthma:
Allergen entering the airways may cross-link IgE on mast cells(or basophils) to release
histamine, lipid mediators and cytokines. Antigen is also processed by airway dendritic cells
and macrophages for presentation to T helper cells. During this process local release of
histamine and cytokines may also occur. Resultant histamine from these processes can act at
a variety of cells and levels. Histamine can facilitate the recruitment of inflammatory cells
via direct chemotaxis of additional dendritic cells, eosinophils and mast cells to the airways
via action at H4R, whilst also aiding the chemotactic and inflammatory process through
effects at H1R on the airway epithelium and vascular endothelium. Release of cytokines,
such as IL-8, from the airway epithelium and increased vascular permeability in response to
H1R activation enrich the inflammatory milieu. Constriction and proliferation of airway
smooth muscle via H1R also contributes to the asthma phenotype.
Histamine has diverse effects on the activation of leukocytes via H1R and H4R. Complex
autocrine and paracrine processes in response to dendritic cell histamine and cytokine
release control the priming and education of T cells via cytokines that are released from
dendritic cells in response to H1R and H4R ligation, during antigen presentation. Histamine
may additionally affect the cytokine release from CD8+ cells via H4R and from mast cells,
eosinophils and neutrophils through multiple histamine receptor activity.
Eosinophils:
Eosinophils are traditionally one of the major cell types implicated in the pathology of
asthma, due to their observed increase in asthmatic lungs and the plethora of cytotoxic and
pro-inflammatory mediators, linked with disease progression, that they are able to release.
Although a direct causative role in disease has been difficult to prove, recent pharmacologic
intervention has suggested their importance in at least a subset of asthma patients.
Therefore, it is provocative to note that, as for mast cells, histamine has been shown to be a
potent chemoattractant and enhancer of chemokine mediated chemotaxis, once more
through the recently described H4R. H4R dependent upregulation of adhesion molecules
was also reported. H1R antagonists have been investigated for their role in eosinophil
function, but effects via nonhistaminergic pathways at irrelevant concentrations confuse the
interpretation of these studies. As with mast cells and basophils, these activities appear
independent of H1R activity and are not discussed here, as they do not contribute to an
understanding of the role of histamine in asthma.

Neutrophils:
The presence of neutrophils in the airways has been demonstrated during asthma
exacerbations, after treatment withdrawal and in a sub-set of chronic asthma patients with
severe disease. In patients that die suddenly from asthma, they are also increased, with the
same study demonstrating elevated histamine levels. Interestingly, neutrophils have also
recently been identified as a source of histamine in the lung, so this correlation may be
effect rather than cause. The role of histamine in neutrophil function is also unclear, with
any function appearing limited to the H2R, which may in fact have a negative regulatory
role on their function. Histamine may have an indirect effect on neutrophil chemotaxis via
its H1R-dependent ability to stimulate release of proneutrophilic cytokines and chemokines
from airway tissue. Similarly, an indirect role for H4R has been reported in mast cell
dependent models of neutrophilia.
Monocytes and Macrophages:
Expression of the H1R, H2R and H4R have been demonstrated in human monocytes yet
their expression on different macrophage lineages and at different levels of activation may
vary and has not been well studied. Alveolar macrophages are the most abundant
inflammatory cell in the human lung, yet their association with asthma is not clear, likely
due to the difficulty in studying this highly heterogenous population. However, alveolar
macrophage suppression of T-cell proliferation is reduced in asthma and after allergen
challenge and some alterations in specific subpopulations have recently been described in
asthma. In vitro studies have demonstrated that histamine may have modulatory effects on
LPS stimulated monocytes via the H2R, including a reduction in the production of IL-12
and an increase in IL-10 release, which could conceivably promote Th2 cell development.
The constitutive production of MCP-1 (CCL2) has also been reported to be inhibited via an
action at the H4R. In alveolar macrophages, specifically, H1R mediates histamine induced
β-glucuronidase and IL-6 release, which may indicate a role for histamine in
macrophage-mediated remodeling processes.
Dendritic Cells:
Dendritic cells are professional antigen presenting cells that may develop from cells of
either lymphoid or monocytic lineage. They are intimately associated with the pathogenesis
of asthma through their initiation and maintenance of T-cell responses, particularly Th2
type. Polarization of naïve Th0 cells to Th2 and other T helper sub-sets may be
differentially controlled at the level of the interaction between dendritic cells and

antigen-specific T cells. Such interaction can be directed by a variety of cytokines,
chemokines, toll-ligands and biogenic amines, such as histamine. These are released at sites
where antigen is encountered or presented and may sequentially modulate both the dendritic
cell and subsequent T helper phenotypes. All four histamine receptors have been identified
on immature and mature dendritic cells. Histamine, released from dendritic cells or more
traditional sources, may act in an autocrine or paracrine fashion to modify their phenotype,
as measured by alterations in surface markers, or in cytokine release. Cytokine secretion,
including inhibition of IL-12 and enhancement of IL-10 and IL-6, may be modulated by
histamine with H1R, H2R and H4R all involved in these Th2 promoting processes. The
autocrine activation of dendritic cells by histamine deserves additional discussion, since it is
indicative that low levels of locally released histamine are able to define and control
immune responses that may be important in asthma, via actions at high affinity histamine
receptors, such as H4R and at concentrations where low affinity H1R and H2R may not be
engaged. This may suggest that the high levels of histamine frequently cited as correlating
with asthma severity are unrelated to the underlying immunology of asthma and therefore,
at best, may only have a relationship to the physiological sequalae previously described.
T Cells
As described above, T cells are pivotal cells in the initiation and perpetuation of adaptive
immune response associated with allergic asthma. In addition to being modulated by the
effects of histamine on dendritic cells, they may be directly affected by histamine. H1R,
H2R and H4R are all expressed on CD4+ and CD8+ cells and have been shown to
demonstrate reciprocal responses to histamine, based on the preferential expression of H1R
on Th1 cells and H2R on Th2 cells, for example.63 Most recently, H4R has also been
described to be functionally active on human Th2 cells64, with upregulation of H4R in
response to IL-4 reported. H4R agonism of these cells resulted in activation of the pro-Th2
transcription factor, AP-1 and the induction of the Th2 cytokine, IL-31. H1R on Th1 cells
appears to enhance Th1 type responses, with deletion in mice leading to a consequent
skewing to Th2 type responses after T-cell dependent antigen immunization and resultant
enhanced production of IgE and IgG1. H2R appears to negatively regulate both Th1 and
Th2 responses, with the surprising net effect of H2R deletion in mice resulting in decreased
IgE in response to immunization, at least in the Th2 predominating system tested. This was
in spite of the predicted increase in IL-4 and IL-13 production from H2R deficient mice,

suggesting that the concomitant overproduction of IFNγ had a dominant effect on the
humoral response.
In vitro, T-cell proliferation has also been reported to be affected by histamine, with once
more a pro- inflammatory enhancement of proliferation associated with H1R activation and
an inhibitory effect on proliferation via H2R activation, reported. On CD8+ T cells deletion
of either H1R or H2R has been shown to increase their capacity for IFN-γ release whilst
reducing IL-2 and IL-10 secretion. Activation of H2R and H4R on CD8+ cells also leads to
IL-16 release, a potent T-cell cheomattractant associated with asthma. Direct effects of
histamine on the chemotaxis of T cells are also apparent, via activity at either H1R or H4R.
Fig. No. 3.16: Role of histamine H4R on dendritic cell and T-cell function
iNKT Cells:
Although still somewhat controversial, invariant NKT cell are considered to be a potentially
important initiator of allergic and inflammatory responses, via their ability to rapidly
produce primary cytokines such as IL-4 and IFN-γ. Allergens such as pollen may also act at
the invariant TCR to cause their activation, further linking them to allergic conditions.
Modulation of iNKT cells by histamine has recently been reported, whereby histamine
deficient mice demonstrated reduced IL-4 and IFN-γ production in response to in vivo iNKT

activation. This could be reconstituted with histamine and blocked by a selective H4R
antagonist (Paul J. Dunford et al., 2010).
Pyridone compounds:
The 2(1H)-pyridone ring system and the corresponding dihydro and tetrahydro derivatives are
found abundantly in a wide variety of naturally occurring alkaloids. Militarinone A, a pyridone
alkaloid is known to show pronounced neurotrophic effect. Lyconadin A, a Lycopodium alkalo
was demonstrated to possess modest anticancer activity. Harzianopyridone, a 4-hydroxy-2-
pyridone analogue is representative of the atpenin class of penta-substituted pyridine
based natural product that was reported to be potent inhibitor of SQR (Dorigo P et
al.,1993).
Substituted 2-pyridones represent useful scaffolds for drug discovery and are also versatile
synthetic building blocks and constitute important core units in a large number of
pharmaceuticals, agrochemicals and functional materials.
Phosphodiester inhibitor Amrinone, antifungal agent Ciclopirox, an anticancer antibiotic
Diazaquinomycin A, a cardiotonic agent Olprinone, L-696,229 and L-697,66 were
identified as specific HIV-1 inhibitors used in clinic are a few selected examples.
2-Pyridones constitute an important type of hyterocycles which have shown variety of
biological activities. In particular 2-pyridones containing H-bond acceptor substituent in
position-5 constitute a relatively new class of specific phosphodiesterase 3 (PDE3)
inhibitors.
The 2-Pyridone derivatives amrinone VI (Farah A.E. et al., 1978) a n d milrinone VII
(Alousi A.A et al., 1983) are considered good alternatives to classic digitalis glycosides
for the acute treatment of congestive heart failure (CHF)
Pyridones have been reported to possess non-nucleoside HIV type I specific reserve
transcriptase inhibitors (Paulvannan K et al., 2000) and anti-inflammatory (Bristol-Myers et
al., 2005) activities besides wide range of pharmacological activities. 2-Pyridones have been
also reported as fungicidal agents were also reported as tissue factor VIIa inhibitors
(Lawrence et al., 2007)

Cyclopenta[b]pyridin-2,5-dione constitutes also an interesting tensor of pharmaceutics
exemplified by the antibacterial product and a building-block for the access to 2-
cyclopenta[b]pyridin-5-one as seco analogues of 8-azasteroids (Pemberton N e t a l. ,
2 0 0 7 ) and antiviral activity (Prakash H.S. et al., 2004). This is nitrogen containing
synthetically designed scaffold with a broad spectrum of biological activities and play an
important theoretical and practical role in heterocyclic chemistry (Forlani L et al., 2003).
5- Hydroxy 2-pyridone is an intermediate in the bacterial metabolism of a number of
pyridine derivatives including nicotinic acid (Kaiser J.P et al., 1996) It has been linked with
damage to DNA and show activity as an antitumor agent (Kim S.G. et al., 1990).
A wide range of biological activities were also observed in compounds possessing a 2-
pyridone motif which includes anti-cancer, antifungal, antitumor, anti-inflammatory,
antiviral and ant insecticidal properties (Semple G et al., 2003). Fused 1,2,4- triazoles
express antifungal, bactericidal,anxiolytic, anticonvulsant , herbicidal activities and can
act as antidepressants, analgesic and anti-inflammatory agents (Suresh.M et al., 2011).
Li and his co-workers have discovered series of 3-urea-1- (phenylmethyl)-pyridone as
novel EP3 antagonists via high throughput screening and subsequent optimization and
reported as selective EP3 receptor antagonists (Li Y.A et al., 2010).
N
O
H
N
H
N
O
O
Fig.No.3.17: 3-urea-1- (phenylmethyl)-pyridone
Pemberton et al have synthesized dihydroimidazolo and dihydrooxazolo ring-fused 2-
pyridones and biological evaluation revealed that these compounds inhibit pilus assembly
in uropathogenic E. Coli (Tipparaju S.K., 2008).

Fig. No. 3.18: Dihydroimidazolo and dihydrooxazolo ring-fused 2-pyridones
N
H
N
O
R
COOLi
Tipparaju has synthesized 2-pyridone derivatives and evaluated them for their BaENR
inhibitory and antibacterial activities (Pfefferkorn J.A et al., 2009).
Fig. No. 3.19: 2-pyridone derivatives
N
O
R2R3 R1
Smyth has reported 3-amino-1H-pyrazolo [4,3-c]pyridin-4(5H)-ones as potentially
attractive heteroaromatic scaffold suitable for screening against kinases and other cancer
drug targets (Abadi A.H et al., 2010).
Fig. No. 3.20: 3-amino-1H-pyrazolo [4,3-c]pyridin-4(5H)-ones
NH
O
R1
R2
N
N
H2N
R3

M.Suresh has synthesized a series of substituted triazolo pyridinones and evaluated them
for antibacterial activity (Suresh.M et al., 2011).
Fig. No. 3.21: Triazolo pyridinones
N
N
N
S
NC
NC
O
O
Wahid M. Basyouni has reported the synthesis of various novel substituted {1,2,4}triazolo
pyridinones and evaluated their herbicidal activity (Magdy A et al., 2014).
Fig. No. 3.22: Various novel substituted {1,2,4}triazolo pyridinones
N
N
NH
NC
NC
O
O
N
N
NH
NC
NC
O
O
CH3
N
N
NH
NC
NC
O
O
CH2
CH3

4. MATERIALS AND METHODS 2014
SHRI VISHNU COLLEGE OF PHARMACY Page 31
MATERIALS AND METHODS:
From the literature survey it is evident that the fused [1,2,4 ] triazolo substituted pyridinones
posses varied pharmacological activities . This gave us an impetus to synthesize some novel
fused [1,2,4] triazolo pyridinones and evaluate them for H1antagonistic activity.
This study was performed in 2013–2014 in the animal house (Regd.No:439/01/a/CPCSEA)
at Shri Vishnu College of Pharmacy, Bhimavaram, and Andhra Pradesh, INDIA.
Materials:
The general structures of the compound is given below,
N
N
N
O
NC
H2N
R
Test compounds:
Fig.No. 4.10: Structure of A1:7-Amino-5-oxo-2-phenyl-5,8-dihydro-[1,2,4]triazolo[1,5-
α]pyridine-6-carbonitrile
N
N
N
O
NC
H2N
7-Amino-5-oxo-2-phenyl-5,8-dihydro-[1,2,4]triazolo[1,5-a]pyridine-6-carbonitrile
C13H9N5O
Mol. Wt.: 251.243

Fig. No. 4.11: Structure of A2: 7-Amino-2-(4-chloro-phenyl)-5-oxo-1,5dihydro-
[1,2,4]triazolo[1,5-α]pyridine-6-carbonitrile (C13H8ClN5O).
N
O
N
H
N
NC
H2N
Cl
7-Amino-2-(4-chloro-phenyl)-5-oxo-1,5-dihydro-[1,2,4]triazolo[1,5-a]pyridine-6-carbonitrile
C13H8ClN5O
Mol. Wt.: 285.689
Fig. No. 4.12: Structure of A13: 7-Amino-2-(4-bromo-phenyl)-5-oxo-5,8-dehydro-
[1,2,4]triazolo[1,5-α]pyridine-6-carbonitrile (C13H18BrN5O).
N
N
N
O
NC
H2N
Br
7-Amino-2-(4-bromo-phenyl)-5-oxo-5,8-dihydro-[1,2,4]triazolo[1,5-a]pyridine-6-
carbonitrile
C13H8BrN5O
Mol. Wt.: 330.140
All the compounds evaluated were synthesized and characterized by the Department of
Pharmaceutical Chemistry, Shri Vishnu College of Pharmacy, Bhimavaram. The compounds
were given the codes A1, A2 and A13.

Animals:
Animals Swiss albino mice (20–25 g) of either sex were kept under standard environmental
conditions (i.e.12:12 hour light and dark sequence; at an ambient temperature of 25±2.c;
3560% humidity). They were housed in cages and fed with standard pellet diet and water ad
libitum.
Methods:
Acute toxicity studies:
Acute Oral Toxicity Testing-. OECD -423
Principle of the test:
It is the principle of the test that based on a step wise procedure with the use of a minimum
number of animals per step, sufficient information is obtained on the acute toxicity of the test
substance to enable its classification the substance is administered orally to a group of
animals at one of the defined doses. The substance is tested using stepwise procedure, each
step using three animals of single sex (normally females). Absence or presence of compound-
related mortality of animals dosed at one step will determine the next step, i.e.; (i) no further
testing is needed, (ii) dosing of three additional animals, with same dose (iii) dosing of three
additional animals at the next lower or the next higher dose level.
Description of the method:
Selection of the animal species:
The preferred rodent species is rat, although other rodent species may be used. Normally
females are used. This is because the literature surveys of conventional LD50 tests show that,
although there is little difference in sensitivity between sexes, in those cases where
differences are observed females are generally slightly more sensitive. So in the present study
female rats are selected. Healthy young adult animals of commonly used strains were
employed. Females were nulliparous and non pregnant. Each animal, at the commencement
of the dosing, were between 8 and 12 weeks old.
Housing and feeding conditions:
The Temperature in the experimental room was maintained at 22oc (±3oc). Although the
relative humidity was maintained at least 30% and preferably not exceeded 70% other than
during room cleaning it was 50-60%. Lighting was artificial, the sequence being 12 hours
light, 12 hours dark. For feeding, conventional laboratory diets was used with an unlimited
supply of water. Animals were grouped, such that the number of animals per cage must not
interfere with clear observations of each animal.

Preparation of animals:
The animals were randomly selected, marked to permit individual identification, and kept in
their cages for at least 5 days prior to dosing to allow for acclimatization to the laboratory
conditions.
Preparation of the doses:
In general test substances should be administered in a constant volume over the range of
doses to be tested by varying concentration of the dosing preparation. The maximum volume
of the liquid that can be administered at once depends on the size of the test animal. In
rodents volume should not normally exceed 1mL/100g body weight. However in the case of
aqueous solutions 2mL/100g body weight can be considered with respect to the formulation
of the dosing preparation, the use of an aqueous solution/suspension/emulsion is
recommended whenever possible, followed in order of preference by
solution/suspension/emulsion in oil and then possibly solution in other vehicle. Doses were
prepared shortly prior administration.
Procedure
Administration of the doses.
The test substance was administered in a single dose by using a stomach gavage needle. In
the unusual circumstances that a single dose is not possible, the dose may be given in smaller
fractions over a period not exceeding 24 hours. In the present study it was not required as the
dose was administered at once. The animals had been fasted overnight during period of drug
administration with complete access to water all the time. Following the period of fasting, the
animals were weighed and the test substance was administered. After 3 hours diet was given
to the animals.
Number of animals and dose levels
Three animals were used for each step. The dose level to be used as the starting dose selected
from one of four fixed levels 5, 50, 300 and 2000 mg/kg body weight. The starting dose level
should be that which is most likely to produce mortality in some of the dosed animals. The
time interval between treatment groups was determined by the onset, duration, and severity of
toxic signs. Treatment of animals at the next dose, delayed until was confident of survival of
the previously dosed animals. The dose level 300 mg/kg was selected.
Observations:
Animals were observed individually after dosing at first 30 minutes, periodically during
the first 24 hours, with special attention given during the first 4 hours, and daily

Thereafter, for a total of 14 days, it should be determined by the toxic reactions, time of
onset and length of recovery period, and may thus be extended when considered
necessary. All observations were systematically recorded with individual records being
maintained for each animal.
Body weight
Individual weights of animals were determined shortly before the test substance was
administered and weekly thereafter. Weight changes were calculated and recorded.
Data and reporting:
Individual animal data were provided. Additionally, all data is summarized in tabular
form, showing for each test group the number of animals used, the number of animals
displaying signs of toxicity, the number of animals found dead during the test or killed for
humane reasons, time of death of individual animals, a description and the time course
of toxic effects and reversibility.
Fig. No. 4.13: Test procedure with a starting dose of 5mg/kg body weight OECD-423

Screening models:
Isolated Goat tracheal chain preparation: Isolated adult goat tracheal tissue was obtained
from slaughter house. Trachea was cut into individual rings and tied together in series to form
a chain. Trachea was suspended in bath of Kreb’s solution and was continuously aerated at 37
± 0.5oC. Dose response curves of histamine was obtained in Kreb’s solution and in Kreb’s
solution containing 1 µg/mL and 10µg/mL of test compounds A1,A2,A13. Percent of
maximum contractile response were plotted to record dose response curves of histamine in
the absence and presence of test substances (Mahajan et al., 2011)
Fig. No. 4.14: Isolated goat tracheal chain preparation

Effect on Clonidine induced catalepsy in mice: Bar test was used to study effect of test
substances on Clonidine induced catalepsy to determine antihistaminic (H1) activity. Mice
were divided in six animals per each group. Clonidine (1 mg/kg, s.c) was injected to mice
pretreated with vehicle (10 ml/kg, i.p.), Chlorpheniramine maleate (10 mg/kg, i.p), test
compounds (500µg/kg, 1000µg/kg) respectively. The forepaws of mice were placed on a
horizontal bar (1 cm in diameter, 3 cm above the table). The time required to remove the
paws from bar was noted for each animal. Duration of catalepsy was measured at 15, 30, 60,
90, 120, 150,180 and 210 min interval (Tote et al., 2009).

Fig. No. 4.15: Clonidine induced catalepsy in mice
Milk induced leukocytosis and eosinophilia: Mice were divided into eight groups and six in
each group. Blood samples were collected from tail cut off. Group I served as control and
received distilled water (10mL/kg), groups II received milk (4mL/kg s.c) group
III,IV,V,VI,VII,VIII treated with test A1,A2,A13 (500µg/kg, 1000µg/kg respectively). All
the groups injected boiled and cooled milk (4mL/kg, s.c.) 30 min after treatments. Total
leukocyte and eosinophile count was done in each group before administration of test
compound and 24 h after milk injection. Difference in total leukocytes and eosinophile count
was calculated (Dnyaneshwar J Taur et al., 2012).
Fig. No. 4.16: Images of milk induced eosinophilia:

In-vitro anti inflammatory activity (Membrane stabilization by HRBC):
HRBC method was for the estimation of anti inflammatory activity in-vitro. Blood was
collected from the healthy volunteers and was mixed with equal volume of sterilized Alsevers
solution (composition Glucose 20.5g, sodium chloride 4.2, Tri-sodium citrate 8.0g, citric acid
0.55g, distilled water 1000 mL). This blood solution was centrifuged at 3000 rpm and the
packed cells were separated. The packed cells were washed with isosaline (0.85%; pH 7.2)
solution and a 10% v/v suspension was made with isosaline. This HRBC suspension was
used for the estimation of anti-inflammatory property. Difference concentration of test
compounds, reference sample and control were separately mixed with 1mL of phosphate
buffer (0.15M, pH 7.4), 2mL of hyposaline (0.36%) and 0.5mL of HRBC suspension. All the
assay mixtures were incubated at 37°c for 30 min and centrifuged at 3000 rpm. The
supernatant liquid was decanted and the hemoglobin content was estimated by a
spectrophotometer at 560nm. The percentage hemolysis was estimated by assuring the
hemolysis produced in the control as 100% (TK Mohamed Saleem et al., 2011).
Instead of hyposaline 2mL of distilled water was employed as control.
The percentage inhibition of hemolysis was calculated by using the following formula:
Percentage protection= (Abs control – Abs test) / Abs control × 100
The diclofenac sodium (5mg/mL) used as a standard.

5. RESULTS 2014
Table 5.10: Acute toxicity studies: OECD-423 Guide lines:
GROUPS BEHAVIORAL STUDIES MORTALITY
I (5mg/kg) Normal Nil
II (50mg/kg) Normal Nil
III (300mg/kg) Normal Nil
IV (2000mg/kg) Normal Nil
The test compounds A1, A2, A13 did not shown any sign of toxicity up to 2000 mg/kg body
weight and hence they were considered to be safe.

5. RESULTS 2014
Isolated goat tracheal chain preparation:
Table 5.11: Effect of A1(1ng/mL and 10ng/mL) on histamine induced contraction of isolated
goat tracheal chain preparation
S.no Concentration
of histamine in
µg
Histamine
induced%
maximum
contraction
D.R.C of
histamine in
presence of A1-
1ng/mL
D.R.C of
histamine in
presence of A1-
10ng/mL
1. 10 16.96 ± 0.35 11.99 ± 0.03* 2.16 ± 0.10*
2. 30 26.62 ± 0.32 18.66 ± 0.31* 7.08 ± 0.07*
3. 100 69.10 ± 0.51 40.05 ± 0.55* 31.33 ± 0.56*
4. 300 86.13 ± 0.31 57.99 ± 0.38* 53.86 ± 0.47*
5. 1000 100 ± 0.71 78.63 ± 0.41* 67.33 ± 0.31*
Values in Mean ± SEM, n=6.
Statistical analysis done by using student ‘t’-test.
*p<0.05 significantly different from Histamine induced percentage maximum contraction.
Fig. No. 5.10: Effect of A1 (1ng/mL and 10ng/mL) on histamine induced contraction of
isolated goat tracheal chain preparation.

5. RESULTS 2014
Table 5.12: Effect of A2(1ng/mL and 10ng/mL) on histamine induced contraction of isolated
goat tracheal chain preparation
S.no Concentration
of histamine in
µg
Histamine
induced%
maximum
contraction
D.R.C of
histamine in
presence of A2-
1ng/mL
D.R.C of
histamine in
presence of A2-
10ng/mL
1. 10 22.75 ± 0.27 17.35± 0.15* 3.85 ± 0.07*
2. 30 38.34± 0.55 30.38± 0.25* 11.58 ± 0.14*
3. 100 68.18± 1.50 56.99 ± 0.26* 27.84 ± 0.38*
4. 300 81.77± 0.44 63.67 ± 0.36* 60.20± 0.40*
5. 1000 100± 0.63 84.80 ± 0.35* 66.72± 0.52*
Fig. No. 5.11: Effect of A2 (1ng/mL and 10ng/mL) on histamine induced contraction of
isolated goat tracheal chain preparation

5. RESULTS 2014
Table 5.13: Effect of A13 (1ng/mL and 10ng/mL) on histamine induced contraction of
S.no Concentration
of histamine in
µg
Histamine
induced%
maximum
contraction
D.R.C of
histamine in
presence of A13-
1ng/mL
D.R.C of
histamine in
presence of
A13-10ng/mL
1. 10 16.13 ± 0.08 8.13± 0.03* 6.38 ± 0.05*
2. 30 31.39± 0.29 17.26± 0.06* 9.05 ± 0.04*
3. 100 47.42± 0.39 28.52 ± 0.31* 24.8 ± 0.17*
4. 300 73.35± 0.19 52.55 ± 0.18* 30.66± 0.24*
5. 1000 100± 0.46 75.47 ± 0.21* 45.97± 0.16*
Fig. No. 5.12: Effect of A13(1ng/mL and 10ng/mL) on histamine induced contraction of

5. RESULTS 2014
Table 5.14: Effect of CPM (1µg/mL) on histamine induced contraction of isolated goat
tracheal chain preparation.
S.no Concentration of
histamine in µg
Histamine induced%
maximum contraction
D.R.C of histamine in
presence of CPM-1µg/mL
1. 10 16.96 ± 0.35 4.09± 0.10*
2. 30 38.34± 0.55 8.00± 0.05*
3. 100 47.42± 0.39 12.06 ± 0.09*
4. 300 73.35± 0.19 28.04 ± 0.27*
5. 1000 100± 0.10 44.15 ± 0.23*
Statistical analysis done by using student‘t’-test.
Fig. No. 5.13: Effect of CPM(1µg/mL) on histamine induced contraction of isolated goat
tracheal chain preparation.
In the study, histamine produced dose dependent contraction of goat tracheal chain
preparation. The modified physiological solution containing A1, A2, A13 (1ng/mL and
10ng/mL) and CPM (1µg/mL) significantly inhibited (p<0.05) the contractile effect of
histamine.

5. RESULTS 2014
Effect on Clonidine induced catalepsy in mice:
Table 5.15: Effect of A1, A2, A13 (500µg/kg and 1000µg/kg) on Clonidine induced
catalepsy in mice:
Values of Mean ± SEM, Where n=6
Group I: Positive control, Clonidine (1mg/kg); s.c.,
Group II: Chlorpheniramine maleate (10mg/kg); i.p.,+ Clonidine (1mg/kg s.c.,)
Group III: A1 (500µg/kg p.o.,) + Clonidine (1mg/kg s.c.,)
Group IV: A1 (1000µg/kg p.o.,) + Clonidine (1mg/kg s.c.,)
Group V: A2 (500µg/kg p.o.,) + Clonidine (1mg/kg s.c.,)
Group VI: A2 (1000µg/kg p.o.,) + Clonidine (1mg/kg s.c.,)
Group VII: A13 (500µg/kg p.o.,) +Clonidine (1mg/kg s.c.,)
Group VIII: A13 (1000µg/kg p.o.,) +Clonidine (1mg/kg s.c.,)
Group Duration of catalepsy (sec) at Mean ±SEM
15mi
n
30min 60min 90min 120min 150min 180mi
n
210min
I 36±2.
15
86±1.45 177±5.5
9
318±1.9 337±16.4
7
438±8.48 142±6.
16
96±3.47
II 13±1.
0**
30±1.54
**
75±1.78
**
43±2.24
**
38±1.65*
*
32±0.80*
*
20±1.4
**
4±0.98*
*
III 30±1.
52*
72±1.7 154±5.4
8*
202±5.2
8**
258±11.9
7*
118±7.07
**
89±2.6
7**
11±1.14
**
IV 14±1.
11**
20±2.41
**
107±5.5
3**
180±2.9
3**
121±6.28
**
98±3.87*
*
60±3.4
2**
5±0.60*
*
V 13±1.
01**
55±1.92
**
130±3.6
9**
197±3.3
3**
38±2.46*
*
25±1.97*
*
14±1.5
4**
6±1.21*
*
VI 31±1.
37*
43±2.27
**
86±2.56
**
109±5.3
2**
66±3.86*
*
23±4.32*
*
16±2.4
4**
10±1.68
**
VII 28±3.
27**
44±2.77
**
101±3.7
3**
203±7.8
7**
40±4.75*
*
31±2.19*
*
24±2.8
2**
20±2.48
**
VIII 28±3.
24**
46±5.82
**
68±5.81
**
115±7.8
2**
39±3.38*
*
28±3.19*
*
18±1.4
7**
16±2.20
**

5. RESULTS 2014
Fig. No. 5.14: Effect of A1, A2, A13 (500µg/kg and 1000µg/kg) on Clonidine induced
catalepsy in mice:
Group I: Positive control, Clonidine (1mg/kg); s.c.,; Group II: Chlorpheniramine maleate
(10mg/kg); i.p.,+ Clonidine (1mg/kg s.c.,); Group III: A1 (500µg/kg p.o.,) + Clonidine
(1mg/kg s.c.,); Group IV: A1 (1000µg/kg p.o.,) + Clonidine (1mg/kg s.c.,); Group V: A2
(500µg/kg p.o.,) + Clonidine (1mg/kg s.c.,); Group VI: A2 (1000µg/kg p.o.,) + Clonidine
(1mg/kg s.c.,); Group VII: A13 (500µg/kg p.o.,) +Clonidine (1mg/kg s.c.,); Group VIII: A13
(1000µg/kg p.o.,) +Clonidine (1mg/kg s.c.,).
Statistical analysis done by ANOVA followed by Dunnett test.
**p<0.05, *p<0.01 compared to positive control group.
Clonidine induced catalepsy in mice, which remained for 3hr the vehicle treated group
showed maximum duration of catalepsy (438 ± 8.48 sec) at 150 min after the administration
of Clonidine. There was significant inhibition (p<0.05) of Clonidine induced catalepsy in
animals pretreated with A1, A2, A13 (500µg/kg and 1000µg/kg, p.o.,). Chlorpheniramine
maleate (10mg/kg, i.p.,) significantly inhibited (p<0.01) catalepsy in mice at 150 minutes
after the administration of Clonidine.

5. RESULTS 2014
Milk induced leucocytosis:
Table 5.16: Effect of A1, A2, A13 (500µg/kg and 1000µg/kg) on milk induced leucocytosis
in mice:
Groups Treatment Number of
leucocytes
before treatment
per cu mm
(Mean ± SEM)
Number of
leucocytes after 24
hours drug
treatment per cu
mm (Mean ±
SEM)
Difference in
number of
leucocytes per
cu mm (Mean ±
SEM)
Group-I
(Normal
control)
Distilled water
(10mL/kg., p.o)
7456 ± 2.01 7506 ± 1.66 50 ± 2.71
Group-II
(Positive
control)
Milk-4mL/kg
(s.c.,)+Distilled
water
7449 ± 5.72 21050 ± 1.11 13600 ± 5.52#
Group-III A1-500µg/kg+
milk
4mL/kg(s.c.,)
8050 ± 1.82 20500 ± 5.96 12450 ± 6.67**
Group-IV A1-1000µg/kg+
milk
7020 ± 3.65 13150 ± 3.65 6130 ± 6.32**
Group-V A2-500µg/kg+
milk
8020 ± 7.30 17520 ± 4.83 9500 ± 11.40**
Group-VI A2-1000µg/kg+
milk
10750 ± 56.33 11750 ± 3.65 1000 ± 56.80**
Group-VII A13-500µg/kg+
milk
9200 ± 7.30 20900 ± 36.51 11700 ± 40.66**
Group-VIII A13-1000µg/kg+
milk
9100 ± 38.64 10050 ± 13.16 950 ± 39.83**
Values of Mean ± SEM, Where n=6

5. RESULTS 2014
Fig. No. 5.15: Effect of A1, A2, A13 (500µg/kg and 1000µg/kg) on milk induced
leucocytosis in mice
Group I: Distilled water (10mL/kg., p.o) ; Group II: Milk-4mL/kg (s.c.,)+Distilled water;
Group III: A1-500µg/kg+ milk 4mL/kg(s.c.,); Group IV: A1-1000µg/kg+ milk; Group V:
A2-500µg/kg+ milk; Group VI: A2-1000µg/kg+ milk; Group VII: A13-500µg/kg+ milk;
Group VIII: A13-1000µg/kg+ milk.
Statistical analysis done by using student-t-test (Group II were compared with group I)
#p<0.05 significantly different from normal control.
Test groups were compared with group II (positive control), statistical analysis done by using
ANOVA followed by Dunnett test.
**P<0.05 significantly different from positive control.
*P<0.01 significantly different from positive control.
Subcutaneous injection of milk at doses of 4mL/kg produced a significant (p<0.05) increase
in leukocyte count after 24 hour of its administration. In the group of mice pretreated with
A1, A2, A13 there was significant (p<0.01) inhibition of milk induced leucocytosis.

5. RESULTS 2014
Milk induced eosinophilia:
Table 5.17: Effect of A1, A2, A13 (500µg/kg and 1000kg/mL) on milk induced eosinophilia
in mice:
Groups Treatment Number of
eosinophils
before treatment
per cu mm
(Mean ± SEM)
Number of
eosinophils after
24hours drug
treatment per cu
mm (Mean ±
SEM)
Difference in
number of
eosinophils per
cu mm (Mean ±
SEM)
Group-I
(Normal
control)
Distilled water
(10mL/kg., p.o)
149 ± 0.04 150 ± 0.03 1 ± 0.05
Group-II
(Positive
control)
Milk-4mL/kg
(s.c.,)+Distilled
water
148 ± 0.11 842 ± 0.04 693 ± 0.11#
Group-III A1-500µg/kg+
milk
4mL/kg(s.c.,)
161 ± 0.03 410± 0.11 249 ± 0.13**
Group-IV A1-1000µg/kg+
milk
140± 0.07 263 ± 0.07 122 ± 0.12**
Group-V A2-500µg/kg+
milk
160 ± 0.14 350 ± 0.09 190 ± 0.22**
Group-VI A2-1000µg/kg+
milk
215 ± 0.12 235 ± 0.07 20 ± 1.13**
Group-VII A13-500µg/kg+
milk
184 ± 0.14 418 ± 0.73 234 ± 0.81**
Group-VIII A13-1000µg/kg+
milk
182 ± 0.77 201 ± 0.26 19 ± 0.79**
Values of Mean ± SEM, Where n=6.

5. RESULTS 2014
Fig. No. 5.16: Effect of A1, A2, A13 (500µg/kg and 1000kg/mL) on milk induced
eosinophilia in mice
Group I: Distilled water (10mL/kg., p.o) ; Group II: Milk-4mL/kg (s.c.,)+Distilled water;
Group III: A1-500µg/kg+ milk 4mL/kg(s.c.,); Group IV: A1-1000µg/kg+ milk; Group V:
A2-500µg/kg+ milk; Group VI: A2-1000µg/kg+ milk; Group VII: A13-500µg/kg+ milk;
Group VIII: A13-1000µg/kg+ milk.
Statistical analysis done by using student-t-test (Group II were compared with group I)
#p<0.05 significantly different from normal control.
Test groups were compared with group II (positive control), statistical analysis done by
using ANOVA followed by Dunnett test.
**P<0.05 significantly different from positive control.
*P<0.01 significantly different from positive control.
Injection of milk (4mL/kg, s.c.,) produced a significant increase (p<0.05) in the total
eosinophil count. In the groups pretreated with A1, A2, A13 there was significant (p<0.01)
inhibition in the total eosinophil count.

5. RESULTS 2014
In-vitro anti inflammatory activity (Membrane stabilization by HRBC):
Table 5.18: Percentage inhibition of hemolysis of A1, A2, A13
S.no Compound Concentration Abs at 560 nm Percentage
inhibition of
hemolysis
1. A1 5 mg/mL 0.728 54.30 %
2. A1 10 mg/mL 0.340 78.60 %
3. A2 5 mg/mL 0.581 63.50 %
4. A2 10 mg/mL 0.540 65.60 %
5. A13 5 mg/mL 0.604 62.10 %
6. A13 10 mg/mL 0.375 76.47 %
7. Diclofenac 5 mg/mL 0.619 61.16 %
Fig. No. 5.17: Percentage inhibition of hemolysis of A1, A2, A13

5. RESULTS 2014
Test compounds A1, A2, A13 at different concentrations (5,10 mg/mL) showed significant
stabilization towards HRBC membrane. The percentage protection at concentration 10
mg/mL was higher than that of concentrations. However the percentage protection was found
to be increased at higher concentrations. Within this A1(10mg/mL) shown higher
stabilization towards HRBC membrane

6. DISCUSSION 2014
Discussion:
 Histamine is an autocoid having profound physiological effect in the body. The
contraction of tracheal or bronchial smooth muscle in-vitro has been utilized for the
study of contractile / dilator responses of agonists as well as antagonist. Both goat
tracheal chain and strip preparations are suitable for screening the activity of a drugs
acting on respiratory smooth muscles.
 Spasmogens such as histamine, acetylcholine and barium chloride produce dose
dependent contraction of bronchial smooth muscle. Goat tracheal smooth muscles are
contracted by histamine through the H1-receptor stimulation. This leads to activation
of IP3 and DAG pathway. This increased increased IP3 is responsible for releasing
the microsomal calcium, leads to phosphorylation of actin-myosin fibers of goat
tracheal or bronchial smooth muscle in-vitro has often been utilized for the study of
contractile / dilator responses of agonists as well as antagonist (Suralkar Anupama A
et al., 2012)
 In isolated goat tracheal chain preparation, histamine produced dose dependent
contraction of goat tracheal chain preparation while there was right side shift of dose
response curve of histamine was observed in the presence of the fused [1,2,4] triazolo
pyridinones A1, A2, A13 indicating antihistaminic activity (Table 5.11, Table 5.12 &
Table 5.13).
 Histamine is the major inflammatory mediator in asthma, causing hyper
responsiveness and bronchial airway inflammation. Most allergic and non-allergic
asthmatics, including those with mild asthma, having bronchial eosinophilia and there
is significant association between eosinophil activation and asthma severity as well as
bronchial hyper-responsiveness.
 In the present investigation fused [1,2,4] triazolo pyridinones, A1, A2, A13 at doses
of (500µg/kg, 1000µg/kg p.o) was evaluated for management of H1 mediated asthma
using milk induced leukocytosis and eosinophilia in mice.
 Asthma involves various types of mediator in pathology. It was demonstrated that
subcutaneous administration of milk produces a marked increase in the leukocytes
and eosinophils count after 24 h of its administration (Table 5.16, Table 5.17).
 Leukocytes during asthmatic inflammation release the inflammatory mediators like
cytokines, histamine, and major basic protein, which promote the ongoing of

6. DISCUSSION 2014
inflammation. The infiltration of leukocytes potentiates the inflammatory process by
the release of reactive oxygen species into the surrounding tissue, resulting in
increased oxidative stress and associated with many pathogenic features of asthma.
 In this study observed that leukocytes count was decreased in mice treated with fused
[1,2,4] triazolo pyridinones A1, A2, A13 at doses of 500 and 1000µg/kg significantly
as compared to positive control group.
Result suggests that fused [1,2,4] triazolo pyridinones decreases milk induced leukocytes
count by normalizing oxidative stress. An abnormal increase in peripheral eosinophil to more
than 4% of total leukocytes count is termed as eosinophilia. In asthmatic patient there is an
increase in eosinophil count and mucus hypersecretion and airway hyperreactivity were
stimulated (Mr. Dnyaneshwar J Taur, et al., 2011).
 Clonidine induces catalepsy via H1 receptor. The prior treatment with the fused
[1,2,4] triazolo pyridinones, A1, A2, A13 at doses of 500 and 1000µg/kg significantly
inhibits the catalepsy compared to positive control group (table ). Which may be due
to its H1- antagonistic activity.
 Inflammation is a common phenomenon and it is a reaction of living tissues towards
injury. Here HRBC method was selected for the in-vitro evaluation of anti-
inflammatory property because the erythrocyte membrane is analogous to the
lysosomal membrane and its stabilization implies that the test compounds may as well
stabilize lysosomal membranes. Stabilization of lysosomal membrane is important in
limiting the inflammatory response by preventing the release of lysosomal
constituents of activated neutrophil, such as bactericidal enzymes and proteases,
which cause further tissue inflammation and damage upon extra cellular release (TK
Mohamed Saleem et al., 2011).
 The result indicted that the fused [1,2,4] triazolo pyridinones, A1, A2, A13 at various
concentrations (5mg/mL and 10mg/mL) has significant anti-inflammatory property
(table 5.18).

7. CONCLUSION 2014
Conclusion:
 The fused [1,2,4] triazolo pyridinones, A1, A2, A13 showed significant antagonistic
effect on histamine induced contraction of isolated goat tracheal chain preparation.
 The two doses 500µg/kg, 1000µg/kg of the fused [1,2,4] triazolo pyridinones, A1, A2,
A13 showed significant antagonistic effect on clonidine induced catalepsy and milk
induced leucocytosis and eosinophilia.
 The two concentrations 5mg/mL, 10mg/mL of the fused [1,2,4] triazolo pyridinones,
A1, A2, A13 showed significant percentage protection on membrane stabilization by
HRBC.
 This [1,2,4] triazolo pyridinones, A1, A2, A13 compounds may be providing a scope
for use as H1 antagonistics. Further work should be conducted for unvelling the
receptor mediated action for H1 antagonistic activity.

8. REFERENCES 2014
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