Unit 5 Pharmacology 2 Carewell Pharma

Page 1 of 42
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Unit | Pharmacoloq-2
Phamacoloq of drs ach on Cudocriul Sqstem
Andio qu ondAonlolic seroids

Fcboqn progasrona
ma
Cd Ora Covracopthiu
Sex hormonus
Drugs achng on UCWS.
ar
ioassay
Piiplus nd appliahans obioasscu
Ty p a
Bio ass0u
of
Ph
of
bioass
Lnsulin, Oxytocin, Vasopessin , ACTH
Aat d-tubocuraninl, dgualis, Histamia

nd S HT
ell
mPu
on Oral covtTaupti
rew
Wrik a detouls not
nok DI drugs achng on (UeWE
Wrie on
a Ahortnolk
yAndhogtnd Ohd fAvabalic stsolds

Ca
Ecbogns Prqusteron
Dufin Ond dasithy BioaAa wrik u bloavcan

of dyet
Tnsulin d- tubo wvanina
Oxytoun Dugetceis
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Page 2 of 42
ISaslc Rnolldo
Sex hovmnmU
hovmons wic
pro duud
plaay
ma
Qud
aud eluased nunon becu
COxual develbpmour f epoabuMm,
ImportautF
rol
Onadol Aeslds
Sex eaids 0d ocodicojds,
Typy
ar
Sex - Hormaus
Ph
femada
Mala
ell
Atndvogun
ESboguu oqoteron
Testosjenone
rew
hormonuy
Dasialy elua fom femoe y
Elase pom
otqaud Oudho llps on
bosically tstes
developmusctofAxua) orgous
in
aul bu elps
ud aluo lp in (ovulolm
Ca
duelepmuF of Sexuc orqaus

Cud
d up n elua o
OVUm
(SpOumeldgu
(fsHfollida Atimulahin
hrmmus
deuelopma of (oOvUm) seCrehion of eshauS
prodwchin also Support spamatoq auuis
perm
(LH evtini 2inq hermos stimuat
- piggn ovvlahom, gulermu Stcnhon , etos teon
OChon
Page 3 of 42
wrie bhort noe on
Andogun Anobolíc slvoids
Atudvogns male sSux hormonus
Testostvou u andvdqn. Tt is
ma
main otural
Mauny studhusizic 1estitial cells Of -H testis,
nd Amaler Qmount y OVaies d adkenal cortex)
S4x AjcAGds
ar
TLL Avbstan (auA developmN of SCondary
chaa ce in Min, inthdung focial aud bod 4

-8
hain qraw And
PhvOtCA Choa
give hein achon b binding H Ovedvoge

Rept
ell
Auunogus MO Suuthh
dgans
rew
Sudhun'e
Natua
MeHlealo tjerona ona
Hut ofF adulF mau produu
tlvoxy musJors
(S-12g of tapskm dady
Ca
14-0lkyl Auelfitvtd dadlvadjvts of

Adrenal cory producps Amalu
testo tvmL (oaly ochve).
quovdtties of deluydroepiaudrostonu
f andro ShenadiMA
weaR audvoguns
youan
OvBAY
do produdsAlu
oHeatpseonuy
.
Page 4 of 42
huulay glauds
O Requlabiou Aa
oacdohopin homonaConk
relanq, hormanc
HypotnalonU osteuio
Aei
ma
KiDnodo hopiw
/fu Pno
leutenizlvg homp/ 1ollick stimulaing
ar
hormovu)
Ph 1
Testes (ONBles
qovedls inki bin
Testortron hogen
ell
|Progustevc
fAuhin
rew
Anabeli q enten ino

pvprl
Respond or al chaug fatosern Apoulsla
Ho t
Ca
Polata Apurt of q
puloudt in boys
0udo q Amallu ex
in gil
groun oF quutalu-pLnu
Rapad bou qouth, bsth in
Uotvm, Sewwnal eiclus,
prostale. uudknL well a n leath
qiouwm of hai Pubic bad, pronmote muscl buulduug,
moUs tod body, espaciallp 4 adid by exuerti
han Cud
Page 5 of 42
of a
pattn Appetie Improve an
male
diubibuhon asuns T e- bLing
giows voícL prevails
cup
e e2
ma
. pcolciniy
. USually
a4ieDa e cte
ar
2nd win ptamoa Prettin globulin
10-20 mUnURA
playma V2
Anabolit stooids
Ph
(Analboli'c ondhogunic sta4ds)
Thy mthuic Qndrogens with suppos e

S
ell
gh anaoolic Qnd ower audvoqtnic) actiuw
grouwHh .Sexval qyowHh

Rape bon
rew
promoe mucda
buildno
hore Chuical Aubsan whih

T anR
muscluy ono lps to qrow thu bod
Ca
Sonuim4 n llugal by ploy ports.
Naudrolon, Muhandaenons. ek.
Anhiando GLNS Tnos arvs whih octagonds adhom df

0udrogens USac to deceaM CKCAsie
Saual desi e ulamida yprokona

e.
Page 6 of 42
hegns
Geshegsn
pumay emala Sx homon

C C
divelopmeiUano
ma
po nsilblu fou Hu
Equlaion df u fenalt Hpioduch e sem Caud
SPCndoy Stxual chanaclerishs»
ar t
Sex skuetd invlved InHu Hqulatin of
an aL
Cycle changs
mpo vtaudin
Ph
expksucd
pegnonc
meuva Cycus
Sythuzid y OvO and placonda

ell
smaly avmouvts
bu tis Oncadenalcorte
rew
Th
he ajoendendogenw (exbogns
malu- esdone (oesbme) Eshadial (oesradisl)
Oud uiol &esiol)
Ca
(Ethadiol is tu mos
pincipo sboqun sece

ed
Sqwtri
Cthiylehadiole, MUubandl
ek
CReguloh
abtody diuswa
Page 7 of 42
Man tmchom
pubupujal Cha ) -fcmalus

, pub
qrowh
qrouwlh of Ueus,alopion tubu aud vaaaMa,
cproductivê organ.
ma
gcna epuhulum guts -hchenus
- elponsiblu proluferahn peovulotoy

of endomim in he
pha
ar
eyogen Incea Coaquhl OF Dlood nd Inteahuu
t risk OF
Ph
omboembolicm.
linical U
Overt OVanlan (eaurnur's Cuhduaa)
fad
ell
Secgmdantga ovclan fail for fiuskinq, Vaqjinal dyws and
to pesevve bonu mass
Couta cupthion in cobinahin pills
rew
proae ad
baCoht.
Proqarton
Ca
serts lay Corpvs luttUm in th SeCom
po of mnshv yc, and b plocata during

pregnont
amounts aho SAcetto by Hestis ahd adena
mal
cort
Tu ae AubAantag uhich Convent u (eAbogn paim
Page 8 of 42
Maun n chy
cic Joy. ChON.gtsn -T uhgen

Pon
t baings abou
endoruhmm
CavsL cevixb bicmahik
Ahnnd Ou muCUS in
ma
gain whih pevent Apwm pon paysng 1uh
T inducu pkgnanuy
mamma glands
h
ar
proliftuahionm
FaSL
pituilay qonadobopins
allo Inhub
PhStLehions of
wich pheven Ovulahom
wwrik a derails note on Oral (ontvacoptioe
Orad Conbhaaphioei
ell
HO nouwn gir control Bl"
Thu hot dvvgs ohlch

rew
pven pkgnony Pesuphon mudicahions
udici Cotain hormons lR hogun

progsthin( sqpthuhic bu similm. 0s(pogusbsm)
Ca
Rou tue
oF hovmonu
paanty Cnovmal munavnism

thoguu
T OVones product tu egg
teus. Ovulahon) (fs)
Callnd ovum (ova)tun
transpore antd falUopain tut. YLH
eluaj
Page 9 of 42
(UJcrus uleri Cauly
MAVMwY allopas
u
ma
Evdomublum
Covix va
Ceviol
Cana
ar
Vaapna
Now
e
Ph
fetilizd y ApAm in fallopian fubA
Now, tu moves UerUs Wh th Uteim unng

endombium
pep Hegele a
ha ichinLd and t
ell
featiua eg.
(Esbegen
1)
rew
Phoqusterai
Rísing level of pragujevon tha CauSe glands in ining

edomalvm
to relea fluds 4that (teds) tu egg-feuilzad eg9).
Ca
T also (ou ths tonnud ou mucus in t

Cevix to becm tuR which peven Spm pom
pasun hrough.
on 99 hosn' bun fetiliud t level of both ehagin

which (usR mwstuanm.
proguslerm bgn ofa
Page 10 of 42
Ora Cohacptives
Conbaceptivey
Cmbaoptivej
ypus of oral
Oral
wo maun
Combinahim pus
)8
(mini pil)
ma
Progustin- Only pill
Combinahim pulls
and a iesi(progishin
ar
Cousalns Q eshogen
MoA C phoqute).
Ph (Hpitholan
Cowbinahn ply
nP
eL1/fSH (Liutove
glaud
ell
(esrogen Proouhin
Ovonles
rew
waintain Comsa
InbuF Secveion
lvele Ethogin
OF FSH ( ugahve
fudbadk o Prbguttu
System)
SupeAA(Ovulahon) Jaei nubu Secrehn
Ca
Of LH
pevent ovulalim)
alle tu (udomuhm u
Such as Way T tutken ng of ceviy muvS
discovrag implaahon.
Ovvlahm pRgnont
Page 11 of 42
mosl popvlar avd most efficaciou mulo
Hhiny lespadiole Noguubol ee
ma
Adre gain Vomui hg ee
Mausta
wise IOdhie pall tha
ar
Coctain hormonus
) Mini pil (proquhin mly pal)

Thy
Ph
(ajain only proguhin
MOA hickening of cwical muwS

preuFovulahm 4
ell
pRven pHgnanty
tn ehicaty ower than Cwlolnahon pils

rew
Writ no On dug ah on ORS
.Ueuinl mUSc Contracs vhuthmicaly both in uho anel

Ca
wwo, Tu Coram onginrin n th mutd itult.

myomhiel cellb fundaa Ac 0) (poumoker)and
qive Cmducte Ochion potthal.
NOW
achu of paumgakar equlatd by
(Sex homOnu
Page 12 of 42
Cahacts
spnjans y)
Codachom
ulein
tunlng mnsbualim.
duing.
(partutitim v DLh
fu Coodinatd
ma
Classupohiom
Uleune
ar
|URani
Ph
stimulaw J
Uteun claxavds
Calcitonj
Oxytocin
Prostaglandins B2 adnoceptri
ell
(adauvalina )/salbuan
Erga dRal ids
frgamuhnnn)
rew
OOntocin hormm
ptxkn secretd by u posejon
pihutany aong
ithVaopesunADH)
Ca
T 1notam h Cotrachm of ut mUIclly
GytoinO Oxytocin C-PCR

Yeeuptor TP -DnG)
Contra dchm
Drpalalsanion (by infux)
1a
Page 13 of 42
.tko Thu
1 numbtr Of oxytocin eceptorsInCeaU mokea
mkea
during atr paF ofF PAcg nany
CtrachionP Hel n child bity)
ma
Usus
ch
th
USd oIndua Or auomin lalbour wn
ar
Uterin muscll is not funchon tmq aduqvaclely
AKo V sed
tohea postpartum hamorvhogi-
Ph biuth
oft imudialely chuld
(exive blodd toss)

ell
Brcast nttai u SeCrehim Of mulk from h
mMammo qlond
rew
(Aavo etfilh)-
n huh
CoUse Sussaind Cohadhon thaF inlnfm wih olodd
Ca
AO Miavgh pocuta Ond Cawse fctu dath
P'oinaic
ven (TV) o.M bu mosHy Irtvaveox nfvsim.
Inachvattd in UveL 4
d a
Page 14 of 42
hostaglondint
IS-utuyt PGFu pole

PCE,PGLa ad
CudoonoUS Ukint Atimulats
ma
Cahacin of ulavs
relaxahm ofCewix/
ar
Eigs Akalsud
Caouds u human
on
Ph
(onrocted terus ( normal Atat fpllowina dlive),
Crqoman ha elaively luttlu etfcty.
ell
UEWS inappo plakly elaxed,
trgbmuhint inuho stoen Cotracin ,tvs vedUun
bludg from plautal leid.
rew
yddn
Ca
Utun elaxants|
Seltdively
2- odsenocaptou agmsti, such as (vitadun)
or Salbutomo), inbut outocin-Inducud Cotrachion) o 4u
pHcgnovt Utrus.
USado peven pematu lobour )(dloy deluy upb
Page 15 of 42
RIOASSAY
(Bio+-(fA ssay protus procus of

cleler nunin -u (pohcncy) Of t
ma
in liv Hes
ti dvug by usin SUitab blologial
Sysjem uke ,
avimols, iss , ioou ek..
ar
Outeunina relohive poitnty of acubttona (droq) by COrnpaing
àrs biological
attiul
Ph With tho of a reftnce sJavndands
ell
AlSo ULdto Sandovdyz Peparalion Ao that
rew
ench pepurahon Cocpin Speeifiesl phoavmocolog4 Qctiuu-

Octiun-
Pindiple oL
Tu boMc principa Of
blosa to tnpa t tt
Ca
Substonu with inlernationga Adord pHpMahMs of

TIP,USP, BP ek
So ad find ou how mvch
Svbstanu Is vequied fo produ Sam bloloaical effrctr,
produca by Sondord
Page 16 of 42
Applicohiny
oF a dvu
dekrmiu Thpolency)
deleimins (doce (quanlily) of a drug equied to
ma
pioduad a huaptulic Or tonit epons, e Colcoloim
(EDSDoso) cke.
effeche eha
ar
dog doso
Alo ps dvelopmaa ot nd drugs

in th
Ph
Thu maind ypus (Acc. to th klpn us
ell
) Qvovtal ioossay
) Grndud oiosa
rew
1QUatal) bloassay
AlSO R10.0 rAaons (No)

Evd piut)
*kespons Al or no", if. euhIr

Mximum esponse no esponsA w
Ca
Or
uermudiole Hesponses
eg. Digatals Codiac onus

n una pg /pouon
-fubocurarim hvabbir uad

cbop umod
Dste
- compani slonm of threshold esponsacompislon of
Page 17 of 42
td poi
(Anima
Tk qoup Ihcl group
wuch Akou
ma
threshold dose
Thshold dou tve effrcs
Spundavd Test
ol so
ar
COmpore
Tweshold clou oF sauolor

Conourahon of
Unkvoun
Ph Thesnold dose of te
Conc. of sjaudovd
i) Gradus bioassau
muans Aponsa proportionmal to
ell
t do Ond Epons may e eetuwen no HEponse
tu moaimUm Hspon so
rew
ceiling effects
Responsa
Ca
DOTR
T graduo dote espons Curve

dANd
plottiny qhaph wih dose X-O1S Ono
EApon&e On T-axis
Tt USually sig mold in Slapa
Page 18 of 42
T ponss 0 ud n Hu Obsuwatioms u
tha u CUn inueasomon In Hpmsu wih
Inoeasemunt An u Conceurdim of dose
ma
Typus of qrodid aay
a) Matclung
ar
b) 8tockehing
Tnerpolatton
Ph thHee poln
d) Multipu pointk bioa:sy Tur four poinr
i poin
igh 1gh poin
ell
rew
Ca
Page 19 of 42
ioassoly OF inulin
Insulin
a hormom mods by panRatic P-ulls
ma
.T SyntaMzid 0 po-inulhin In endoplasmic etiiullum
prousd t bioloqially acthiv form insucda

Aetory hanuA
ar
fnchon
lps to ontvol tha blood sugar 1vel by Cmveiv
glutor itd glyogn
Ph and akso InCRaAe t glutoSt uptuR
clls
Uptake
ell
quca
glucogn
rew
Also we duf. OF byassay (Principl
Fxpujmt model
Ralolai mumoc
Ca
mumad
prepanatn Camplus
puR, d 0nd ostalinu insulin.
u
vna 0.04092 mg spuified by nuaishy of uath
Govt: oF Indua f also eqinal
o iatemahon Uni)
Page 20 of 42
Isulin 2o vnu+ Injulin
Hcoeuey wigu
Qnd dusole ormal Saun. (NOVmal Salin
Audihy wih H to pH S
HU ph 2)
OSphuol pRservaives
ma
0S phno
Add 147. - 1-9 yuin +
final volu Ahould otan ou/m
Atou so1 in a Col plau
ar
oU/m
wi thin AX mot 0-Smgm
Cappaov.)
Te bum
Ph 8tamdond
Rabler mh
Selechion of Robkeas (animal) - Ty Anould a haty4,
ell
Eig about (23 Ka). A ttaia Ahauld
-follos Otc.to Chovt. $VM ax (CPCsEA) ele-

rew
for pefovn tost

Roblis mould bL maintoinud On Unifovm die bu
fastcd for 19s efou 0may (tt)
Ca
standond and Aanplu (dluhms)

use uy upan by dlutguHhnormial
Nau Aol A0 a to Cotain (1/) +(2U
-
SImilay,, tsF ampl Sol
plutiou adso Apwdard
ppaucd.
Page 21 of 42
prinup T potny OF a Het Campl is esfinate
by CompaA
ypogoN effe ct oF u
Saumplu wm tha of ppav ahn of insulin.
td.
ma
proedUR >
enc
Aymals diudsd qusps of3 radbis
Jnko
TM roldoets 0L T pu udo awa nolaay

noldar
avoa
ar
Dhuy Ahoul4 ehdedaanel handle wth a
exutma
pa - frs paat+ pa
Tpfoun
Ph
tup ucond
O fin paa
-A sampla of bloc tapan fromt martna
0
ell
Co veln oF raho
pe lopmd of
Ptsena of ecucing Sugar simatd
rew
blooc by Sutabl chunal muthod
Conc blood sugor

Thus alludnhia level").
Thun four groups of rablils qiven

Ca
subtwtauous (C) thjecim of Incoln a follou
Pabb Croup SapRe
3 Std-1- I/
Std -2 2U/
Test-L Ju/
3
3 Test-2 - 2U/n
OfsL
Page 22 of 42
amplt of bl6dd J WIhda
wIhdrau
from tad rabeu, a
nerval ot hr
upto
Shrs a eauh,
Now Slood Sugan dijen mlnnd

blodd suqay level
Hi is allud "-inal
ma
SeCnd pant
A tL O eek Chodse Aamt aumals
ar
agaln fusted fo 19 hrs Qud Inuhal blcod cuga
u deteminud.
cKd qoub
NOu, May
Ph
Chanaa
tMans- host animals which Hceived th Atamdad

OAL wen nd Hoived
ell
now given L Audand
Also
thos aimals Heceived
les
do ofF Atandad quen a
rew
hou doz o
tf Sampla Od viIa -VerA.
Ths taF nown a Twin osS over tutP.

pary pan
ist |Sutnd cutk sugan
Ca
Level ^an
Sarpla Cop Sapla (u fin
Std-L Test-2 pant.
Std-2 Tet L
I
Std-
Tut IT
TV
Test- 2)T -L
Page 23 of 42
uan purconT of decease

.
blaod SLga o -firsF
uud eLndpant u Calwlattd
MOUR muthod
ua houd choractevisti convulsias atu (sc.echom
ma
of insulin a elevufd emperctues
Th pirAnlag Comvul sloma proclucad and
Aandard pparatioma COnpanL
ar
phocecu Aimalulana)
munimum J00 m uegnig beuen 19-22gm of
Ph
sama srain USAd
Tu
Thuy ou ld bt maintain M (msant jek.
ThLy Ahould hsttd 18 hs Pio p H

ell
expeai munt
Seunpl
rew
PLpua
luhions AL pepaLd with ule Ao, o 0 to
Contan O.064 urr/m (std. ) ane 0.096 Unus/ (sta T),
SImalanly ts amplu o
Ca
diwdd four groups
Crp-1 SaumbL
Tnsulin ieckd sc.
4 fol low 11-
sd.2
L
Test
T Test 2
Page 24 of 42
IncoDajoL at
m pu
a halFhouv.
oburv fo M nd
SSCand
ovt of
which lnvulsa) o de
ma
tu InwbalorY Qud obsevved
favtaaga Cmvulsis protut y tf Aampus
Compand of Btandna onpl

wth -hose
ar
Convulcive mice
Ph b Aaved
O.S m of S dextroce sol"
ThoCA SUWIv may 2 Used aga4

animals
fo Ovnottn expumr aft Infeava of Onl

ell
wek .
1
rew
Ca
Page 25 of 42
- TubOLUranin
claxatian of skelulal muscus

CaUSLA
t
.Biocassay USIng Rabeil head- drop mutha
ma
Principl
Eis ineced o manginal vein of
Vablbiks ea l t ralos uck mUs cis a
ar
tloxed Such a har Qmal Cannor hold u
wad
Ph
ambUnf of He samplu Hauirtdto
produc u endpoint COmpoed wth tu total
CumOUt of standand Aampla Kquidto
ell
produa siilan efecr.
Selechon ofF Rabeiy
rew
ueigh2Kg
houd
- Chsore pom a uathy Colony
Should be CUstonid uwith 4u

Ca
expimrdal
procLduR
procedue
-Rableit plaCac in a hold wi i wad

probuding ovtiud. Tha Aheuld laa paly
ovabls
Page 26 of 42
- Min. able anl USed T aAl duuidd
eoh Coutann
8 rabbit
ma
rablaus 4 saax
T
Spucdand col
ar tjeced at Citan spud

sieon
Ph appautv bragh tu mangua in
Spu
SpAA o.4 /min (Him aken abou lo min)
DosL 0.o127. wfU Sali n
In
ell
Ihfuslm Coinued hl t rabitt wid no
rew
pein apositionm hold

had ecc
Aaau CrOT-er tut Comied ou
bíological
evorsduu to anmal Volahion,
hosA aubleits whl teceivtdt standndamplu n
Ca
da given tof Sampl m AL

CeCand
do of expnl ma n MCR
eKa
MLAn doe ich produceS uad f tu
drop tea
Samipl Compann with t nuan dbA C
auclend puponahion.
Page 27 of 42
Dwlna t RXptalma n
hl possibiluie of kupaoy
emb lass nmun jething heostigm
Lohih htattd by
utuyl sulphalt (o.o0 ma) anc abopmt Sulpkate fmmudle
mmgtnal ear .
hough ein
ma
. Roassay of Dugtaiis
sorce (ohachon
Indta t
ar
of Lar
p nupt
Poknty
Ph
sSamplu is COmans withho
of standoud PLpanahm tuteaunlgaght achon
(ondiac murd
ell
Canple
Atandond PLpaNm u a xtUH Of dried auol
aves
rew
pousduned dgutalu (1 % ng)

xoc amOm of extrackd WIh
dahydiattd alcohol SOxulu appanatu sIx hovrs
final m Sn.
Ca
- exrud hauld Cauain lo allohol + S

wat)
p lo F_digstals pouwd.
hovld 21 stoud
(Animall
UI I wa- pig muthod CEnd pol)
Cgem muthwd
Page 28 of 42
Gqula - ptq nuthod
g dugclali powin u dlukd to Po n

Sanpll
ma
(GUlnLa-p
anaustizid
ar
discchtel on
t
opuahion phl. Ph
Jgulan Vein d pawd out
by imOI adingtiscun
and Cannulattd y ans otF
ell
venovS Cannula )
insevted n t
rew
pin
hu (n tu app Of hat) St
we Ca observe ha
ua ey p +
Ca
doum movtmvts
o a pin.
Irekd Ltinved haugh

a avsted In Systol
(tu amoVn of shad kqujnd to produu huis
Page 29 of 42
effceF Is aeen -u Lethal dos of h

exud)
hot HA San
SA OF
19animals oF
Sp.rdes DCed expeumo aud

-fo
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aveag LeHhal deleumlnt
Nap
u Savy to utmn tu uhal do
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standond dusng eauh
Ph (but hichud)
and
finally potenty Cakolaltd in clehntd tnd
dividinq aveuge ehal do of thu
of test expHsu nik pu qum

Sanp CLd
ell
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Page 30 of 42
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BIOASSAY OF OXYTOCIN:
Oxytocin is a peptide hormones and Neuropeptide. Oxytocin is normally produced by
the paraventricular
Ph
nucleus of the hypothalamus and released by the
posteriorpituitary.
Oxytocin is a natural hormone that causes the uterus to contract. Oxytocin is used to
induce labor or strengthen labor contractions during childbirth, and to control
ell
bleeding after childbirth.
Oxytocin is also used to stimulate uterine contractions in a woman with an incomplete
or threatenedmiscarriage.
rew
Different bioassay methods used for oxytocin are

o By contraction of the rat uterus
Ca
Page 31 of 42
o By depression of the blood pressure in chicken
o By measurement of milk ejection pressure in a lactating rat etc.
Principle:
>The poteney of oxytocin injection is determined by comparing its activity with that of
the standard preparation of oxytocin under the conditions of the followingmethod of
assay
ma
Standard preparation:
The standard preparation is consisting of a freeze dried preparation of oxytocin with
human albumin and citric acid (supplied in ampoules containing 12.5 units) or any
other suitable preparation, the potency of which had been determined in relation to the
ar
International standard.
The Unit is the specific oxytocin activity corresponding to that yielded by 0.0005 g of
the Standard preparation.
The
established in 1978.
Ph
standard preparation is as per the 4h international standard for Oxytocin,
Experimental Methods:
1) By contraction of the rat uterus:
Use female rats weighingbetween 120 and 200 g. Immediately before the assay
ell
confirm the rat is in oestrous or preoestrus by vaginal smear.
Inject 100 microgram of oestradiol benzoate intramuscularly 18-24 hours before the
assay.Kill the rat and suspend one horm of the uterus in a bath containing a solution of
rew
the following composition:

Composition (% w/v)
Sodium chloride 0.662
Potassium chloride 0.045
Calcium chloride 0.007
Ca
Sodium bicarbonate 0.256

Disodium hydrogen phosphate 0.029
Sodium dihydrogen phosphate 0.003
Magnesium chloride 0.010
Dextrose 0.050
Maintain the bath at a temperature of 32° C so that spontaneous contraction of the
uterus are abolished and preparation maintain its sensitivity.
Oxygenate the solution with a mixture of 95% of oxygen and 5% of carbon dioxide.
Page 32 of 42
Record the contractions of the muscles produced by the addition to the bath of two
doses of the Standard Preparation suitably diluted with the above solution. The doses
should be such as to produce clearly discriminated, submaximal contractions.The
required doses normally lie between 10 and 50 micro Unitsper ml of bath liquid.
When maximal contraction has reached replace the bath liquidby a fresh solution.
The doses should be added at regular intervals of 3 to Sminutes depending upon the
ma
rate of recovery of the muscle.
Dilute the preparation being examined so as to obtain the responses on the addition of
two doses similar to thoseobtained with the Standardpreparation.
The ratio between the two doses of the preparation being examined should be the
ar
same as that between the twodoses of the Standard Preparation and this ratio should
keptconstant throughout the assay.
The two doses of Standard preparation and the two doses of the test preparation
Ph
should be given according to a randomized order or Latin square design and at least
six responses toeach should be recorded.
Measure all the responses and calculate the result of theassay by statistical methods.
2) By depression of the blood pressure in chicken:
ell
Anaesthetize a young healthy adult cockerel weighing 1.2 to 2.3 kg with an anesthetic
that will maintain aprolonged and constant high blood pressure.
Expose the gluteus primus muscle in one thigh and cut and retract it to reveal the
rew
popliteal artery and cruralvein.

Cannulate the popliteal artery and record the bloodpressure.Cannulate the crural or
brachial vein.
Prepare standard solution with saline so that the volume to be injected is between 0.1-
0.51ml.
Ca
Inject 2 doses of standard solution into cannulated vein and record blood pressure.
The doses should be such that as to produce clearly discriminated, precipitious,
submaximal decreases in B.P.
The required doses normally lie between 20 and 100 milli units.
The interval between injections should be constant and lie between 3-10 minutes
depending on the rate at which B.P. returns tonormal.
Dilute test sample before use with saline solution so as to obtain responses similar to
those obtained with the standard preparation.
Page 33 of 42
The ratio between the two doses of the test preparation being examined should be
same as that between the two doses of the standard preparation and the ratio should be
kept constant throughout the assay.
The two doses of Standard preparation and the two doses of the test preparation
should be given according to a randomized order or Latin square design and at least
ma
six responses toeach should be recorded.
Ifanimal rapidly become insensitive due to repeated injections of the solutions
another animal must be used. Measure all responses and calculate the result by
standard statistical methods.
3) By measurement of milk-ejection pressure in a lactating rat:
ar
Select alactating rat, in the third to twenty-first day after parturition and weighing
about 300 gseparate it from the litter and 30 to 60 minutes later anaesthetise (for
example, by theintraperitoneal injection of a solution of Pentobarbitone Sodium).
Tie
Ph
the rat to anoperating table, maintained at 37, by its hind legs leaving the front
legs free. Cannulatethe trachea with a short polyethylene tube of internal diameter
about 2.5 mm in such amanner so as to ensure a free airway; apply artificial
respiration only if necessary.
ell
Cannulate an external jugular or femoral vein with a polyethylene tube of
internaldiameter about 0.4 mm which is filled with saline solution and closed with a
pin.
rew
Shave the skin surrounding the inguinal and abdominal teats and excise the tip of
oneteat, preferably the lower inguinal teat. Insert a polyethylene tube of internal
diameterabout 0.3 mm and external diameter about 0.6 mm, to a depth sufficient to
obtainappropriate measurement of pressure (3 to 10 mm depth), into the primary teat
ductwhich opens onto the cut surface and tie firmly in place with a ligature.
Ca
Connect thiscannula with a suitable strain gauge transducer (such as that used for
recording arterialblood pressure in the rat) and fill the whole system with a 3.8% w/v
solution of sodiumcitrate or saline solution containing 50 Units of heparin sodium per
ml to prevent clottingof milk.
After cannulation, inject a small volume (0.05 to 0.2 ml) of this solution into theteat
duct through the transducer to clear the milk from the tip of the cannula.
(Thisprocedure may be repeated durin the assay should obstruction arise from milk
ejectedinto the cannula).
Page 34 of 42
Clamp the strain gauge so that a slight tension is applied to the teat andits natural
alignment is preserved and connect the gauge to a potentiometric recorderadjusted to
give full-scale deflection for an increase in milk-ejection pressure of about 5.3kPa.
Inject all solutions through the venous cannula using a 1-ml syringe graduated in0.01
ml and wash them in with 0.2 ml of saline solution.
>Prepare a solution of the Standard Preparation and a solution of the preparation
ma
beingexamined in saline so that the volume to be injected is between 0.1 ml and
0.4ml.
Choose two doses of the Standard Preparation such that the increase in milk-
ejectionpressure is about 1.35 kPa for the lower dose and about 2.7 kPa for the higher
ar
dose.
As aninitial approximation, a lower dose of between 0.1 and 0.4 milliUnit and an
upper dose ofl.5 to 2 times this amount may be tried.
Choose two doses
Ph
of the preparation beingexamined with the same inter-dose ratio,
matching the effects of the doses of theStandard Preparation as closely as possible.
Inject the four doses (two doses of theeStandard Preparation and two doses of the
preparation being examined) at intervals of 3to 5 minutes.
The two doses of Standard Preparation and the two doses of the preparationbeing
ell
examined should be given according to a randomised block or a Latin squaredesign
and at least four responses to each should be recorded. Measure all the responsesand
calculate the result of the assay by standard statistical methods.
rew
BIOASSAY OF VASOPRESSIN:
Biological Assay for Vasopressor Activity
Principle:
The vasopressor activity is estimated by comparing the activity of thepreparation
being examined with that of the Standard Preparation of arginine vasopressinunder
Ca
the conditions of a suitable method of assay.

Standard Preparation:
The Standard Preparation is the Ist International Standard forArginine
vasopressin,established in 1978,consisting of freeze-dried synthetic
argininevasopressin peptide acetate with human albumin and citric acid(supplied in
ampoulescontaining 8.20 Units),or another suitable preparation the potency of which
has beendetermined in relation to that of the International Standard.
Suggested Method:
Page 35 of 42
Inject slowly into the tail vein of a male albino rat weighing about300 g a solution of
a suitable alpha-adrenoceptor blocking agent.for example 10 ml perkg of body weight
of a solution prepared by dissolving 5 mg of phenoxybenzaminehydrochloride in 0.1
ml of ethanol(95%),adding 0.05 ml of IM hydrochloride acid anddiluting to 5 ml with
saline solution.
ma
After 18 hours,anaesthetise the rat with ananaesthetic that will maintain a prolonged
and uniform blood pressure.After 45 to 60minutes,tie the rat on its back to the
operating table by its hind legs.
Cannulate thetrachea with a short polyethylene tube of external diameter about 2.5
mm and dissect acarotid artery ready for cannulation.Then cannulate the femoral vein
ar
close to the inguinalligament.
Retract the abdominal muscles to expose the inguinal ligament.Retract thesuperficial
pudendal vein to one side and dissect the femoral vein towards the inguinalligament
rom the corresponding artery.
Ph
>When dissecting,a deep branch reaching thefemoral vein must be found and tied off to
prevent bleeding during cannulation.Tie ashort polyethylene cannula of external
diameter about mm into the femoral vein by twoligatures and join by a short piece
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of flexible tubing to a 1-ml burette with an attachedthistle funnel containing saline
solution at about 37.
Firmly fix a wet absorbent cottonswab to the thigh so as to cover the incision and
rew
cannula.
At this stage inject through thevenous cannula 200 Units of heparin,dissolved in
saline solution,per 100g of bodyweight.
Then tie in a carotid cannula of external diameter about 1
mm and connect by
acolumn of saline solution containing heparin with a suitable pressure measuring
devicesuch as a mercury manometer of internal diameter about 2 to 3 mm.
Ca
The central and peripheral nervous system including both vagus and
associatedsympathetic nerves is left intact. No artificial respiration is necessary.

Taking care that noair is injected, inject all solutions through the venous cannula by
means of a 1-ml syringegraduated in 0.01 ml and wash in with 0.2 ml of saline
solution fronm the burette.
Dilute the extract of the Standard Preparation and the preparation being examined
withsaline solution so that the volume to be injected is between 0.1 ml and 0.5 ml.
Choosetwo doses of the Standard Preparation such that the elevation of the blood
Page 36 of 42
pressureisabout 4 kPa for the lower dose and about 7 kPa but always submaximal for
the higherdose, the ratio of low to high dose being determined by the response and
usually being 3to 5.
As an initial approximation doses of 3 and 5 milliUnits may be tried. Choose
twodoses of the preparation being examined with the same inter-dose ratio, matching
ma
theeffccts of the dose of the Standard Preparation as closely as possible.
Inject doses atintervals of 10 to 15 minutes. The two doses of the Standard
Preparation and the twodoses of the preparation being examined should be given in a
randomised block or aLatin square design and four to five responses to each should be
recorded.
ar
Measure allthe responses and calculate the result of the assay by standard statistical
methods.
BIOASSAY OF ACTH:
ACTH
Ph
(Adrenocorticotropic hormone, corticotropin) is polypeptide tropic hormone
(39 amino acids) secreted by the anterior pituitary gland.
ACTH stimulates the production of cortisol, a steroid hormone important for
regulating gucose, protein and lipid metabolism, suppressing the immune system
ell
response, and helping to maintainblood pressure.
Official Preparations
Corticotropin injection: Is a sterile solution , in a suitable diluent, of the polypeptide
from the pituitary glands of mammals. Potency range should be 80.0- 120.0 % of
rew
USP cartiotropinunits.
Corticotropin for injection, antimicrobial agent. Repository corticotropin injection is
corticotropin in a sterile solution of partially hydrolyzedgelatin and is intended for
subcutaneous and intramuscular use. This solution has beenadopted as the reference
standard for the bioassay.
Ca
o Packing: Preserve in single-dose or multiple-dose containers of Type-1glass.

o Storage:Store in cold place.
o Labeling:njection recommends intravenous administration
Purpose and rationale
This is a historical assay method. Administration of pituitary ACTH decrease the
ascorbic acid present in the adrenals. The depletion of adrenal ascorbic acid is a
function of the dose of ACTH administered. This relationship has been used for a
quantitative assay ofACTH.
Page 37 of 42
Solutions:
Solution A: Five units of test or standard dissolved in 0.25 ml of0.5% phenol solution
and diluted with 8.1 ml of 15% gelatin solution (Now 0.5ml contain 300 mU ACTH).
solution B: Three ml of solution A diluted with 6 ml gelatin solution.
Nowconcentration reduced to 100 mU ACTH/ 0.5 ml.
solution C: Again 3 ml of solution B diluted with 6 ml of gelatin solution, theresulting
ma
solution contains 33 mU ACTH/ 0.5 ml.
Procedure
Male Wistar rat (100-200 g) are hypophysectomized (pituitary gland removed by
surgery) one day prior to the test.
ar
For one test with 3 dose of test preparation and standard solution used for the study.
Number of hypophysectomized rats required: at least 36(preferably 60).
The hypophysectomized rats are randomly distributed in to sixgroups. Each rat
Ph
receives subcutaneous 0.5 ml of the variousconcentrations of test or standard.
Three hours after injection, the animals are anesthetized andboth adrenals removed,
freed from extraneous tissue andweighed. The rats are sacrificed and the scull opened
to verifycompleteness of hypophysectomy.
ell
The adrenals are homogenized in glass tubes contains 200 mgpure sand and 8.0 ml of
4% trichloroacetic acid and the ascorbicacid determined. (Roe and Kuether 1943).
The potency ratio including confidence limits is calculated withthe 3+3 point assay.
rew
Ascorbic acid determination:

Reagents
0.02% ascorbic acid solution
85 % sulfuric acid (9N H2S04)
.0.02 g/ml of dinitrophenolhydrazine in 9N H2S04

Ca
0.06 g/ml of thiourea are dissolved in distilled water

Charcoal
Preparation of 0.02% ascorbic acid solution
100 mg L-ascorbic acid are dissolved in 100 ml of 4% trichloroacetic acid (lmg/ml
solution)(Solution A=1% solution)
2 ml of Solution A diluted in 10 ml of 4% trichloroacetic acid to achieve a 0.2%
ascorbic acid solution (solution B)
Page 38 of 42
1
ml of solution B diluted in 10 ml of 4% trichloroacetic acid to achieve a 0.02%
ma
ascorbic acid solution (solution C)
Preparation of other solutions
Sulfuric acid (85%) is obtained by adding 900 ml concentrated sulfuric acid to 100 ml
distilled water.
ar
Two g dinitrophenolhydrazine are dissolved in 100 ml 9 N H2S04 (75 ml distilled
water and 25 ml concentrated sulfuric acid).
Ph
Six g thiourea are dissolved in 100 ml distilled water.
Calibration
> Trichloroacetic acid (4%) is added to 0.0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0 ml of the
0.02% ascorbic acid solution (solution C) and 1.0, 1,5 and 2.0 ml of the 0.2% ascorbic
ell
acid solution to reach a final volumeof 8.0 ml (Solution B).
>100 mg charcoal is added to cach sample and thoroughly mixed byshaking for 1 min.
After 5 min the solutions are filtered.
rew
An aliquot of 0.1 ml of the 6% thiourea solution is added to 2.0 mlof the filtrate
followed by 0.5 ml dinitrophenylhydrazine solution.
> The mixture is shaken and heated for 45 min at 57°C in a waterbath.
The solutions are placed in an ice-cold water bath and withfurther cooling 2.5 ml of
Ca
the 85% sulfuric acid are added.

The calibration curve is established at a wave length of 540 mmusing the solutions
without ascorbic acid as blank.
Page 39 of 42
ar ma
BIOASSAY OF HISTAMINE:
Ph
is present in almost all the animal tissues mostly within mast cell and
Histamine
basophil granules. Tissues rich in histamine are skin, gastric and intestinal mucosa,
lungs, liver and placenta.
ell
Non-mast cell histamine is present in brain, epidermis, gastric mucosa and growing
regions. Histamine is also present in blood, most body secretions, venoms and
pathological fluids. It is now known to play important physiological roles. Histamine
rew
Ca
Page 40 of 42
produces effects by acting on the histamine receptors (H1, H2, H3 and H4) present on
target cells.
Bioassay of histamine on isolated guinea pig ileum can be determined by graded
bioassay procedure i.e. i) Matching bioassay, i) Interpolation bioassay, ii) Bracketing
assay, iv) Multiple point assays.
Bioassay of histamine in biological samples can be studied by using different bioassay
ma
methods. Depending upon pharmacological action, histamine can be assayed by:
o Contractile effect of isolated ileum of guinea pig,
o Contractile effect of uterusof guinea pigand
o Fall in blood pressure of anaesthetized and atropinized cat. Guinea pig's uterus
ar
Bioassay of histamine using guinea pig ileum:
Principle:
Guinea pig ileum is very useful preparation for the bioassay methods. It is more
Ph
sensitive to histamine. Contractile response of histamine to ileum is due to presence of
H1 receptor.
Preparation of Standard and other solution:

Prepare the stock of Tyrode solution. Also prepare the standard stock solution of
histamine (l mg/ml) and then different concentrations of histamine by serial dilution
ell
method.
Procedure:
Sacrifice the 24 hr fasted guinea pig by stunning on the head and carotid bleeding. Fix
rew
the animal on the dissecting board by tying its legs. Open the abdominal cavity by a
small horizontal cut followed by vertical midline incision and expose the abdominal
organs.
Trace the ileocaecal junction by lifting the caecum, then go upwards up to 8 cm from
the junction and cut the 2-3 cm long segment of ileum muscle (excluding the terminal
Ca
5-8 cm, contains excess of excitatory a-adrenergic receptor, which may interfere in
the study).
Immediately place it in a petri dish containing aerated warm Tyrode solution. Slowly
remove the mesentery attached to the muscle and then gently clean the lumen of ileum
by passing luke warm Tyrode solution through it with a pipette or syringe.
Mount the preparation in the inner organ bath containing Tyrode solution (20 ml)
maintained at 37°C. Tie the bottom end of the muscle to the hook of aeration tube and
the upper end to the isotonic frontal lever by a thread without closing the lumen.
Page 41 of 42
Adjust the magnification of response to 7-10 fold. Aerate the tissue with 02 or
carbogen slowly (40-60 bubbles per minutes).
Stabilize the tissue for 30 min by applying a tension of 0.5 g weight attached to the
lever, during which wash the tissue with fresh Tyrode solution once in every 10 min.
Use 5 min time cycle with contact time of 30s for recording the contraction
dependent responses of tissue due to histamine on the smoked drums.
ma
Record the responses of standard and test compounds.
Record the responses of test compound i.e. unknown concentration of histamine with
gradually increasing volume, till obtaining a response (T) which would lie on the
linear portion (30-70%) of the CRC. Fix the response obtained due to volume of T.
ar
Record the graded responses of standard solution and test solutionof histamine.
The potency of the test sample is calculated in relation to that of the std. preparation
by dividing the average lethal dose of the sample to the test and expressed as units per
gram.
BIOASSAY OF 5-HT:
Ph
5-Hydroxytryptamine (5HT) is the biologically active local hormone, low molecular
weight, and also an important neurotransmitter in the brain and periphery.
It was first detected in serum (serotonin) and enterochromaffin cells of gut mucosa
ell
(enteramine), and later both were identified to be SHT. Today the terms 5HT and
serotonin are used interchangeably.
About 90% of body's content of 5-HT is localized in the intestines; most of the rest is
rew
in platelets and brain. It is also found in wasp and scorpion sting, and is widely
distributed in invertebrates and plants (banana, pear, pineapple, tomato etc.).
The concentration of 5-HT in biological samples can be assayed by:lsolated fundus
strip of rat stomach, Isolated terminal colon of rat,Isolated rat uterus,Perfused rabbit
ear.
Ca
Isolated fundus strip of rat stomach

Principle:
Rat fundus preparation is a very sensitive tissue for the bioassay methods and is useful
to study the action of several substances like 5-HT, ACh, PGE2 and bradykinin.
Fundus strip preparation is slow contracting muscle. Fundus part of stomach can be
easily identified by its grey colour and situated above the pink coloured thick pyloric
part. A zig-zag preparation of the fundal strip is prepared so as to expose maximum
portion of the tissue to drug.
Page 42 of 42
Preparation of Standard and other solution:
Prepare the stock of Krebs solution. Prepare the standard stock solution of serotonin
(1 mg/ml) and then different concentrations of serotonin by serial dilution method.
Procedure:
ma
Sacrifice the 24 hr fasted rat by stunning on the head and carotid bleeding. Fix the
animal on the dissecting board by tying its legs. Open the abdominal cavity by a small
horizontal cut followed by vertical midline incision and expose the abdominal organs.
Identify the stomach and separate it from the abdomen by gently cutting its cardiac
ar
and pyloric end. Place it in a petri dish containing aerated warm Krebs solution.
> Incise the fundus of the stomach (upper grey part) from pyloric part (pink and thick
Ph
part). Cut the fundus from the lesser curvature and open it longitudinally. Then cut the
fundus at the midline into two equal parts. Give alternate transverse cuts (zig-zag cut)
on opposite sides of the muscle to make a fundal strip preparation (as shown in fig.
9.1).
ell
Mount the preparation in the inner organ bath containing Krebs solution (20 ml)
maintained at 37°C. Tie the bottom end of the muscle to the hook of aeration tube and
rew
the upper end to the isotonic frontal lever by thread. Adjust the magnification of
response to 10-15 fold. Aerate the tissue with 02 or carbogen slowly (40-60 bubbles
per minutes).
> Apply 1g load and stabilize the tissue for 30 min during which wash the tissue with
Ca
fresh Krebs solution once in every 10 min.

Use a 5 min time cycle with contact time of 90s (since the fundus strip muscle
contracts slowly and relaxes slowly) for recording the contraction due to serotonin.
Record the graded responses of standard solution and test solutionofserotonin.
The potency of the test sample is calculated in relation to that of the std. preparation
by dividing the average lethal dose of the sample to the test and expressed as units per
gram.

Unit 5 Pharmacology 2 Carewell Pharma

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Page 1 of 42

Carewell Pharma - A Family of Learning

Phamacoloq of drs ach on Cudocriul Sqstem

Andio qu ondAonlolic seroids

Drugs achng on UCWS.

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