DEPARTMENT OF PHARMACOLOGY AND THERAPEUTICS FACULTY OF MEDICAL SCIENCES

COLLEGE OF MEDICINE UNIVERSITY OF NIGERIA

ENUGU CAMPUS

PRACTICAL MANUAL FOR MEDICAL STUDENTS

EXPERIMENTAL AND CLINICAL. IF

DEPARTMENT OF PHARMACOLOGY AND l'HEKAYE:Url'lCS

FACULTY OF MEDICAL SCIENCES COLLEGE: OF MEDlClNE UNIVERSITY OF NIGERIA

ENUGU CAMPUS

INTRODUCTION . . . . . . . . . . . . 1 - 4

ACTION OF DRUGS ON THE ISOLATED GUINEA-PILEUMEUM 6 - 7

BIOLOGICAL ASSAY HISTAMNEINE USING THE ISOLATED

GUINEA PIG ILEUM , . . . . . . . . . . . 8 - 9

THE ACTION OF DRUGS ON THE ISOLATED RABBIT INTESTINE

THE ACTION OF DRUGS ON THE ISOLATED TOAD RECTUS

ABDOMINUS MUSCLE .. . . . . . . . . .. 1 0 - 1 1

THE ACTION OF DRUGS ON THE ISOLATED RAT UTERUS 1 2

THE ACTION DRUGS ON THE SLEEPING TIME OF MICE .. 13

I ! I

VALIDATION OF EXPERIMENT PROCEDURES 14

CLASS PARTNER'S NAME - . - -

FIRST TERM: MAY - JULY 2000

FINAL COMMENT:

DATE: . . - - -- -

REG. NO.

day of

EXPERIMENTAL PHARMACOLOGY

PREFACE

Laboratory exercises in pharmacology are meant t o i l lustrate t he act ion of

drugs t o t he s tudent . Our exper imental pharmacology w i l l consist o f group

exper iments and demonstrat ions. It is very essential, fo r t he s tuden t t o be famil iar

w i t h t he pract ica l schedule thoroughly before coming t o each exper imental class.

The results o f each experiment should be recorded careful ly anti honest ly

Kyrnograph tracings where relevant, should be ful ly labeled The traclng s h o i ~ l d

s h o w the date o f t he exper iment, k ind of operation, t he temperature t he p o ~ n t o f

app l~ca t ion o f any d rug used, and any other relevant in format ion. Where on ly one

tracing is available for t w o students, a copy of t he tracing shoulcJ be rnacle for onc of

the s tudents and a referrmce t o original t racing made.

One o f t he m o s t c o m m o n causes for t he fai lure o f pharmaco log~ca l

exper iments is mishandl ing o f l iving t issues. Greatest care s l ~ o c ~ l d therefore be

exercised in handl ing the t issue in order t o avo id stretching, crushing or o therw ise

damaging t he l iv ing cells. Dry ing o f t he t issue m u s t also be avoided.

Isolated t issue preparations are crs~rally bathed in the pI1ysioloqic;ll sol i l t ions

in w h i c h they have found t o per form opt imal ly, and remain viable.

PRACTICAL REPORT

EXPERIMENT?

(1 ) Aim: To demonstrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

(2) P r o c e d u ~ As in the schedule, in addition to any methods used but not

contained in the schedule.

( 3 ) Results and Observation:

Description of the results of your particular experiment and any

observation made

(4) CommeenJs: What the experiment is supposed t o convey including the

expected results. If expected results differ from those in No. ( 3 )

above, comment briefly giving possible reasons and suggestions as to

discrepancy or new discovery..

(5 ) Discussion and conclusion:

Theory and principles underlying results e . g . mechanism or site of action

of drugs used.

The following table shows the types and composition of some physiological solutions

COMPOSITION OF PHYSIOLOGICAL SALT SOLUTIONS

MgSo4:71 1:O 201111 20mI 10% Soln.

The student must therefore note the physiological solution he is being supplied with. A whole experimental class can be ruined by using the wrong solution Oxygen is very necessary for the survival of living tissues especially when isolated. Refer to table 1 above. In all cases, oxygenation must be maintained throughout the experiment. Living tissues are very sensitive to changes in temperature. While amphibian tissues will survive for long periods at room temperature, mammalian tissues require heat up to body temperature. But most of these preparations last longer at lower temperatures e.g.32OC. Students should endeavour to maintain the required temperature throughout the experiment.

Guinea-pig ileum.

2. Plasticine ,

6. Physiological

Svlulion e.g .

Guinea-pig ileum

8. Inflow of

I~liysiologicd

Solufiori

Y. Uulfluw of Pliysiu-

Ivgical solution.

b'ig. 2 Example uf a

Iyyicd rcuorcl of

lhe responec lo an

.\go&l c.g

Acetykholine

(Alh)

I , I - I'Ilil~lll i~~OIO~}~ 1'1 ;lcIic,11 h'Iill111~1I -

I Preparations last longer at lower temperatures e.y 32oC. Students should endeavour to maintain the required temperature throuyhout the

I I ' ' -

experiment t

9 a

Calculations of drugs volumes and concentrations

1

Img (Milligramme) - - 111000 or 0 001gm (gram)

1 - , lug or I (Microgramme) - 1/1,000,000 or 0.001my or 10"ym

I ng (nanogram) - - 111,000,000,000; 0.001g or 10"gm

I mglml Acetylcholine means I ml of solution provided contains I mg of acetylcholine Ad ,

Making dilutions:- ',

1 Given 1Omglml acetylcholine, how would you prepare 10ml of the fol1owmg:-

(a) Imglml

(b) 1OOglml

(c) 20glml

Sample Solution

, Let x represent no of mls (vol.) To be taken from stock solution containing 10mg/rnl.

To prepare 20uglml solution of 10mls; the following can be used. ,

',

x mls x lo4 glml

V,.C,=V,C,.

X x 10000=200 X =0.02ml of 10mg Iml. I

Take .02ml of stock and add 9.98ml of distilled H,O on salirie to make up to 10ml. I

INTRODUCTION

The introduction is a two part-

PART I Demonstration of techniques in experimental Pharmacology,

isolated organ procedures and whole animal pharmacological methods

will be emphasized. The student is expected during this period to learn

and be able to inject animal and human subjects.

PAR-TI! To familiarize the students to the use of the organ bath, the smoked drum

recording device and to perfect the technique for setting up tissues In

organ baths for the use of assay of drugs

PROCEDURE 1. Measure 5Oml of Tyrode's solution into the inner vessel of the - .- - . -- .-

organ bath.

Mark [PART 11) The level of the solution in the bath and always fill the bath to this

mark. Check the temperature of the cuter bath and make sure it is

about 3 5 ' ~ (The temperature must not exceed 37" for Rabbit

intestine).

By means of a thread using a "rift-knot", tie one end of the 2cm

piece of intestine provided to the hook on the aerating tube. Tie a

"rift-knot" to the other end of the tissue allowing one end of the

thread to be very long. Suspend the tissue in Tyrode's solution in

the inner bath.

Balance the lever with plasticine such that it moves with small

increases in tension, and tie the long thread to the lever. Adjust the

lever to a horizontal position in the first instance. Ref. Fig. I.

3. Allow the preparation to rest in the solution for about 1 Srnin, aAt the

right temperature and aerating rate. Set drum at the lowest speed.

4. Make a recording of the spontaneous rhythm of the intestine for

3mins: Wash bath, add 0.1 nil of solution 'B' provided Record for

30sec. Stop drum and wash bath.

Discuss briefly the spontaneous movement of the rabbit intestine.

ACTION OF DRUGS ON THE ISOLATED GUINEA-PIG ILEUM

To denionstrate t l ~ e effect of acetylclmline ori sriiootli rni~scle; and develop a

dose response curve to acetylcholirie.

70 see tlie ariticl~oli~ier y ic effect of At1 opir ~ e .

The extent to which a piece of smooth rriirscle contracts in response to drily is a

function of \lie concelltration of t l ~ e drug. 1 he smallest dose or coricentrat~on of a drug

wliich produces and observable respollse is the tl~lesliold dose.

'I he srnallest dose of drug wliicli prodirces tlie largest lesponse of which the

s~l loott i rnuscles is capable is tlle rnaximal dose

Althouyll quantitative ptiarrnacological work is corllplrcated by "biological

variation", if all other sources of errors are reduced to a rninir~ium, this "biological

variation" 'an be pa~t ly obviated by repeating a given dose and so determine at1

average response, and by giving the various doses arld their replicates in a randoni

ade ro r a suitable pattern.

The t i rw interval between the various procedures must be as constant ;is

poss~ble. Wllcn you have determined approplialc t m c intervals, adhere to t l l c ~ r ~

tl lrouyl~out the exper rmerit.

Irl this experirrient, a suilable procedure is as follows.

I ime - . --- Pi o-ced UI-e ,U.~nin Wash Imtli

1 rnin Stat t Kyniograpli

I 'I1 niin Add dr a!]

2 rnin Wash Stop Kylnograpli

PROCEDURE:

1. Set up a piece of l l ~ e gi~irlea-pig i le i ln~ as in Inti-ocluction.

Part II

WORKING TEMPERATURE 3 2 " ~ MAXIMUM

Repeat (2) but before addition of acetylchol~ne, add 6 ug. of atropine sulphate.

Label the tracings and varnish. Measure the responses and tabulate results. Plot

a graph of the responses as percentage of maximal contraction against dose of

acetylcholine. Plot another graph of the responses against log dose These doses

of Ach could be modified depending on the sens~t~vity of the tissue If the writing

point goes above and of the drum, then the concentration of the drug need to be

decrease by dilution. On the other hand, if the contraction are rather very small,

then you have to increase your drug concentrat~on.

EXPERIMENT 2

Biological Assay of Histamine Using The Isolated Guinea-pig Ileum

OBJECT: To estimate the amount of histamine in an unknown solirtion using

responses of

the guinea-pig ileum preparation.

A. METHOD:

Suspend about 2cm of the guinea-pig ileum in an organ bath containing

tyrod's solution at 32°C and aerated with air. See fig.1

B. PROCEDURE:

1. Prepare a dilution of the standard histamine containing 1 Oug/ml

2. Record responses to 0.2Iug and 0.4ug histamine. This depends on the

sensitivty of the tissue. It may be necessary to increase histamine

concentration until maximum possible recording is obtained.

3. Determine the volume of the unknown histamine solution Test solution which

gives about the same contraction as a known dose of the standard solution. It

may be necessary to make dilution of the unknown solution until a

measurable but submaximal contraction is obtained that can be comparable

to contraction given by a standard solution. Call these doses of the standard

and test solutions Aand C respectively.

(i.e A = low dose of standard

B = high dose of standard

C = low dose oftest

D = high dose of test)

Pharmacology Practical M ; ~ n u a l

The dose should be such as to produce a clearly discriminated submaximal contractions.

4. ' Perforrn a two point (or 2 = 2) assay. The doses should be given in a latine square

randorn order e.g A B C D A B D C C A D B CBAD B C D A B A D C C B DA DCAB

C 9 A B C D B A B D C A ACBD

.' D A B C D C B A B D A C BC AD

During the qssay the following timing must be strictly adhered to start the drum to

seconds berore adding the drug; allow the drug to act for 15 seconds, stop the drum

wash out the drug. Make sure the bath is filled to the mark, and add drug regularly

at 3 minutes interval.

5. Vanish the tracing, measure the height of each contraction and tabulate your result.

I

C. CALCULATION OF RESULTS

Calculate the average response to each dose and draw log-dose response lines for

the standard and the test solution. The Assay is valid if the two lines are parallel The

horizontal distance(M) between the two lines represents the logarithm of the ratio of

the concentration of histamine in the test and standard. If the volumes of the test and

the standard solutions are not the same, the value of M should be modified. Let s, =

volume of low dose and s, that of the high dose of standard. t, = volume of low dose

and t, that of the high dose of test. Strength of test solution = s, x Antilog. (M) times

that of the standard. The value of t, M can also be calculated from the following

formula: let S, and S, represent the responses to low and high doses of standard. T,

and T, represent the responses to low and high dose of test.

M = T, - S , + T2- S2 x log k,) 1

s, I

(See Pharmxological Experiments on isolated preparation 1968, P.18) Strength

of test solution s,x s,

Antilog M times that of standard. Your result as mglml of histamine.

D. Discuss sources of error in biological standardization and steps taken to reduce

Them.

'To study the action of drug on moolh mu.icle and the sympathetic innewation of the intestine

IFinklern,m Preparation 1

?a; 7 3 . 1 ~ 2 r2 : A 2cm piece of rabbit ileum is suspended in Ty~ocle's solution at 3 7 0 ~ earted with air as usual.

The sympathetic nen-es supplying the gut lie alongside the rnesentclic blood vessel. The piece of the

ileum is dissected so that part of the mesentery with its blood vessels remain attached to the issue. The

mensentary is placed owr two electrodes for electrical stirnufation of the sympatlzetic nerves.

The 3ppeed of the drum should be such that each rl~ythmic contraction of the tissue can clearly

be distinguished on the smooked &urn. Satir.f'acto:-y records will be obbhed only if th@ gut i! treated

gently.

?hike a rccord of'nos~nal rlzyth~tnic a~tion of tlrt: gu~, and show how this is modified by drugs.

Control record of the base-line must be obtained before each &-ug is put hto the bath. I)rugs should

bc washed out irnmediatcly after a suilstble rGsponse 11s obtained. Scveral changes of bath fluid may

be needsd to restore norrtrnl movement and tone. Stimulation of the peripheral nerve.

This will he done by the demonstration:

PHOCEDl.rRE:

1. Noradrenake Add 4pg tu the bad1 and record for one min. Wash thorough

2. Aclrert71ine Add 4pg to the bath and record for one rnin. Wagh thoroughly.

3. I~_opr~.rnal~~~ Add 4pg to the bat11 and record for one min. Wash thorough!y.

4. m v d r o g e r g o l (a) Add ZOpg ctiI~ydsogergotarnine to the bath 'and record the

response for 3 minutes.

(b) Miilkout washing oul the clnig and pg. Nomdrenaline

Record for one minute and wash.

5 . Repeat 4 but instend m pg. lsoprtmline after pretreatment with dihydroergotamine. - - ~ ~ ~ ~ ~ ~

6 ,- ' - Stimnlstc the peripKerd s~@ithL.tic n&eSesf& 20 (using 2 5 shocks per second

at 1 . h second pulse duration and 30 votts). Rccosd for 2 minules 'after the stimulation

has been stopped. W:tsh once).

7. Add 100pg or g u a n ~ t h i h e to tht: bafh. Record the effect. After 5 minutes without

response for cnr minulc. LL;-l;n:;h thoroughly.

.-- I>iscuw briefi- the site of action of the dtvgs uacd. IV'hid~ otlw drugs have actions

ElTErnrnNT -.- 5

TIiE AmON OF DRUGS OK THE ISOLAXD TOAD -- ---

KECTUS ABDO -- hdEYIS MClSCLE

OBJECT: To atudy the effect of skeletal muscle relaxants on isolated toad rectus abdominis muscle.

~ ' ~ I Q Q : The rectuq abdominis muwle of the toad $ suspended in an organ bath containing

ncrateil frog-Ringer solution at room temperature. One end of ~ i e tissue i~ ticd to a ,

recording levzr. The Kynognph is adjusted to run at a speed of 4-8mrn per ,

minute. (See Fig 1)

PROCEDC?:

1. Add acetylcholine 1 Ot~g/mt final concentration and ~ I U O W the muvclc preparation to contract

for 9(! sccs. Stop thc drum and wash tl~o~vughly until the lever returns to i(s base-lint

I 2. Lstabtiuh a dose rcsponvc curve to acetylcholine: (Ach) ~ ~ i i l g l,ug.? 4,ug., Spg., Gpg., etc until a

rn~axhurrt rcsponsc is obtained.

I 3. Es~ablish a dose mspoonc c m to t.<;h in the presence of tubowrarine (10,~c~nd) in fin,-

conc.entration; udng a YO sec contact time. The doses of Ach should be as in expt 2 above.

I 4. Soak the rccm muscle in frog-Kinger solution containing (10pgml) of phyaostigmine.

Consrmct s Jose response curve to Ach fn the presence of this eserinised Ringer using thc

snme doses of Ach nu in expa-iments 2 ;md .f above.

I .-.

What explanation can you _give for ihc shifts in the dose rmponse in each case. . '

1

EXPERIMENT 5

THE ACTION OF DRUGS ON THE ISOLATED RAT UTERUS

.'aslen OIIC enel of a strip to llic hook ol'un acralor. silspc~lcl ill I)c .lolon holirlio~l i l l tlic

inner h::rh xrarcd \ v i h osygcn at 30°C.

'l'hc otllcr cntl is tied lo :I lionli~l \wiling I c \ ~ r . 'I'lle prcp;l~ation .;hol~lcl I K Icl'l l o ~ ~ s l liw 2 0 lo

30 minutes w i~h two or l111.cc ringing hcIi)~.c bci~lg I I \ ~ c I , (Sue I:ig. 1 )

l'l<OCl l > [ J l < l . : : I , Osyloci~i 1111 =- I ~ ~ l ~ ~ ~ i i ~ l i o ~ i i ~ I i111its --

Aclcl 0.0 I unils ol'osygen to tlic Iwlli ('l'i~lic I ml ant1 ~nnke up lo I00

and i~!jccl I111l) and rcco~d 111c cl'lkcl.

Making :lppropriarc dilutions. conslrucr a tlosc response curitc to

EXPERIMENT 6 THE ACTION OF DRUGS ON THE SLEEPING TIME OF MICE

PURP0S:- --..- ,-a

1 . To assess the depressant effect of barbiturates on the C.N.S of n ice.

2. To illur,trate the effects of chlorpromazine and caffeine or) barbittrrate induced

sleep.

N.B. The righting reflex is one of the methods of assessing thc depressant

effect of drugs on C.N.S.

PROCECURE: -- .

I. Studerlts will be divided into 3 groups A B and C.

Each jroup will receive 3 mice which shoirld be weighed.

2 , G R O U P Inject thiopentone 35g intraperitonealy into each mouse

GRO!JP B Inject chlorpromazine hydrochloride sirbcutaneously 5mg followed

by injection of thiopentone 35mg inla each mouse.

GROlJP C Inject caffeine 12.5ug weight intraperitonealy followed by - . -- - -

thiopentorie

35ug intraperitonealy into each nlouse.

3 Test wery orie m~nute for the righting reflex by placing the ariirrial on its back

an 1 noting if it rights itself. Note the time when the r~yhtiriy reflex d~sappears.

lndic ~t ing that the anirnal is asleep.

The ! me between the disappearqnce and reappearance of the righting reflex is

thr sleeping titne.

TI)? results from each group will be poked and tabulated.

Plot a graph of sleeping time versus dose of thiopentone.

I-low do chlorpromazine and caffeine rnodify this dose response relationship?

EXPERIMENT SEVEN : .'I

VALIDATION OF AN EXPERIMENTAL PROCEDURE

cnvirollrncrlhl 111airix bii~s.

Rlcll~odology:

Arr~r rw! of soillc pipelks ci111 I)c cv;~luc~lotl 11s cl is l )c-~~s~~~p, i l l l o ;I \ w l r ~ ~ n c ~ ~ i c Ilubh

0bh i11 3, 101111 1)ipcIles. 1:iIl wit11 I0 1111 ol'dis~illcd \ v i 1 [ ~ 1 l i )~ p i I h ~ c s I\ I t , t ',

Void l l ~ c tT:alcr f i un c x h 1c11 l i ~ w s lo ir 100 1\11 V O I I I I I I C ~ I ic I l ;~d\. WII~CII O S 1 1 1 ~ ~ I ~ C I I C I S C O I I ~ C S

close to ch livering I001i1I cxac\lyl? Which is t l ~ c mo:.l ~I(;CLII;IIC"?

1'1-crisior~: oftlclivcry I0 aliquots of IOrl11 pil~cllcs 10 I I I ~ I I ~ L . "1) Iu I O O t ~ l l call I)c vcviliccl I I ~

r c l w l i ~ ~ g 5 r i n w li)r each pipcllc. C'alc~~li~lc lllc ~ o c l licic.111 01' V ; I I ialiou l i ~ r plxxihio~~ ol'cacl~

SECTION II

CLINIC'A!,

I. AKALGL~SICS . , . ... . . . . . . . . . ... . . . 2 4

2. A I S O R I I I N L X C l l l O N O J S .. . . . . 2 6

3. LOCAL ANAESTI 1I:I'ICS . . . . . . . . . ... . . . ... 27

4. ACVI'ION 01: I IIS1'AtVIINC~: O N 1'1 I I S SKIN . . . ... ... ... 25

5. ACTION O F ANTACIDS ON C;AS'I'KIC' J I l I ( ' I ~ . . . , . . . . 2 0

6. AC'SION O F DRLJGS ON '1'1 IF I JlJh?AN II't'I; . . . . . . . . 30

CLASS I --

Analgesics are drugs employed for the rel~ef of pain. .

Pain sensations are of types:-

(1) y~sceral p a b which arises froin diseases of the viscera inflammation of the

organs such as intestine, ureter and bile duct

(2) Somatic pain which arises from diseases of the skeletal muscle and joints,

skin and subcutaneous tissue.

. . .. . . . .

Aoalgesics are a!so of 2 main type:-

(1) Morphine and related substances

(2) Salicylic acid, para-mino-phenol and pyrazolone derivatives. The later group

are effective only in somatic pain while the morphine group are effective in the

more severe visceral pain.

The valuation of analgesic activity is difficult for several of pain -

(a) Drugs differ in the ability to suppress "ariaus types of pain

(b) Art~ficially induced pain is often different iron1 the disease

(c) Pain is a subjective sensation; the threshold therefore varies from individual to

individual even in the same person the threshold may not be fixed or constant.

The evaluation of analgesic activity can be carried out both in experimental an~mals and

on human subjects. Tests carried out on human subjects offer a more precise method of

assessment and the tests carried oqt on human subjects suffering from pain give a true

assessment of the relative potency of the drugs in the different types of pain. Tests

carried out on animals serve only as FI ro~rgh assessment of analgesic properties.

EFFECT OF ANALGESIC O N THE ISCHAEMIC PAIN

We shall assess quantitatively the analgesic potency of Tab. Panadol IC, Tab

Ponstan 100mg and Tab Aspirin IC on artificially induced pain in normal human

subjects.

Pain is produced by execising the muscle when supply is inadequatoe (Ischaemia).

To dc, this a sphygmomanometer cuff is fitted on the upper right arm arid a f~ye.d..prj?ssure_

(ranse-I 10-1 30mgHg) applied throughout. The hand is forcibly clenched at regular rate

(e.g 40-60 per min) until a moderate and easily recognizable pain is felt. The number of

contractions is recorded. The procedure is repeated after rest periods (during which the

presure on the cuff is released), until the number of contractions required to produce

pain is constant.

Acode is used for the three drugs and a place so that the student is unaware of the

ident'ty of the drug he is taking until he has finished his calculations. The drug is taken

oral 2nd the test repeated 30 mins, 60 mins and 90 mins after taking the drug.

The average figure for each period of observation from each group of students

taking a particular type of drug is recorded. The percentage difference for each period

of observation over the control period (zero minutes) is then calculated. The figures for

different drugs serve as an index of the relative potencies of the drugs. Plot graphs to

illustrate this. The onset of actions of the dr.ugs can also be discerned from the plots.

Indicate peak time, approximate duration of action and other pharniacodynamic

para neters.

Pharmacology Practical Manual

CLASS 2

ABSORPTION AND EXCRETION OF DRUGS

The following drugs will be studied.-

(1) Sulphonamides - Solphadimidine;

(2) Salicylate - Asprin;

(3) Purgatives (as a weekend work).

The students shall be divided into three groups. Each student in the first two groups will take just before

breakfast either 1.59 of sulphadimidine (3 tablets) or l g of aspirin (3 tablets). The bladder should be emptied a

few minutes before the tablets. The third group will be given no drugs. All groups should as much as possible

delay emptying the bladder for 2 hours before the class begins.

Test for Sulphonamides:

1. Mix 5mls of urine and add lml of NaOH (80g11), add 2 drops of Copper II sulfate 160g11.

Ts to give greenish blue ppt.

NB: Do these tests using normal urine, urine from students who have taken aspirin and sulphonamide and

normal to which aspirin and sulphonamide had been added.

Salicylate - (Garhardt's Test)

Take some urine (about 3ml) in a test tube and add 10% ferric chloride solution drop by drop.

Appreciate usually forms and disappears again on adding more ferric chloride.

Use the following types of urine:

1. Urine from a student who has taken aspirin

2. Normal urine to which aspirin has been added

3. Normal urine to which aceto-acetic acid has been added

4. Normal urine

5. Urine from student who has taken sulphonamide

6. Normal urine to which, sulponamlde has been added.

Also perform the test after boiling urine 1 and 3.

Record the colour changes. What conclusions do you know from the results?

Purgative - Weekend Work

Each student will be provided with one of the following purgatives:-

Casseara sagrade (2 tablets)

'Dulcolaxw (2 tablets) - - - - - - -

- - - - - - - - -

The tablets should be taken at night. Note the TolhMng poititsani write adescription of yow exf3erier~e-

1. Time of administration of the drug

2. Time of the first evacuation

3. Consistency and colour of stool

4. Griping

5. Number of motions

6. Any other effects

CLASS 3

Local a~laes\lic~ics are drugs which wlict~ al~pliccl locally ill suiliiblc. conccn~ralions havc i~

sclcclivc action in locking the concluelion ol'scnsory ncsve i~~ipulscs. Loss ol 'scnsi~~ion is llie pl'otlllccll. Only Ihc3 action uf thcse C I ~ L I ~ S 011 skin oncl nlucilos 1netn111~1ies \bil l bo studicd hcrc.

(a) C . 11111 1io111ii11 saline (h) (1. I ml procaine Iiydrucl~loricle 2% (c) 0.11nl procaine liydr-ocliluritle conlqining also Aclrc~ialinc Iiyrlrocliloriclt. O.OI'!/;,

( ! : 10.000 solulion) (d) O 11111 Ailrcnalilic hyd~~c~clilot~idc 0.OI1%

3. Aclios of An~c.thoc;tinc I~yd~ocl~lprir lc i111tI I ' ~ w ; ~ i ~ l r I~ydrocltloridt. oil Mucous h I C I I I ~ I I ~ ~ I ~ I C I told a s~iiall piece ol'colloil wool soilkcd ir, 2% Aincll~ocaine Iiyd~~oclilori~lc 10 one hi\((' ol'tlie mucous membrane ol'llle I O L V ~ ~ lip I i ~ r 2 niitlittcs ;111d a ~ ~ l i i ~ l l piece soi~kccl il l 2% pwcaine solution to ~ l i c other lialf Ibr h e s a n e ~ w i o t l New lest fill. :111iilgcsia i ~ t d li)r aj~prcciation of lcmpertlturc (Iiol atid gold) on ci1c11 1i;rlfofllic lip. Vi'li:~( conclusions do you draw fioni your ohscrva~ions? I low tlo y o ~ l ex plait1 llic cl117k1e1ices observed beI\veen Ille hvo dri~gs'?

- Pharmacology Practici~l Mi~nud

CLASS 4

ACTION OF HISTAMINE ON THE SKIN

Studen~s should work in groups of4.

Experiment 1 Inject intradernlally in thc forearm ofonc member ofthe group. 0.1ml of 10,000 solution of histamine acid phosphate. Avoid the neighbourhood of veins. Observ!: carefully the effects of the injection and rccord thc following observations every 2 minute:; for I0 minutes and then every 5 minutes for another 20 n~inutes. Pain Character and intensity.

I'ruritlis Frythernn: Measurc the exact areas al'l'cctcd (bcst scen in in light colourcd skins). W;liIing or Swelling: Measure the area and assess its degree. Make a drawing of the reaction as i t appeared at the heipht ofthe reaction. Experiment I1 When this has been complete the same proccdurc shoulcl he repeatcd on anothcr partncr cxcept that tile skin ol'the forearm has previously been rubbcd with a cream ol'an antihistamine shortly belbre isljecting the histamine. Describe any changes in tlic reaction which you observed.

Expcriment 111 Repeat '.he procedure as in Expcrinwnt I on the 3"' st~rdcnt ctcept that 5 minutes nlier i~~jccting histamirie 0.3nlI of 1 : 1000 solution ofaclrenaline hyclrocl~loridc subcutaneously into the Dcltoicl area. What changes in the reaction do you observe? What are the effects of histamine on tlic skin as clc~nonstratccl l i w l thc esperi~ncnt? Why Joes adrenaline antagonize the action ofhistami~~e?

Action of I-1y;lluronidase I-Iyalurcnidase is an enzyme which decrefises the viscosity of certain tissue matrices. Its main therapeutic application is to increase the absorption'of lli~icls injected into subcutaneous tissues.

Esperirnent: Inject into the subcutanepus tissuc ol'the right l iwxrn~ (of the remaining stuclent of the group) normal saline solution. S t o p i~ljccting when the swelling begins lo heco~ne pni~il'i~l and notc the volun~e irljected. Measure thc swclling am1 assess its tcnsion by palpi~tion. Repcat these okse~wtions every 5 n~inutes for 15 minutes and rccord them.

Repeat the procedure in the opposite forearm, thi.; time injecting normal sali~w to wliicli hyaluronidase has been added (200 Bengel-units to the pint).

What do you notice? . In what type of case would you collsider using liyalilronidase'?

- 4

CLASS 5

ACTION OF ANTACIDS ON GASTRIC JUICE Antacids neuitrnlize thc excess gastric ociclity which is prcscnt in peptic iilccr (ulceration

ol'tlie n!ucous ~iienibranes of the stomach and di~oclcnuni). 'l'lic neutralizing power ol'viuious

antacid\ vary. Moreover some nutacids reduce the clcgrcc ol' acidity by iibsorbing (physical

proccss) sonw ofthe hyclrochloric acid ol'thc gastric juice

I~lXPEKIML<N'f I p-

Add 5ml of N/ 10 Ilyrochloric acicl to each oS 5 LC:;\ tul)cs A,D,C, and note llie pi-l

(using hlulti-range Indicator Paper).

'l'hcn add 50mg (weight) of sodium bicnrbonutc to tube A

50nig .. .. Magnesium oxide lo tube I\

501ng .. .. Calcium carbonate to tube C:

In each case allow the antacid to act Sor 5 mi~iiiks and re~neasl~re the pl I . 'I';lb~~latc

your results. Which is the most cfl'ective antacid'?

i?snerin~enl 2

~\clcl 1 OOmg of Magncsiuln trislicalc inlo 5 bcal\ers A. 13, C', I), ilnd I!. 'I'lic.11 d t l al ~ h c

nubytc ii~tervals 201nl ofNI10 I lyclrochloric acid proviclccl. Slidic well anel allow thc antxiel ill

the beali:rsA, 13, C, D, E to act fol-5, 10, 15,20,25 minutes rcspcctive bel'ort, liltcring, llse 5ml

of the li:tratc ancl titratc with NI10 Soclii~m hydroxide to clctcrmine the nl~iount ol'aciclity in term

oSNo. ol'ml ol'N110 NnCI 1 used. Plot a graph to s l~ow t l~c change i n acidity with timc. Wl~at

c o ~ i c l ~ ~ d i o ~ i s (10 yo11 draw from this? ,

Experin-ent 3

-!'he proccdurc is as in c x I ~ e r i ~ e k 2 except tliat ~ O L I I I S ~ 111;1g11csil11ii ositlc l Onlg \\it11

201111 ol'U10 hydrochloric x i t l .

( I) Plot n graph to show the change in acidity with tlic time

(2) M;hat conclusion clo yo11 draw fro111 this.