PHARMACOLOGY LABORATORY EXPERIMENTS

WRITTEN REPORT
First Semester 2007-2008

Submitted By:
Subsection C6

Pineda, John Michael

Pingoy, Anna Katrina
Piodena, Romeo III
Pioquinto, Fatima
Pioquinto, Khlaire
Pipo, Eugenio III
Ponce, Sharon
Prieto, Rei Joseph
Pua, Jerry
Punzalan, Kristian Anteolin
Quintos, Abigail Marie
Rabago, Maurellen
Ramirez, Joseph Michael
Ramos-Yeo, Andrea
Ramos, Christine Joyce
Ramos, Marie Angelique
Ramos, Ralph Lawrence
Reyes, Joelle Erika
Senoren, Lauren

Dr. Benjamin Bangahan

Facilitator

03 August 2007

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 Experiment 1
GRADED DOSE-RESPONSE CURVE

OBJECTIVES

1. To determine the graded dose-response curve for the following drugs:

paracetamol, aspirin, celecoxib, and prednisone
2. To calculate the potency and efficacy of the following drugs: paracetamol, aspirin,
celecoxib, and prednisone
3. To compare the potency and efficacy of the following drugs: paracetamol, aspirin,
celecoxib, and prednisone

INTRODUCTION

The dose-response curve shows the correlative relationship between the

characteristics of exposure to a drug and the effects caused by the drug. There are two
types the quantal and the graded. For this experiment the graded dose-response curve
was used. It determines the graded response of the individual taking the drug with
different doses of the drug. This is used to determine the efficacy and potency of the
drug. The efficacy measures the ability of the drug to induce a certain action at a certain
dose. From this the maximal efficacy, the maximal response of the drug, can also be
taken which is shown as the highest point in the dose-response curve. On the other
hand, potency is the amount of drug needed to produce a given effect. To differentiate
the graded dose-response curve from the quantal dose-response curve, the quantal
determines the distribution of responses to different doses in a population of individuals.
Quantal dose-response curve also measures the amount of drug needed to induce the
desired specific response thus it can be used to measure EC50 and LD50. EC50 is the
concentration at which a drug can produce the therapeutic effects in 50% of the
population. LD50 is the concentration of the drug at which the drug produces the toxic or
lethal effect at 50% of the population. These measurements are all useful in the
evaluation of the pharmacologic agents that we have.
The use of the graded-dose response curve in this experiment can be seen as
the four drugs efficacy and potency will be compared with each other. In the end, the
experimenters would be able to determine which drug would be able to have the
maximal efficacy and which one would be the most potent.

ANIMALS: 12 wk old rats (32 rats per drug)

MATERIALS: phlogistic agent (Carageenan 1% in NSS)
Gavage tubes
Mercury set-up
Small beakers for drugs
Surgical masks

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 Surgical gloves
DRUGS Paracetamol
Aspirin
Celecoxib
Prednisone
0.9% Normal Saline solution

METHODOLOGY

The animals were fasted overnight prior to weighing. They were weighed using
the triple beam balance and grouped according to drugs and dose levels. (32 rats per
drug, 8 rats per dose). An ink mark was placed on the right or left paw at the level of the
lateral alveolus. The doses of the assigned drug were then computed. All the drugs
were dissolved in 1mL NSS (0.9%)

Paracetamol
Preparation: 500 mg tablet
Doses: 0.5 mg/200 g rat
1.6 mg/200 g rat
5.0 mg/200 g rat
16 mg/200 g rat
Aspirin
Preparation: 200 mg tablet
Doses: 0.5 mg/200 g rat
1.6 mg/200 g rat
5.0 mg/200 g rat
16 mg/200 g rat
Celecoxib
Preparation: 200 mg tablet
Doses: 0.3 mg/200 g rat
1.0 mg/200 g rat
3.0 mg/200 g rat
10 mg/200 g rat
Prednisone
Preparation: 20 mg tablet
Doses: 0.02 mg/200 g rat
0.06 mg/200 g rat
0.2 mg/200 g rat
0.6 mg/200 g rat

The foot volume was then determined using the mercury set-up during the
following periods: (1) before administration of the test drugs, (2) immediately after
Carrageenan injection and (3) every hour for the next three hours after injection. The
test drug was then administered using tuberculin syringe with gavage tube. After 1 hour,
0.1 mL of the phlogistic agent (Carrageenan) was injected into the paw of the animal.

Finally the percent difference of the foot edema was calculated using the following
formula:
reading after- reading before
% difference = (injection of phlogistic agent) x 100
reading before injection of phlogistic agent

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RESULTS

Expected results

In this experiment to determine the graded response curve of different drugs,

potency and efficacy were calculated. It is expected that prednisone, an inhibitor of
phospholipase A2, would be the most potent of the different drugs that were tested
followed by celecoxib, which selectively inhibits COX-2, then paracetamol, a weak COX
inhibitor and lastly by aspirin that inhibits COX. For the efficacious drug it is again
prednisone that is expected to be the most efficacious drug. It is followed by celecoxib
and aspirin both equal in efficacy and lastly by paracetamol being the least efficacious
drug.

Actual results

The actual results were not the same as the expected results. Aspirin, base from
the actual results of the experiment, was the most efficacious of the drugs followed by
celecoxib then prednisone and paracetamol being the least efficacious drug. Regarding
the potency of the drugs that were tested in this experiment, it was not measurable
because the results plotted didn’t show any measurable hyperbolic curve to determine
which among the drugs were the most potent.

DISCUSSION

Before the animals were injected with the four different drugs, they were
administered carrageenan. Other than its industrial uses (thickening agent, cosmetics), it
is also a pharmaceutical agent. It acts by activating macrophages and increasing
cyclooxygenase-2 expression. The increase in COX-2, leads to an increase synthesis of
prostaglandins and thromoboxanes. These eicosanoids induce inflammatory reactions in
the body. Carrageenan was injected to induce inflammation.
Four anti-inflammatory drugs (acetaminophen, aspirin, celecoxib, and
prednisone) were administered to the animals in order to compare potency and efficacy.
These drugs act as inhibitors to the different enzymes in the pathway generating
inflammatory mediators. Acetaminophen (Paracetamol) is a weak inhibitor of COX-1 and
COX-2, therefore having insignificant anti-inflammatory effects. Aspirin is a non-
selective, irreversible COX inhibitor and it also inhibits platelet aggregation. Celecoxib is
a selective COX-2 inhibitor, making it ineffective in the gastrointestinal tract which
contains COX-1 enzyme. Prednisone inhibits phospholipase A2 (PLA2), which is the first
enzyme upstream of the COX enzymes in the inflammatory pathway. The COX-1
enzyme generates products that have physiological functions in the gastrointestinal tract,
kidney, platelets and endothelium. The COX-2 enzyme, which is inducible, produces
inflammatory prostaglandins and proteases that act in the inflammatory response.

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 Potency and efficacy can be demonstrated using the graded-dose response
curve wherein the response of an individual (y-axis) is plotted against the different log
doses of the drug. The potency is the effective concentration of a drug producing fifty
percent of its maximal effect (EC50). Thus, the lower the EC50 of a drug, the more
potent it is. Efficacy refers to the capacity of a drug to induce a specific effect at a certain
concentration. It is usually characterized by the maximal efficacy, which is the maximal
response a drug can produce. It is represented by the highest point in the graded-dose
response curve.
The different curves generated for each drugs after one, two and three hours of
dosing did not produce the typical shape of a logarithmic plot. The relative potencies of
the drugs cannot be compared because no appreciable sigmoid curve was generated for
any of the four drugs. After three hours, the highest maximal efficacy was produced by
aspirin. Celecoxib has the second highest efficacy, followed by prednisone. Paracetamol
displayed the least maximal efficacy. These results deviated from the expected outcome
(prednisone> celecoxib=aspirin> paracetamol). Prednisone should exhibit the greatest
maximal efficacy and potency because its mechanism of action acts on the highest level
of the inflammatory pathway. The efficacy of celecoxib and aspirin is theoretically the
same, but celecoxib is more potent than aspirin. Paracetamol is the least effective and
potent because it only exhibit reversible inhibition to COX enzymes.
The difference between actual and expected results could be due to several
factors such as experimental errors and limitations in the experiment. Experimental error
could arise from the fact that different students administered the various dosages of the
four drugs and the readings of the foot volume were also taken by several students. One
of the limitations is the number of rats used in the experiment. The limited number of test
subjects (thirty two rats per drug, eight rats per dose) could have affected the results. It
was also noted that the weights of the rats were variable. These differences in weight
yields varying pharmacokinetic responses, thus affecting the experimental results.

CONCLUSION
It is therefore concluded that Aspirin has the highest efficacy, followed by
celecoxib, then prednisone, and paracetamol being the least efficacious. The potency
could not be determined because no appreciable hyperbolic curve was generated.

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 Experiment 2a
FACTORS AFFECTING DRUG ACTION
Influence of Route of Drug Administration

OBJECTIVES
General: To determine the influence of route of administration on the latency and
duration of action of Ketamine Hydrochloride injection

Specific
1. To determine the latency and duration of action of Ketamine
Hydrochloride injection when administered intravenously

2. To determine the latency and duration of action of Ketamine

Hydrochloride injection when administered intramuscularly

3. To compare the latency and duration of action of Ketamine Hydrochloride

injection when administered intravenously and intramuscularly

4. To evaluate data obtained by using appropriate statistical tests

INTRODUCTION
The route of administration is the path by which a drug is presented to the body.
It affects the latency and duration of the drug action. The different routes have its own
advantages and disadvantages. Oral route is convenient but the response may be
slowed down by food and increased peristalsis. Rectal route is advantageous if the
patient is unconscious or vomiting but the drug may not be completely absorbed.
Inhalational and parenteral administration causes rapid absorption and accurate dosing
of the drug but has a high risk for infection and is irreversible. Drugs administered
through transdermal and cannulae routes are invasive methods, but may be
advantageous because these are rate controlled and localized respectively.
Ketamine is a phencyclidine derivative synthesized by Stevens in 1962. It is
arguably the most ideal anesthetic agent because of its decreased psychotropic effect
than its parent compound. It can be given by either the intravenous or intramuscular
routes to provide surgical anaesthesia. Excellent analgesia and sedation can be
obtained with smaller intravenous doses.
The righting reflex is an automatic righting reaction integrated in the midbrain that
bring the body into its normal position and resist the forces acting to displace it out of its
normal position. Loss of this reflex indicates the inability to take the body to its normal
position, which is to stand on its feet. Latency of righting reflex loss in this experiment is
the time delay between the administration of the drug and the onset of the loss of
righting reflex. Duration on the other hand is the amount of time that the righting reflex is
lost, from its onset to the time it gains back its reflex.

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ANIMALS: Rabbits (4 per section)
MATERIALS: Rabbit cage
Animal weighing scale
Tuberculin syringe
Stop watch
DRUG: Ketamine Hydrochloride
Preparation: 50 mg/mL
Dosage: 5 mg/ Kg

METHODOLOGY and RATIONALE:

Four rabbits were weighed and recorded. Two rabbits were administered with the
drug intramuscularly and the other two were given intravenously.
The following were recorded: time of injection, time the righting reflex was lost & time the
righting reflex was regained. Righting reflex is a term used to describe various reflexes
that tend to bring the body into normal position in space and resist forces acting to
displace it out of normal position. Evaluating the latency and duration of the loss of
righting reflex demonstrates the difference in effects of the Ketamine Hydrocholoride
when administered through two different routes.
The onset and duration of effect of Ketamine Hydrochloride were compared when
administered IV or IM.
The significance of the results were calculated using the appropriate statistical
test, setting the p-value at 0.05.
COMPUTATION: Amount of Ketamine to be administered
= Wt of rabbit(Kg) x 5mg/kg (dosage) x 1mL/50mg(prep)
Example:
For a 2.65 kg rabbit:
Amt of ketamine administered=2.65kg x 5mg/kg x 1ml/50mg
= 0.27
RESULTS:

Section Intramuscular Intravenous

Latency Duration Latency Duration
(sec) (sec) (sec) (sec)
A 123 1196 46 851
A 239 680 3 1138
B 192 724 69 184
B 388 195 33.5 389
C 147 383 52.3 237
C 73 671 38 106
D 174 1716 156 756
D 98 1032 30 600

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average 179.25 824.625 53.475 532.625

STATISTICAL ANALYSIS: t-test

t= IM - IV - (µIM - µIV)
Sp √(1/nIM + 1/nIV)

Where: IM = mean (intramuscular)

IV = mean (intravenous)
Sp = pooled std deviation
nIM = no. of subjects (intramuscular)
nIV = no. of subjects (intravenous)
µIM - µIV = estimated difference between the means

1) Two- tailed independent t-test

Ho: There is no significant difference between IV and IM (µIM = µIV)
HA: There is significant difference between IV and IM (µIM ≠ µIV)

At 95% confidence interval (α = 0.05), critical value = 2.1448

LATENCY:
IV IM
Mean 179.25 53.475
Std Dev 99.6 45.59

df = 14 sp = 77.46
t = 3.25 > 2.1448
At 0.05 level of significance, there is significant difference between
latency in IV and IM.
DURATION:
IV IM
Mean 532.63 824.63
Std Dev 365.25 481.30

df = 14 sp = 427.23
-2.1448 < t = 1.37 < 2.1448
At 0.05 level of significance, there is no significant difference between
duration in IV and IM.

2) One-tailed unpaired t-test

Ho: Latency in IM is not significantly longer than latency in IV
HA: Latency in IM is significantly longer than latency in IV

critical value = 1.7613

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 t = 1.37 < 1.7613

At 0.05 level of significance, there is no significant difference between

duration in IV and IM.

DISCUSSION:
Intravenous injection is the giving of liquid substances or drugs directly into a
vein and it is the best way to deliver a precise dose quickly and in a well-controlled
manner throughout the body. Bioavailability of the drug is 100% which causes the most
rapid onset of the effects.
Intramuscular injection is the injection of a substance directly into a muscle. It is
used for particular forms of medication that are administered in small amounts.
Depending on the chemical properties of the drug, the medication may either be
absorbed fairly quickly or more gradually. Absorption of drugs into the blood stream
depends on the blood supply to the muscles. Intramuscular injections are often given in
the deltoid, vastus lateralis, ventrogluteal and dorsogluteal muscles. Bioavailability is
from 75-100% due to incomplete extent of absorption.
Latency was measured from the time of injection to the time the righting reflex of the
rabbit was lost.
• Intravascular injection circumvents problems in absorption due to direct
administration into vascular system
• Intramuscular needs to pass through muscle tissue before it reaches site of
action thereby delaying its effects.

Duration of Action was measured from the time the righting reflex was lost to the time
the righting reflex was regained
• Route of administration of a drug does not affect duration of action

CONCLUSION:
• Appropriate routes of administration affect the action of the drug.

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• Intravascular route has a shorter latency than intramuscular route.

• Duration of action does not affect the route of administration.

IV IM

Latency shorter longer

Onset rapid slow

Duration of action same same

Amount of drug smaller larger

Recovery rapid slow

Experiment 2b
FACTORS AFFECTING DRUG ACTION

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 Influence of Chemical Structure

OBEJECTIVES
General

To determine the effects of Epinephrine, Dobutamine, and Terbutaline on heart

rate and amplitude based on their chemical structure

Specific

1. To measure the rate and amplitude of baseline contractions of the turtle’s heart
2. To measure the rate and amplitude of contractions caused by each drug
3. To evaluate the results of each drug with p value set at 0.05 using one-way
ANOVA

MATERIALS and METHODOLOGY

Materials Animals
•Kymograph set-up
• 8 Turtles
•Dissecting set
•Kymograph paper Drugs
•Paste 1. Epinephrine (1:1000 ampule)
•Cotton thread 2. Dobutamine (250mg/20mL vial)
•Surgical thread 3. Terbutaline (2.5mg/mL)
•Heart hook
•Tyrode solution
•Tuberculin syringe

Methods

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 The turtle’s heart apex attached to a
kymograph set up

Baseline rate and amplitude of

the contractions (1 minute)

1mL Epinephrine 1mL Terbutaline

(in 1min) 1mL Dobutamine (in 1min)
(in 1min)

*Wash with Tyrode solution in

between drug administration

Rate and amplitude of

contraction (for 3 mins)

RESULTS

A. Rate of Contraction
Hypothesis
Ho: There is no significant differences on the rates of contraction with Epinephrine,
Dobutamine and Terbutaline.
Ha: There is significant differences on the rates of contraction with Epinephrine,
Dobutamine and Terbutaline.

Table 1. Percentage Difference on Rate of Contraction

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Section
(Subgroups) Epinephrine Dobutamine Terbutaline

A1 -35.7 65 11

A2 40 10 12.5

B1 72.7 40 12.5

B2 8.3 45.4 20

C1 17 117.4 14.8

C2 27.7 -26.3 -18

D1 20 100 -40

D2 9.1 100 -60

Average 15.55 -3.89 -2.735

Based on the results, Epinephrine had the highest rate of contraction with a mean of
15.55 followed by Terbutaline with a mean rate of -2.735 while Dobutamine had the
lowest rate of contraction with a mean of -3.89.
Using ANOVA, the obtained P-value is 0.009893. Since P < 0.05, reject null
hypothesis. There is significant differences on the rates of contraction with Epinephrine,
Dobutamine and Terbutaline.

SUMMARY

Groups Count Sum Average Variance

EPINEPHRINE 8 124.4 15.55 289.0971429

DOBUTAMINE 8 -31.1 -3.89 102.9673143

TERBUTALINE 8 -21.9 -2.735 100.317

ANOVA
Source of
Variation SS Df MS F P-value F crit
Between
Groups 1902.9036 2 951.4518 5.797040808 0.009893 3.4668

Within Groups 3446.6702 21 164.1271524

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 Total 5349.5738 23

B. Amplitude of Contraction
Hypothesis
Ho: There is no significant differences on the amplitudes of contraction with Epinephrine,
Dobutamine and Terbutaline.
Ha: There is significant differences on the amplitudes of contraction with Epinephrine,
Dobutamine and Terbutaline.

Table 1. Percentage Difference on Amplitude of Contraction

Section Epinephrine Dobutamine Terbutaline
(Subgroups)
A1 -35.7 65 11
A2 40 10 12.5
B1 72.7 40 12.5
B2 8.3 45.4 20
C1 17 117.4 14.8
C2 27.7 -26.3 -18
D1 20 100 -40
D2 9.1 100 -60
Average 19.88 56.45 -5.9

Based on the results, Dobutamine had the highest amplitude of contraction with a
mean of 56.45 followed by Epinephrine with a mean amplitude of 19.88 while Terbutaline
had the lowest amplitude of contraction with a mean of -5.9.

ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 15704.89 2 7852.445 5.519044 0.011852 3.4668
Within Groups 29878.61 21 1422.791

Total 45583.5 23

Using ANOVA, the obtained P-value is 0.011852. Since P < 0.05, reject null
hypothesis. There is significant differences on the amplitudes of contraction with
Epinephrine, Dobutamine and Terbutaline.

C. Chemical Structures

The expected results should show that Epinephrine would have the highest rate
and amplitude of cardiac contraction followed by dobutamine and terbutaline where it
would elicit the least rate and amplitude of cardiac contraction. The ability of these drugs
to induce rate and amplitude of contraction depends on the chemical structures of these

14
compounds. Phenylethylamine consisting of a benzene ring with an ethylamine side
chain is the parent compound from which all of the three drugs were derived.

Epinephrine
Epinephrine is a direct acting sympathomimetic drug. It is a potent
vasoconstrictor and cardiac stimulant. It has hydroxyl groups at the 3rd and 4th positions
of the benzene ring causing a maximal α and β activity. The absence of one of the
hydroxyl group would render it as non-catecholamines. However epinephrine is subject
to inactivation by COMT hence the absence of an –OH group will increase its
bioavailability after oral route of administration. It has a positive inotropic and
chronotropic action on the heart as it acts on β1 receptors and vasoconstriction induced
in vascular beds with its action on the α receptors. The activation of β 2 receptors causes
dilation which increases blood flow.

Dobutamine
Dobutamine is a β2 selective synthetic catecholamine which increases cardiac
output. There is less reflex tachycardia as a result of the antagonistic effect on α
receptors. It increases the force and rate of cardiac contraction. Its primary activity is the
stimulation of β receptors in the heart causing an increase in cardiac output and
contractility. It has positive and negative isomers. The positive isomer is a potent β1
agonist and an α1 antagonist. The negative isomer is a potent α1 agonist that causes
significant vasoconstriction. It reduces vasodilation and thus contribute to the positive
inotropic action. Dobutamine is also used in patients with heart failure causing an
increase in contractility and decrease ventricular filling pressure.

Terbutaline
Terbutaline is a β2 selective agonist. It is used mainly as a fast acting
bronchodilator (short-term asthma treatment) and as a tocolytic to delay premature labor.
It has an α substitution on the amino group of the parent compound which enhances its
β receptor activity. The larger the substituting amino group is, the lower the activity at the
α receptor.

The chemical structure of sympathomimetic drug influences the relative affinity

for α and β adrenergic receptors, intrinsic activity and pharmacokinetic properties.
Increasing the size of alkyl substituents on the amino group increases the beta receptor
activity. The β activity is further increased if there is an isopropyl substitution at the
amino nitrogen. As the size of the substituent increases, the activity at α receptors
decrease. Catecholamines have maximal α and β activity. An absence of the hydroxyl
group would increase bioavailability and duration of action because the drug is no longer
a substrate for COMT metabolism while replacing the hydroxyl groups would also make
the resulting drug more widely distributed in the body. A substitution at the α carbon

15
prevents metabolism of a drug by MAO and prolongs the action of non-cathecolamines
while a substitution at the β carbon is typical for direct-acting agonists.
The experimental result of rate of cardiac contraction was different with that of
the expected results. Epinephrine elicited the highest maximal rate of contraction,
followed by Terbutaline and then Dobutamine. Comparing the amplitude of contraction,
Dobutamine gave the highest result which was followed by Epinephrine and the least
effect shown by Terbutaline.
In the experiment, the maximal α and β activity of epinephrine allows it to greatly
enhance the rate of cardiac contraction, but the amplitude didn’t show the expected
result. Dobutamine on the other hand should have exerted the second highest effect on
the activity of the heart because of its β1 selective activity. It elicited the least rate and the
highest amplitude of contraction. Terbutaline, being a β2 selective drug would have the
least effect since the heart has as its predominance receptor of β1 type and a few β2
receptor shown by the amplitude as it ranked least rate of contraction. The experiment
didn’t show the expected result on the rate of contraction. Its effect exceeded
Dobutamine’s rate of contraction.

CONCLUSION

Basing from the results tabulated and evaluated using one way ANOVA it is
concluded that Epinephrine, Dobutamine, and Terbutaline exhibited varying effect on the
rate and amplitude of the turtle’s heart. But comparing the experimental results with that
of the expected result a discrepancy on the effect of Epinephrine and Dobutamine was
observed. On the expected result it is said that Epinephrine should be the most altering
drug on rate and amplitude of the turtle’s heart followed by Dobutamine and Terbutaline
being the least. This action is attributed to the fact that epinephrine activates not only
beta receptors but alpha receptors too adding up to the ionotropic effect of the drug. But
with the experiment it showed that Dobutamine was the most efficacious in terms of
altering the rate and amplitude of the turtle’s heart, the discrepancy is attributed to the
error done during the experimentation process like experimenter error.

Experiment 2c
FACTORS AFFECTING DRUG ACTION
Influence of Metabolism on Drug Action

OBJECTIVES

1. To determine the prothrombin of time of the rats which were not given any drug
interventions

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 2. To determine whether warfarin prolongs or delays prothrombin time of the rats

3. To determine the whether the effect of rifampicin on the action of warfarin is

synergistic or antagonistic

4. To determine whether the effect of ketoconazole on the action of warfarin, is

synergistic or antagonistic.

INTRODUCTION
Biotransformation is the chemical modification (or modifications) made by an
organism. Once a drug enters the body they undergo a process of absorption,
distribution, metabolism and elimination. Biotransformation may lead to inactivation of
drugs or prolong its effect.
Drug metabolism is the metabolism of drugs, their biochemical modification or
degradation, usually through specialized enzymatic systems. Drug metabolism often
converts lipophilic chemical compounds into more readily excreted polar products. Its
rate is an important determinant of the duration and intensity of the pharmacological
action of drugs. Drug metabolism can result in toxication or detoxication - the activation
or deactivation of the chemical. While both occur, the major metabolites of most drugs
are detoxication products.
Drugs are almost all xenobiotics. Other commonly used organic chemicals are
also drugs, and are metabolized by the same enzymes as drugs. This provides the
opportunity for drug-drug and drug-chemical interactions or reactions.
Most metabolic transformation occurs between absorption of the drug into the
general circulation and renal elimination, although there are few transformations
occurring in the intestinal lumen.
There are two major categories of drug biotransformation: phase I and phase II
reactions. Phase I reactions convert parent drug to a more polar metabolite through
introduction or unmasking of a functional group. Phase I reactions (also termed
Nonsynthetic reactions) may occur by oxidation, reduction, hydrolysis, cyclization, and
decyclization reactions. When metabolites produced during phase I reaction are
sufficiently polar, they may be readily excreted. However, many phase I products are not
eliminated rapidly and undergo a subsequent reaction in which an endogenous substrate
combines with the newly incorporated functional group to form a highly polar conjugate.
Conjugation or synthetic reactions are hallmarks of phase II reaction. Phase II reactions,
usually known as conjugation reactions (e.g., with glucuronic acid, sulfonates (commonly
known as sulfation) , glutathione or amino acids) — are usually detoxication in nature,
and involve the interactions of the polar functional groups of phase I metabolites.
The major site of drug biotransformation is the liver. Factors responsible for the
liver's contribution to drug metabolism include that it is a large organ, that it is the first
organ perfused by chemicals absorbed in the gut, and that there are very high
concentrations of most drug-metabolizing enzyme systems relative to other organs. If a
drug is taken into the GI tract, where it enters hepatic circulation through the portal vein,

17
it becomes well-metabolized and is said to show the first pass effect. Through the first
pass effect, the bioavailability of the drug, which is Fraction of unchanged drug reaching
the systemic circulation following administration by any route, is reduced. The liver uses
the CYP450 enzyme system to metabolize drugs.
Enzyme induction results in an acceleration of substrate metabolism and usually
decreases in the pharmacologic action of the inducer and co administered drug;
therefore, Induction of the CYP450 enyzmes would result to increased metabolism of
drug substrates thereby decreasing their pharmacologic effects by increasing their
elimination. On the other hand, inhibition of CYP450 enyzmes would result to decreased
metabolism of drug substrates thereby increasing their pharmacologic effects by
decreasing their elimination.

Warfarin
Warfarin is an anticoagulant that blocks the γ-carboxylation - glutamate residues
(prothrombin, factors VII, IX and X, proteins C and S) and inhibits Vit K epoxide
reductase. It is readily absorbed after oral administration and is 99% bound to plasma
albumin. Warfarin’s elimination depends on metabolism by cytochrome P450 enzymes.

Rifampicin
Rifampicin is an antimycobacterial drug that blocks transcription by interacting
with the β- subunit of bacterial DNA-dependent RNA polymerase and inhibits RNA
synthesis by suppressing the initiation step. Rifampicin induces synthesis of cytochrome
P450 enzymes. It is adequately absorbed (oral) and undergoes enterohepatic recycling.

Ketoconazole
Ketoconazole is an anti fungal drug that interacts with C-14 α- demethylase ◊
block demethylation of lanosterol to ergosterol (fungal membranes) ◊ disrupt membrane
function◊ increase permeability. It inhibits synthesis of cytochrome P450 enzymes
through competitive inhibition to the calcium binding sites. Its absorption impaired (food,
antacids,Rifampin)

MATERIALS
- 8 200g rats
- Rats with the same weight will have less variation with the results of the
substrate. Dosage of the drug is also in direct correlation with the weight of the
individual rat.
- 0.9% NSS
- 0.9% NSS is used as control to see the various effects of warfarin alone, warfarin
with rifampicin and warfarin with ketoconazole.

METHODOLOGY

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 1. Rats were fasted for 8 hours prior to experiment. Fasting was done to ensure that
the metabolism of all the rats to be used were the same. Also to make sure that
there are no extraneous factors that will affect the drug metabolism.
2. The rats were then weighed. It is important for the rats to be individually weighed
and that they have almost the same weight and the same sex to reduce
discrepancy in result (possibly from slower absorption time due to greater tissue
mass). Dosage is calculated to “personalize” amount of drug given to the
subjects
3. The rats were divided into 4 groups: Group I received no treatment – only 1 ml of
0.9 % NSS was given; Group II received Warfarin alone; Group III received
Warfarin and Rifampicin; and Group IV received Warfarin and Ketoconazole.
Dosage computation: Dose = 0.2 mg/200g
0.2 mg = x____
2000 mg wt. of rat
Drugs were given orally using a gavage once daily for 4 days.
4. Prothrombin Time of each rat was determined on the fifth day.

Prothrombin Time Determination

Blood was collected from the tail of the rat by cutting a portion of it and was placed in
a citrated test tube to avoid hemolyzing the blood. The blood was centrifuged for 3
minutes. 0.1mL (100uL) of each rat’s plasma and 0.1mL (100uL) of normal control
plasma was transferred in a test tube. The prothrombin time was measured by adding
0.2mL (200uL) pre-warmed thromboplastic reagent (Simplastin). The prothrombin time is
then measured from the time of the mixing until the appearance of the fibrin strands. PT
determination was done one at a time to be able to observe the fibrin strands very well.
5. Results were then analyzed statistically.

RESULTS AND DISCUSSION

Warfarin is an anticoagulant which is used to prevent tendency for thrombosis or

as a prophylaxis to those who have already formed a blood clot and may reduce the
risks of embolism. Warfarin has a long half life in where in takes days for the
pharmaceutical effect to take place. Its activity has to be monitored by frequent blood
testing, in this case, Prothrombin time. It is also referred to as a Vitmain K antagonist
which blocks Vitamin K dependent clotting factors such as Factor II, VII, IX, X as well as
Protein C, S and Z. It is a Vitamin K antagonist by inhibiting Vitamin K epoxide
reductase. It is readily absorbed after oral administration and is 99% bound to plasma
albumin and its elimination greatly depends on the cytochrome P450 enzyme.
Rifampicin induces CYP450 enzyme induction; therefore, there is an increase in
the metabolism which will further decrease a durg’s pharmacological effect by increasing
its elimination.

19
 Ketoconazole, on the other hand, inhibits the synthesis of cytochrome P450
enzymes through competitive inhibition to the calcium binding sites which would
decrease metabolism and increase their pharmaceutical duration since there is a
decrease in its elimination. Ketoconazole interacts with C-14 alpha-demthylase which
blocks the demethylation of lanosterol to ergosterol found in the fungal membranes
which disrupts the membrane function and increases its permeability.

Table 1.0. Actual Results of the Experiment

Drugs Administered

RAT Group 1 Group 2 Group 3 (Warfarin Group 4 (Warfarin &

(0.9% NSS) (Warfarin) & Rifampicin) Ketoconazole)

1 6 39 127 900

2 3.4 31 279 298

3 3.5 28 334 148

4 2.2 28 245 900

5 2.6 30 307 155

6 8.7 37 455 140

7 3.5 44 238 150

8 9 ---- 179 119

MEAN 4.86 33.86 270.50 351.25

However, in the experiment, some factors were beyond the control of the
experimenters and this resulted in the misinterpretation of experimental results. This
lead to the discrepancy of results observed specifically with the interaction of warfarin
and rifampicin. (See Table 1.0 above). This is due to the fact that the induction of hepatic
enzymes takes weeks to manifest due to the long periods required for hepatic enzyme
synthesis. This in turn would not manifest as increased warfarin clearance and inhibition
of warfarin’s anticoagulant activity would not take effect. Hence, the discrepancy, a
prolonged
400
bleeding time even in the presence of riffampicin, was observed. (See Figure
1.0, Group 3, below) 351.25
Mean PT (seconds)

350

300 Figure 1.0. Comparison of Mean Prothrombin

270.50
Times

250
No effect seen yet with only 4 days of administration

200

150

Ketoconazole
100

50 33.86

4.86
0
Group 1 (0.9% NSS) Group 2 (Warfarin) Group 3 (Warfarin &
20
Group 4 (Warfarin &
Rifampicin) Ketoconazole)
Drugs Administered
Methodology:

CONCLUSION

We can conclude that if warfarin is administered at the same time with

rifampicin or ketoconazole, prothrombin time would then be altered due to the induction
or inhibition of the first pass effect, respectively. Rifampicin decreases the bioavailability
of warfarin by increasing its elimination due to CYP450 induction and is expected to
lessen warfarin’s anticoagulative effect. Ketoconazole,on the other hand, inihibits the
synthesis of cytochrome P450; therefore there will be an expected increase in the
anticoagulative activity of warfarin due to its decreased elimination.

Experiment 2d
FACTORS AFFECTING DRUG ACTION
Influence of Antagonism on Drug Action

OBJECTIVES

General: To show the influence of drug antagonism on drug action

Specific:

1. To determine the effect of morphine on the duration of tail erection in mice

2. To compare the duration of tail erection recorded in rats given morphine alone with
those given with both morphine and nalbuphine

21
3. To perform the appropriate statistical analysis in determining any significant
difference between the two groups

4. To identify and explain the type of antagonism between morphine and nalbuphine

INTRODUCTION

Antagonistic drugs are those drugs which attenuates the effects of an agonist.
Antagonism can be competitive and reversible (i.e. it binds reversibly to a region of the
receptor in common with the agonist.) or competitive and irreversible (i.e.antagonist
binds covalently to the agonist binding site, and no amount of agonist can overcome the
inhibition). Other types of antagonism are non-competitive antagonism where the
antagonist binds to an allosteric site on the receptor or an associated ion channel. An
agonist on the other hand, is a drug which binds to a receptor and activates it, producing
a pharmacological response (e.g. contraction, relaxation, secretion, enzyme activation,
etc.).

Morphine, C17H19NO3, is the most abundant of opium’s 24 alkaloids, accounting

for 9 to 14% of opium-extract by mass. Named after the Roman god of dreams,
Morpheus, who also became the god of slumber, the drug morphine, appropriately
enough, numbs pain, alters mood and induces sleep. Less popular and less mentioned
effects include nausea, vomiting and decreased gastrointestinal motility. (It’s a great
constipator, and in Guerin’s painting, Isis is perhaps bringing Morpheus a laxative.)
Morphine and its related synthetic derivatives, known as opioids, are so far unbeatable
at dulling chronic or so-called “slow” pain, but unfortunately they are all physically
addictive. Both morphine and its hydrated form, C17H19NO3.H2O, are sparingly soluble in
water. In five litres of water, only one gram of the hydrate will dissolve. For this reason,
pharmaceutical companies produce sulphate and hydrochloride salts of the drug, both of
which are over 300 times more water-soluble than its parent molecule. Whereas the pH
of a saturated morphine hydrate solution is 8.5, the salts are acidic. Since they derive
from a strong acid but weak base, they are both at about pH= 5; consequently, the
morphine salts are mixed with small amounts of NaOH to make them suitable for
injection. Morphine acts on a specific receptor of nerve cells. More specifically many
such receptors are found in the spinal cord’s substantia gelatinosa, a region where pain
signals are first processed. The architecture of the morphine receptor is what dictates
the morphine rule. There is a flat part that binds to the aromatic ring, a cavity that
attracts the two carbon atoms and an anionic site that accommodates the tertiary
nitrogen atom. When morphine or another agonist binds to the receptor, the cell
membrane’s affinity for sodium ion changes. This
eventually reduces the release of neurotransmitters
from the affected neurons.

22
 Morphine

Morphine consists of five rings, three of which are approximately in the same
plane. The other two rings, including the nitrogen one, are each at right angles to the
other trio.
Investigators learned about morphine’s mode of action by applying it and other
opiates (including enkephalin) to guinea-pig intestines. In the presence of antagonists,
Na+ affinity was restored and intestinal contractions which had dropped precipitously
shot up again.

Nalbuphine

Nalbuphine

Nalbuphine is an opioid analgesic and a narcotic agonist-antagonist, meaning

that it is a synthetic narcotic analgesic with agonist and weak antagonist properties.
Analgesic potency is approximately equal to that of morphine at equivalent doses. On a
weight basis, produces roughly the same level of respiratory depression as morphine
but, unlike morphine, doses > 30 mg produce no further depression. On the other hand,
antagonistic potency is approximately 1/4 that of naloxone. Its onset is within 2 to 3
minutes. And duration lasts for 3 to 6 hours.

The drug is indicated for chest pain associated with myocardial infarction,
moderate to severe acute pain, may be necessary in some chronic pain syndromes and
pulmonary edema, with or without associated pain (morphine is first-line medication in
this class). It should not be taken in patients with hypovolemia, hypotension,
hypersensitivity to narcotics and head injury or undiagnosed abdominal pain. Side effect
such as hypotension, bradycardia, facial flushing, respiratory depression, CNS
depression, euphoria, paradoxical CNS stimulation and blurred vision may be present. It
could be supplied by10 mg in 1 ml ampule or 20 mg in 1 ml ampule. The dosage for
administration in adults is IV: 2-5 mg slow push; may be augmented with 2 mg doses prn
and it is not recommended in pediatrics.

23
ANIMALS: Thirty two (32) mice of the same sex and about the same weight (16 for
morphine alone, 16 for morphine + nalbuphine

MATERIALS: animal weighing scale

tuberculin syringe
animal cages
Asbestos gloves
Stopwatches

DRUGS
Morphine (10mg/mL or 1%)
Nalbuphine (0.5mg/mL or 0.05%)

METHODOLOGY

Thirty two male white mice of similar weight were used in the experiment. They
were divided into 2 groups. Dosage of the different drugs was computed based on the
weight of the different subject specimen. A dosage of 0.5 mL of drug per 20 grams of
mouse in morphine and 0.1 mL of drug per 20 grams of mouse in nalbuphine.
Group A was given with morphine. Upon administration, tail erection was waited
to be exhibited by the mice and one of the group members was assigned to record the
time of tail erection and the duration of it.
Groub B was also given morphine. But when tail erection was exhibited,
nalbuphine was given. Duration of tail erection was also observed and recorded.
Independent t-test was used for the statistical analysis of the different data
gathered and also for the significance of the data tested, specifically for the duration of
tail erection in mice.

RESULTS AND DISCUSSION

MORPHINE ALONE Morphine + Nalbuphine

A1 1849 1690
A2 1598 382
A3 2010 316
A4 1717 289
B1 3475 3371
B2 1873 1630
B3 2268 1931
B4 2074 1683
C1 564 343
C2 1499 786

24
 C3 1653 1398
C4 1478 819
D1 845 900
D2 981 1176
D3 1535 1440
D4 450 353

Statistical analysis
Null Hypothesis:
There is no significant difference between the observed means of the
morphine group and the morphine with nalbuphine group.

Alternative Hypothesis:
There is a significant difference between the observed means of the
morphine group and the morphine with nalbuphine group.

Reject null hypothesis if t value is greater than t critical value.

• t value = 1.68; p value = 0.103

• t critical value = 2.04
• Since t value < t critical value, then;
o Accept null hypothesis

Result The time of tail erection was decreased when nalbuphine was added.

Interpretation: There is no significant difference between the observed mean of the

morphine group and the morphine with nalbuphine group

Possible sources of error

For some of the mice in group B, in which the mice injected with morphine-
nalbuphine did not exhibit a decrease in time but rather longer tail erection when
compared to those with injected with morphine alone may have had improper
administration of drugs or inaccurate dosage.

DISCUSSION

Straub tail reaction

It is the S-shaped dorsiflexion of the mouse tail based on the contraction of the
sacro-coccygeal dorsalis muscles induced by a long-lasting stimulation of the muscle's
motor innervation at the level of the lumbosacral spinal cord, brought about by
administration of morphine.

Principles involved

25
 The morphine – induced tail reaction (straub tail reaction) was due to the
contraction of the sacrococcygeal dorsalis muscle in rats. The receptor for the tail
erection of white mice is the mu receptor. As morphine acts on the presynaptic mu
receptors, there is decrease calcium influx, decreasing the transmitter release leading to
increase potassium conductance. Nalbuphine, a synthetic narcotic analgesic with
agonist and weak antagonist properties, should also decrease the effect of morphine.
This was due to the fact that nalbuphine acts as an agonist primarily on the kappa
receptors (opiod/ analgesis effects) and a partial antagonist on mu receptors (anti-
analgesic effecs). But data showed that upon administration only little decline in duration
of tail erection was observed.

CONCLUSION

Based on the data, there is no statistically significant difference on the duration of

tail erection in mice administered with morphine alone, and morphine and nalbuphine.
According to t-test values between Group A and B, there is no significant
difference between the duration of tail erection in mice administered with morphine alone
and morphine with nalbuphine. This may be due to the limitation of the experiment such
as erroneous computation of dosage and improper administration of drugs.

Experiment 3
DETERMINATION OF LD50

OBJECTIVES
General
To determine the Median Lethal Dose (LD50) of intraperitoneally administered
Lidocaine HCl on mice

Specific

1. To determine the number of deaths at each preset dose of Lidocaine HCl one
hour after administration.

26
 2. To calculate the percentage of deaths for each dose from the number of deaths
observed.

3. To obtain the LD50 of Lidocaine HCl by plotting the doses and their respective
probit number (percentage of deaths) on log-probit paper (Miller-Tainter graphical
method).

4. To obtain LD50 of Lidocaine HCl by plotting the log dose and probit number
(percentage deaths) using Linear Regression

5. To compare the experimentally determined LD50 with the clinical standard value.

INTRODUCTION
Lidocaine
Pharmacodynamics

Anti-arrhythmic Effect (Heart)

Other than being an anesthetic, lidocaine also acts as an antiarrhythmic agent.

Arrythmia is a result of abnormal pacemaker activity or impulse formation in the
conduction system of the heart. It can also result from a disturbance in impulse
propagation or conduction. Such is the case in reentry circuits wherein a part of the
conduction system of the heart is blocked. Though the tissue is depolarized, the
blockage prevents the passage of a forward impulse. The reexcitement of the tissue
happens when an impulse coming from another direction reenters the area of blockade.
When this happens, arrhythmia results.
Antiarrhythmic drugs aim to reduce ectopic pacemaker activity and modify
conduction or refractoriness in reentry circuits. The mechanisms by which these drugs
achieve this are via sodium channel blockade, sympathetic blockade, prolongation of the
refractory perios, and calcium channel blockade. Lidocaine is a sodium channel blocker
(Class 1B). It blocks both activated and inactivated sodium channels. When sodium
channels are blocked, the threshold for excitability is decreased or there is greater
membrane depolarization required to open up sodium channels. Its therapeutic use is
mainly for terminating ventricular tachycardia and preventing ventricular fibrillation.
This drug has greater effects in ventricular and purkinje cells than in atrial cells because
the former have longer action potentials.

Anesthetic Effect of Lidocaine

Lidocaine alters depolarization in neurons, by blocking the fast voltage gated

sodium (Na+) channels in the cell membrane. With sufficient blockade, the membrane of
the presynaptic neuron will not depolarize and so fail to transmit an action potential,
leading to its anesthetic effects.

Local anesthetic drugs act mainly by inhibiting sodium influx through sodium-
specific ion channels in the neuronal cell membrane, in particular the so-called voltage-

27
gated sodium channels. When the influx of sodium is interrupted, an action potential
cannot arise and signal conduction is inhibited. The receptor site is thought to be located
at the cytoplasmic (inner) portion of the sodium channel. Local anesthetic drugs bind
more readily to "open" sodium channels, thus onset of neuronal blockade is faster in
neurons that are rapidly firing. This is referred to as state dependent blockade.

CNS effect of Lidocaine

Lidocaine has an excitatory or depressant effect depending on tissue

concentration. If there is low concentration, there will be generalized convulsions.
However, high concentration of this drug can lead to coma, respiratory arrest and death.

Pharmacokinetics

Lidocaine is given parenterally to by-pass the first-pass effect in the liver, where it
is extensively metabolized when given orally. Hepatic metabolism is rapid and 90% of
the given dose is dealkylated. It has 3% bioavailability. The half-life of the drug is 1-2
hours. A loading dose of 150-200mg is delivered in a single administration within 15
minutes. This is followed by maintenance with 2-4mg / minute. This loading dose
mechanism allows the drug to reach a concentration of 2-6mcg/mL.
There are variations in treatment with Lidocaine for patients of special cases.
Myocardial Infarction patients require higher concentrations of the drug. This is because
plasma α1-acid glycoprotein binds Lidocaine, allowing less free drug available. In
patients with heart failure, there is decreased volume distribution and total body
clearance of the drug. Loading and maintenance dose should then be decreased to
avoid toxicity. In liver disease, there should be a decrease in the maintenance dose and
a usual loading dose. This disease causes a longer time to achieve steady state. Drugs
such as Propanolol and Cimetidine causes decrease in liver blood flow, reducing
Lidocaine clearance with an increased risk of toxicity.

Toxicity

The toxicity of Lidocaine depends mostly on its metabolites. Dealkylation in the

liver produces monoethylglycine xylidide and glycine xylidide. These metabolites have
longer half-lives and the accumulation of which is the chief cause of toxicity.
Lidocaine is the least cardiotoxic of the currently used sodium-channel blockers.
But with large doses and heart failure, Lidocaine causes hypotension, bradycardia,
arrhythmia and cardiac arrest, by decreasing myocardial contractility.
Toxicity in the CNS has 2 classifications: excitation and depression. The
excitation effect includes tremors, nervousness, seizures, paresthesia, tinnitus and
convulsions. A depressed state, on the other hand, includes nausea of central origin and

28
lightheadedness. It has a dose-related effect and levels exceeding 9mcg/mL are
avoided.

ANIMALS: 80 mice of the same sex and weight (20 per section)
MATERIALS: Animal weighing scale
Tuberculin syringe
Animal cage
Asbestos gloves

DRUG: Lidocaine HCl (0.1,1%, 2%) preparation)

METHODOLOGY

Eighty mice of the same sex and approximately the same weight provided by the
Department of Pharmacoloy were divided into seven groups corresponding to the
following Lidocaine HCl doses: 1 mg/ 100 g; 2 mg/ 100 g; 4 mg/ 100 g; 8 mg/ 100 g; 16
mg/ 100 g; 32 mg/ 100 g; 64 mg/ 100 g. Freshly prepared 0.1%, 1%, and 2% Lidocaine
HCl solutions were also provided.
Each mouse was weighed and the volume to be injected into it was calculated
from its weight, the assigned dose, and the appropriate concentration of Lidocaine HCl
to be used. The calculated volume must be large enough to be measured accurately but
should still be small enough so as not to adversely affect the hemodynamics of the
mouse.
Each mouse was injected intraperitoneally with its appropriate volume of
Lidocaine HCl in order to achieve its assigned dose. An hour after injection, the number
of deaths resulting from the administration of the drug were determined and converted
into percentages. The percentage of deaths per group were plotted against the dose on
log-probit paper.

RESULTS AND DISCUSSION:

Table 1: Number of Deaths Recorded After An Hour of Lidocaine HCl Administration

(Intraperitoneal)

Section Lidocaine dose Log No. of No. of Percentage of Probit

(g/100 mL) Dose Mice Deaths death (%) Value
D6 1 0.0 10 0 2.5 3.04
D6 2 0.30 10 0 2.5 3.04
Faculty 4 0.60 10 1 10 3.72
C6 8 0.90 10 3 30 4.48
C6 16 1.20 10 8 80 5.84
B6/A6 32 1.50 10 10 97.5 6.97
A6 64 1.80 10 10 97.5 6.97
Faculty 128 2.10 10 10 97.5 6.97

29
A. Dose vs. Percentage Death After An Hour of Lidocaine Administration

Mortality of Mice After An Hour of Lidocaine

Administration

100
80
B. Determination of Percentage Death
% Death

60
After
40 an hour of Lidocaine administration, the numbers of deaths were noted and
converted into percentage death.
20
a. For
0 each dose: % death = no. of deaths x 100
total no. of mice
1 2 4 8 16 32 64 128
Dose: 4g/100mL Dose: 8g/100mL Dose: 16g/100mL
Dose
% death = _1_ x 100 % death = _3_ x (mg/100g)
100 % death = _8_ x 100
10 10 10
% death = 10% % death =%30%
death % death = 80%

b. 2.5 was added to the percentage of death reported as 0

Dose: 1g/100mL, 2g/100mL
0% + 2.5 = 2.5 %

c. 2.5 was subtracted to the percentage of death reported as 100

Dose: 32g/100mL, 64g/100mL, 128g/100mL
100% – 2.5 = 97.5%

C. Conversion of Percentage Death to Probit Number

Each percentage death has a corresponding probit number. These values can be
obtained using the table provided by the Probit. Probit numbers are more accurate to
use than the percentage deaths. These numbers are standards use in quantal dose and
in getting the LD50.

Section Lidocaine Dose No of Sample No of % death Probit

(mg/100g) Mice Deaths
D6 1 10 0 2.5 3.04
D6 2 10 0 2.5 3.04
Faculty 4 10 1 10 3.72
C6 8 10 3 30 4.48
C6 16 10 8 80 5.84
B6/A6 32 10 10 97.5 6.97
D6 64 10 10 97.5 6.97
Faculty 128 10 10 97.5 6.97

Probit Table

30
 In
getting
the
probit

numbers, the percentage deaths were used. The first column corresponds to every tenth
digits of the percentage while the first row corresponds to every one digits of the
percentage.
a. 10% = probit value of 3.72

b. 30% = probit value of 4.48

c. 80% = probit value of 5.84

d. For 2.5 %: If the percentage in which the probit number cannot be found in the
table, we can use the interpolation method. For instance,

2 = 2.95 2 – 2.5 = _2.95 – x_

2.5 = x 2 – 3 2.95 – 3.12
3 = 3.12 x = 3.04

In 1 and 2 mg/100 g dose, we obtain a percentage of 2.5. Since it has no

exact probit number, we use the probit number of 2% and 3%. The interpolation
method was then done.

e. For 97.5 %
97 = 6.88 97 – 97.5 = _6.88 – x_
97.5 = x 97 – 98 6.88-7.05
98 = 7.05 x = 6.97

D. Dose vs. Probit Number by Miller-Tainter Method

31
 6.97 6.97 6.97

5.84

4.48

3.72

3.04 3.04

The Miller-Tainter method is the standard use in getting LD50. Plot the dose
against the probit value. By trial and error method, find the best-fit line. To know the best-
fit line, measure the vertical distances from the line you made to the plotted points below
and above the line. When the vertical distances of the points above the line are added,
this should be equal to the sum of the vertical distances of the points below the line.
Based on the graph, the LD50 was 10 mg/100g. Dose
E. Determination of LD50 by Linear Regression

1. Determination of Log Dose

Determination of log dose was important in analysis of the data by linear

regression. The log dose was plotted against the probit number.

Log Dose = log (dose)

a. 1: log (1) = 0
b. 2: log (2) = 0.30
c. 4: log (4) = 0.60
d. 8: log (8) = 0.90
e. 16: log (16) = 1.20
f. 32: log (32) = 1.50
g. 64: log (64) = 1.80
h. 128: log (128) = 2.10

2. Determination of LD50 by Extrapolation

In getting the LD50 using the Miller-Tainter Method, there may be possible
sources of error since the graph was manually made. To support the LD50 obtained, the
Linear Regression method was also done. The LD50 was obtained by extrapolation
method.

32
 Log Dose vs. Probit Value

8
7
6
Probit Value

5
4
3
2
1
0
0 0.5 1 1.5 2 2.5
Log Dose

Probit Value Linear (Probit Value)

Based on the graph, the LD50 obtained was antilog 1 or 10 mg/100g which is the
same as the one obtained by the Miller-Tainter method.

F. Theoretical LD50 vs Experimental LD50

Theoretical LD50 = 8 mg/100g

Experimental LD50 = 10mg/100g

The experimental LD50 obtained was higher compared to the theoretical LD50.
The theoretical LD50 is more potent since at 8 mg/100g it could already elicit a lethal
effect on 50% of the population while the experimental LD50 would elicit the same effect
at 10 mg/100g. Also, the experimental LD50 obtained is less toxic.

CONCLUSION:

Based on the probit graph the LD50 for Lidocaine is 10mg/100 g body weight. A
linear regression was also used in data analysis. Based on the LD50 result of
10mg/100g, it can be interpreted that the Lidocaine used is less toxic and less potent
than the theoretical value of 8mg/100g. Lidocaine given at a dose greater then
10mg/100g body weight will elicit a higher mortality rate.
Therefore, it can be said that Lidocaine 10mg/100g body weight is less toxic and
less potent then the given theoretical value for LD50.

33
34