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WRITTEN REPORT
First Semester 2007-2008
Submitted By:
Subsection C6
03 August 2007
1
Experiment 1
GRADED DOSE-RESPONSE CURVE
OBJECTIVES
INTRODUCTION
2
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
3
RESULTS
Expected results
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
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
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average 179.25 824.625 53.475 532.625
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.
8
t = 1.37 < 1.7613
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.
IV IM
Experiment 2b
FACTORS AFFECTING DRUG ACTION
10
Influence of Chemical Structure
OBEJECTIVES
General
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 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
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.
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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
D1 20 100 -40
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
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
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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.
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
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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.
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
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.
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.
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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.
Drugs Administered
1 6 39 127 900
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
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 &
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Group 4 (Warfarin &
Rifampicin) Ketoconazole)
Drugs Administered
Methodology:
CONCLUSION
Experiment 2d
FACTORS AFFECTING DRUG ACTION
Influence of Antagonism on Drug Action
OBJECTIVES
Specific:
2. To compare the duration of tail erection recorded in rats given morphine alone with
those given with both morphine and nalbuphine
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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.).
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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
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.
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ANIMALS: Thirty two (32) mice of the same sex and about the same weight (16 for
morphine alone, 16 for morphine + nalbuphine
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.
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.
Result The time of tail erection was decreased when nalbuphine was added.
DISCUSSION
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
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
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-
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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.
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
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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
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.
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A. Dose vs. Percentage Death 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%
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.
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
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,
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
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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
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.
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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
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.
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