Download Group 1 - Random Sampling

Pharmacology Laboratory Experiments
First Semester SY 2006 – 2007
Submitted by:
Subsection D3
Salazar, Nathania
Saludo, Antonio
Salvador, Louie
Salvanera, John Vincent
Sampayan, Sigrid
Sanchez, Jackyline
Sanchez, Rommel Ray
Sandoval, Denize Grace
Sanghilan, Jasmine
Santiago, Ma. Theresa
Santos, Caroline
Santos, Charlene
Santos, Gian Carlo
Santos, Jenn Rachelle
Santos, Joanne Grace
Sarip, Sittie
Sarthou, Ma. Katrina
Senoren, Lauren
Servas, Jerlyn
07 August 2006
Table of Contents
Experiment Title
Page
…………………………..
3
Factors Affecting Drug Action: Influence of Route of
Administration
…………………………..
12
Factors Affecting Drug Action: influence of Chemical
Structure
…………………………..
17
Factors Affecting
Potentiation
…………………………..
23
Factors Affecting Drug Action: Influence of Drug
Antagonism on Drug Action
…………………………..
33
…………………………..
39
Biostatistics
Drug
Determination of LD50
Action:
Influence
of
2
BIOSTATISTICS
GENERAL OBJECTIVE
To establish the average height and weight of 2 nd year UST medical students who
are 75-100% Filipino by blood
SPECIFIC OBJECTIVES
1. To determine if the sample population can represent the general population of
75-100% Filipino by blood
2. To determine gender as a variable for height and weight
3. To be able to plot the frequency distribution curve
4. To determine position of an individual in the given data
5. To determine if the individual is usual or not based on standard deviation
6. To be able to derive conclusions regarding homogeneity of a population or
variability of a sample
INTRODUCTION
Statistics
is
a
mathematical
science
which
involves
collection,
analysis,
interpretation, and presentation of data. On the groundwork of its analysis, conclusions
and recommendations for further studies are made.
Biostatistics, used in medicine is of great importance due to its numerous
advantages when it comes to medical applications. As such, biostatistics can be applied
in public health, which includes epidemiology, nutrition and environmental health, and
also in designing and analyzing critical trials. In genomics, population genetics and
statistical genetics, biostatistics is used in populations in order to link variations in
genotypes with variations in phenotype. This can be of assistance in finding candidates
for gene alleles that can cause or influence predisposition to disease in human.
Biostatistics is also used in ecology and biological sequence analysis.
3
The most basic procedure in analyzing data are through computations and
subsequently interpretations of the mean, standard deviation, standard error of the
mean, and coefficient of variation. Mean is defined as the average of a given set of
data. The standard deviation which is always non-negative shows how dispersed values
are on a given data set. Coefficient variation is defined as the ratio of the standard
deviation to the mean. It is dimensionless and allows comparison of the variation of
populations which have significantly different mean values. The standard error of mean
is the standard deviation of the sampling distribution of the mean, and is inversely
proportional to the square root of the sample size.
METHODOLOGY
Materials
Meter stick
Foot scale
Calculator
Procedure
All second year medical students who are 75% Filipino by blood were identified. Their
height and weight were obtained. After then, the following were computed:


o

o
Mean = sum of observations
number of observations
Standard Deviation
square root of the variance
= √s2
Standard Error of the Mean
measure of how far your sample mean is likely to be from the true
4
population mean
SEM = SD
√N

Coefficient of Variation
measure of relative dispersion which expresses the standard deviation as
o
a percentage of the mean
CV = s x 100
X
Interpretation of individual heights based on standard scores (SC) were as follows:

SC of 2.5 = usual or normal

2.5 – 3.5 = unusual

3.5 = outer, very unusual
The frequency distribution curve was then plotted for the height and for the weight for
each sex. Conclusions were then derived regarding the homogenicity of the population
or variability of the samples based on the statistical analysis.
RESULTS:
Group 1 - Random Sampling
MALE
MEAN
SD
SEM
CoV
Height
169.71
11.48
2.57
6.76
Weight
74.69
13.42
3.00
17.92
Height
156.93
6.37
1.42
4.06
FEMALE
Weight
56.24
14.34
3.21
25.51
Group 2 - Frequency Distribution
MALE
MEAN
MEDIAN
MODE
Height
170.25
169.35
166.00
Weight
74.80
74.17
63.50, 64.80
FEMALE
Height
Weight
157.68
54.45
157.00
52.30
158
51.50
5
(unimodal)
SD
6.69
66.00, 66.22
67.50, 81.00
81.20, 81.90
82.50, 84.50
86.64
(multimodal)
6.00
(unimodal)
54.50
(bimodal)
12.23
11.13
T- test Result
MALE
MEAN
VARIANCE
T VALUE
P VALUE
DF
Height
169.71
131.79
FEMALE
Weight
74.69
180.12
Height
156.93
40.60
4.35
<0.0001
38
Weight
56.24
205.76
4.20
<0.0002
38
Section D - Standard
Scores
CN
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
% Filipino Ht (cm)
50% 161.50
100% 157.00
50% 161.50
100% 163.50
100% 164.50
100% 160.00
100% 172.00
75% 156.50
0% 155.00
100% 152.50
75% 157.00
75% 166.00
100% 163.00
100% 161.00
75% 159.70
100% 153.00
100% 174.50
100% 158.00
100% 174.50
100% 161.00
100% 175.50
25% 172.00
100% 152.50
100% 152.00
100% 172.50
100% 171.50
100% 155.00
100% 178.00
75% 165.50
75% 157.00
100% 166.00
Standard
Score (Ht)
-1.31
-0.11
0.64
0.97
1.14
0.39
0.26
-0.20
-0.45
-0.86
-1.99
-0.64
0.89
0.55
0.34
-0.78
2.80
0.05
0.64
0.55
2.97
0.26
-0.86
-0.95
0.34
0.19
-0.45
1.16
1.30
-0.11
-0.64
Wt
(kg)
69.50
61.50
57.00
58.00
57.80
45.50
111.00
54.00
45.00
53.00
81.50
81.00
48.50
56.50
54.50
46.70
67.70
54.20
88.50
51.10
60.50
101.70
53.50
46.80
74.50
67.00
85.60
75.90
62.70
65.70
73.50
Standard
Score (Wt)
-0.43
0.45
0.05
0.14
0.12
-0.98
2.96
-0.22
-1.03
-0.31
0.55
0.51
-0.71
0.00
-0.18
-0.88
1.01
-0.20
1.12
-0.48
0.36
2.20
-0.26
-0.87
-0.02
-0.64
2.62
0.09
0.56
0.83
-0.11
Sex
M
F
F
F
F
F
M
F
F
F
M
M
F
F
F
F
F
F
M
F
F
M
F
F
M
M
F
M
F
F
M
6
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
100%
50%
100%
100%
100%
100%
100%
100%
100%
75%
100%
50%
100%
100%
100%
100%
100%
100%
75%
75%
100%
100%
100%
50%
100%
100%
100%
100%
100%
75%
50%
100%
25%
100%
100%
100%
0%
50%
100%
100%
100%
25%
75%
75%
75%
100%
50%
100%
50%
75%
100%
100%
75%
100%
100%
157.50
162.50
171.00
165.00
167.50
151.00
183.00
168.80
179.00
164.00
184.00
161.50
169.00
169.00
162.00
153.50
158.00
160.00
153.00
173.00
152.00
149.50
165.00
161.00
167.50
159.70
167.60
173.50
171.00
178.00
162.00
149.50
159.00
161.00
165.00
169.00
173.50
171.00
179.00
166.50
155.50
165.00
163.00
163.50
151.50
161.50
164.50
165.00
157.00
178.00
173.00
158.00
158.00
172.00
170.50
-0.03
-1.16
0.11
-0.79
-0.41
-1.11
1.91
1.85
1.31
1.05
2.06
0.64
1.89
-0.19
0.72
-0.70
0.05
0.39
-0.78
0.41
-0.95
-1.36
1.22
0.55
1.64
0.34
1.65
2.64
2.22
1.16
0.72
-1.36
0.22
0.55
1.22
-0.19
0.49
2.22
1.31
-0.56
-0.36
1.22
0.89
0.97
-1.03
0.64
1.14
1.22
-0.11
1.16
0.41
0.05
0.05
0.26
0.04
57.20
88.00
62.60
84.00
67.50
51.00
73.80
49.90
77.50
74.40
82.50
53.00
65.70
87.00
40.00
54.50
49.50
49.20
63.50
63.90
50.50
41.00
48.40
49.10
60.00
47.90
59.10
58.00
40.10
79.70
62.00
41.50
56.20
57.60
48.60
67.20
87.00
51.60
83.00
72.50
51.70
56.80
52.00
48.70
45.00
57.20
49.00
53.50
54.50
48.50
62.00
48.30
49.80
76.30
81.00
0.07
1.08
-1.00
0.75
-0.60
-0.49
-0.08
-0.59
0.22
1.61
0.63
-0.31
0.83
1.00
-1.48
-0.18
-0.62
-0.65
0.63
-0.89
-0.53
-1.39
-0.72
-0.66
0.32
-0.77
0.24
0.14
-1.47
0.40
0.50
-1.34
-0.02
0.10
-0.71
-0.62
1.00
-0.44
0.67
-0.19
-0.43
0.03
-0.40
-0.70
-1.03
0.07
-0.67
-0.26
-0.18
-2.15
-1.05
-0.73
-0.60
0.12
0.51
F
M
M
M
M
F
M
F
M
F
M
F
F
M
F
F
F
F
F
M
F
F
F
F
F
F
F
F
F
M
F
F
F
F
F
M
M
F
M
M
F
F
F
F
F
F
F
F
F
M
M
F
F
M
M
7
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
100%
100%
100%
100%
0%
50%
25%
25%
100%
100%
100%
100%
100%
100%
75%
100%
100%
100%
100%
75%
75%
75%
75%
50%
100%
0%
0%
100%
75%
171.70
167.00
158.70
147.40
160.40
161.00
166.20
156.20
176.50
169.80
150.80
165.00
142.50
155.78
165.00
150.00
174.50
163.50
163.00
168.40
151.00
159.70
161.90
159.80
153.80
170.00
172.20
145.00
156.00
0.22
-0.49
0.17
-1.71
0.45
0.55
1.42
-0.25
0.94
-0.07
-1.15
1.22
-2.53
-0.32
1.22
-1.28
0.64
-1.01
0.89
-0.28
-1.11
0.34
0.70
0.35
-0.65
-0.04
0.29
-2.11
-0.28
75.70
94.00
44.30
40.00
67.00
46.50
50.40
55.70
86.10
67.50
45.00
55.20
39.70
74.80
46.90
47.10
90.80
65.00
50.90
69.00
54.70
47.90
52.50
86.00
50.40
73.00
63.10
43.90
46.30
0.07
1.57
-1.09
-1.48
0.95
-0.89
-0.54
-0.07
0.92
-0.60
-1.03
-0.11
-1.50
1.65
-0.86
-0.84
1.31
-0.80
-0.50
-0.47
-0.16
-0.77
-0.35
2.65
-0.54
-0.15
-0.96
-1.13
-0.91
M
M
F
F
F
F
F
F
M
M
F
F
F
F
F
F
M
M
F
M
F
F
F
F
F
M
M
F
F
DISCUSSION
In this experiment, 20 male and female 2nd year medical students were randomly
selected to test if their heights and weights represent the population of the 374 2 nd year
medical students.
Mean, median and mode are measures of central tendency. Mean is equal to the
sum of individual observations, divided by the total number of observations. Median is
the middlemost observation in a set of observations put in numerical order. Mode on the
other hand is the most frequently occurring value in a set of observations. It can be unimodal, bi-modal or multi-modal.
Variance is measure of variability with the mean as the reference point. It is computed
by dividing the number of observations from the sum of squared deviation from the
8
mean. Standard Deviation on the other hand, is the square root of the variance.
Coefficient of Variation measures of relative dispersion which expresses the standard
deviation as a percentage of the mean. Standard error of the mean is the measure of
how far your sample mean is likely to be from the true population mean.
The average height of males from the sample population is 169.71 cm, and 170.25 cm
from the total population. From these values, we can see that the average height of
randomly selected males is quite near to the total male population. The same is true
with the average height of females, 156.93 cm and 157.68 cm, from the random
samples and from the population respectively.
The middlemost value for male height from the random sample is 171.00 cm and for
the females, 156.75 cm. It implies that half of the population male and females from the
random sample is less than or equal to 171.00 cm and 156.75 cm. While the other half
have heights greater than or equal to 171.00 cm and 156.75 cm. From the total
population, the middlemost values for males and females are: 169. 35 cm and 157.00
cm. Comparing it to the sample population, again we can see that the values are quite
near or close to each other.
The frequently appearing height for males is 166 cm. For the females, it is 158 cm.
Mode for the sample population was not obtained.
For the weight, average weight of males from the sample population is 74.69 kg,
and 74.80 from the total population. From these values, we can see that the average
weight of randomly selected males is quite near to the total male population. The same
is true with the average weight of females, 56. 24 kg and 54.45 kg, from the random
samples and from the population respectively.
The middlemost value for male weight from the random sample is 70.45 kg. and for
the females,53. 95 kg. It implies that half of the population male and females from the
random sample weigh less than or equal to 70.45 kg and 53.95 kg . While the other half
have weights greater than or equal to 70.45 kg and 53.95 kg. From the total population,
9
the middlemost values for males and females are: 74.17 kg and 52.30 kg. Comparing it
to the sample population, again we can see that the values are quite near or close to
each other.
The frequently appearing weight for males are 63.50, 64.80, 66.00, 66.22, 67.50,
81.00,, 81.20, 81.90, 82.50, 84.50 and 86.64 kg. Because there are a lot of values, it is
considered as multi-modal . For the females, 51.50 and 54.50 kg appeared frequently.
Hence, it is bimodal.
For standard deviation, the larger the value obtained, the more dispersed the
samples are. In the data, female weight with a value of 14.34 is the most dispersed and,
female height, having a value of 6.37 is the least dispersed.
For standard error of the mean, the larger the value, the farther it is from the true
mean, and vice versa. Again, the female weight again has the highest value, with 3.21
and 1.42 for female weight has the least value.
For the coefficient of variation, the larger the value, the more variable the values are
and vice versa. Again, female weights are the most variable, with a value of 25.51 and
female height is the least variable, with a value of 4.06.
Aside from the measures of central tendency and variability, unpaired t-test was
also done. Through the T-test we can compare the means of two groups of data in
which the data from the groups are not matched. To check whether the ratio is large
enough to say that the difference between the groups is not likely to have been a
chance finding.
The computed T value for height is 4.35 with a p-value of <0.0001. for the weight, it
is 4.208 and the p-value is 0.0002. The degrees of freedom is 38 for male & female
height and also for male and female weight.
10
CONCLUSION
In conclusion, we can say that the height and weight of males are definitely larger
than of the females both in the random sample group and the total population. The
weights of males and females are variable as compared to the heights. Factors such as
nutrition and genetics affect a person’s weight and height.
11
FACTORS AFFECTING DRUG ACTION
Influence of Route of Drug Administration
OBJECTIVES
1. To determine the loss of righting reflex after injection of intravenous and
intramuscular ketamine on rabbits.
2. To statistically compare the latency and duration of the loss of righting reflex
upon administration of intravenous and intramuscular ketamine in rabbits.
3. To review and observe the pharmacokinetic and pharmocodynamic principles of
Ketamine in rabbits.
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
12
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.
METHODOLOGY
Materials
A. Test Animals
8 rabbits (2 per section)
B. Instruments
rabbit cage
animal weighing scale
tuberculin syringe
stopwatch
C. Drugs
Ketamine Hydrochloride
-
Preparation: 50mg/mL
-
Dosage: 5mg/kg
Procedure
The group was supplied with 2 rabbits which were weighed with the animal
weighing scale. The weights of the rabbits were then recorded and used to compute for
the amount of drug to be administered. One of the rabbits was injected with the
Ketamine Hydrochloride intravenously while the other rabbit was injected with the drug
intramuscularly. For the rabbit injected intravenously, the ear was selected as the site of
injection due to its accessibility, specifically because the veins in rabbits’ ears are easily
seen. This is also the reason why rabbits were selected as test animals for this
experiment. For the rabbit injected intramuscularly, the lateral part of the thigh was
selected as the site of injection also due to its accessibility. The time of injection was
then recorded together with the time the righting reflex was observed to be lost and the
13
time the righting reflex was regained for both rabbits. The data was then gathered and
subjected to statistical analysis.
Calculation for Amount of Ketamine Hydrochloride to be Administered
Preparation: 50mg/mL
Dosage: 5mg/kg
Amount of drug = (weight of rabbit in kg) x (5mg/1kg) x (1mL/50mg)
Results and Interpretation of Statistical Analyses
Table 1 shows the result of intramuscular (IM) and intravenous (IV) route of
administration of Ketamine. Theoretically, there should be shorter time for Ketamine to
show onset of action (latency) upon IV route and longer time before its reversal
(duration) for the IM route. Ketamine has low bioavailability because of the extensive
first pass effect. IV or IM therefore is the preferred route of administration for ketamine.
Section
Intramuscular Ketamine
Intravenous Ketamine
Latency (s)
Duration
Latency
Duration
(s)
(s)
(s)
A5
360
597
45
270
B5
117
4020
1800
660
C5
191
598
172
731
D5
176
870
30
485
Table 1. Results of different mode of administration of ketamine
Latency was measured from the time of injection to the time the righting reflex of
the rabbit was lost. Administration of the drug, Ketamine, upon IV route implies that the
drug readily enters the blood stream so it will immediately reach to the desired site of
action and rapidly show its effect. IM route, however, will still have to perfuse to the
muscle tissue before reaching the site of action, therefore, taking longer time to show
onset of action.
Duration of action was measured from the time the righting reflex was lost to the
time the righting reflex was regained. Theoretically, there should be longer duration for
IM route because of its slower clearance. The IV route, on the other hand, is readily
absorbed so it has faster metabolism and thus more rapid clearance.
Statistical analysis (one tailed t-test) of latency and duration is shown in Table 2
and 3. Upon statistical analysis, there is no significant difference for both latency and
14
duration between IV and IM route. The t value for latency is 0.693 which is less than
2.45 (at p=0.05). The t value for duration is 1.19 which is less than 2.45 (at p=0.05).
Both the t values are less than the p values at 0.05.
IM
IV
21
511.7
1
5
Sample size
4
4
Degrees of freedom
6
6
Standard deviation
10
861
Mean
4
Standard deviation for 613
both
T value (p=0.05)
0.693
Table 2. Latency statistical analysis
IM
Mean
1.521
IV
x 52
103
2
Sample size
4
4
Degrees of freedom
6
6
Standard deviation
1.671
x 19
103
5
Standard deviation for 0.199 x 104
both
T value (p=0.05)
1.19
Table 3. Duration statistical analysis
The deviation from the theoretical result may be due to several factors. One may
be due to the inaccurate injection of site of administration, meaning that it may not really
be intravenously or intramuscularly. Also, there may be errors on computation of the
dosage to be given for the rabbits. Another is that the determination of the loss of
righting reflex was not properly performed and so making the end point not constant.
Finally, there are different observers and experimenters for this experiment adding to
the variability in the experiment.
15
RECOMMENDATIONS
After performing the experiment, it is recommended that:
1. The righting reflex be clearly defined and the manner of ascertaining that the
righting reflex has been lost and regained be established for those performing the
experiment.
2. Only one person would perform the observation of the loss and recovery of the
righting reflex for all rabbits.
3. The calculations for the amount of drug to be administered be performed
masterfully to reduce error to nil.
4. The experimenter responsible for the administration of the drug be skillful and
knowledgeable on the proper way and location of injecting the drug.
16
FACTORS AFFECTING DRUG ACTION
Influence of Chemical Structure
OBJECTIVES:
General Objective: To determine the influence of chemical structure of diazepam and
midazolam on drug action
1. To observe the latency and duration of loss of the righting reflex upon
administration of Diazepam
2. To observe the latency and duration of loss of the righting reflex upon
administration of Midazolam
3. To compare the latency and duration of loss of righting reflex between
Midazolam and Diazepam using statistical analyses
4. To review and observe the pharmacodynamic and pharmocokinetic principles of
Midazolam and Diazepam in white mice
INTRODUCTION:
Each drug has a particular chemical structure unique to it. This chemical
structure affects its action whether it is the drug’s duration of action, site of action, or its
mechanism. A single change in a drug’s chemical structure can already alter its action.
It can prolong or shorten its duration of action.
Diazepam is a 1,4-benzodiazepine derivative, which possesses anxiolytic,
anticonvulsant, sedative and skeletal muscle relaxant properties. It affects mainly the
central nervous system. It binds to specific benzodiazepine-receptors, which is part of
the GABA-receptors. When diazepam binds to this receptor, it causes an increase in the
inhibitory effects on GABA. The effect of diazepam does not last long, partly because it
is fairly quickly redistributed into tissues and fat depots, and partly because of the
adaption of the receptors.
Diazepam is used to treat anxiety and tension and muscle spasms. It is also
used as a sedative premedication and to treat alcohol withdrawal symptoms.
Midazolam is a benzodiazepine with an imidazole structure. It is used commonly
as an anxiolytic and sedative. It is water soluble in its acid formulation but highly lipid
soluble in vivo. It provides an effect similar to diazepam, but with a quicker onset and
shorter duration. This medication is frequently used by anesthesiologists for sedating
patients prior to surgery.
Righting reflex is a reflex, which will tend to bring the body into normal position in
space and resist forces acting to displace it out of normal position. It ensures that the
head and body are correctly oriented to each other and the environment. Loss of this
17
reflex is the time at which the subject is unable to stand on its feet and maintain
balance.
Latency is defined as the time in seconds from administration of drug to the time
the righting reflex is observed. Duration is the time in seconds from loss of righting
reflex to the time it is regained.
MATERIALS:
Animal:
4 Mice (same sex & weight)
2 assigned to Diazepam
2 assigned to Midazolam
A total of 16 mice were used for the entire experiment, with four coming from
each section
Instruments:
- Animal weighing scale
- Tuberculin syringe
- Animal cage
- Asbestos gloves
Drugs :
Diazepam
Preparation: 0.5 mg/ml
Dosage: 0.5 mg/20g mouse
Midazolam
Preparation: 0.5 mg/ml
Dosage: 0.5 mg/20g mouse
METHODOLOGY:
a. Mice of the same sex and weight were used in the experiment.
b. For diazepam administration, .75mg/1.5 ml is injected intraperitonially to the two
mice
c. For midazolam administration, same dosage was injected via same route.
d. The time of injection was noted. The mice were observed and the time the righting
reflex was lost and the time it was regained were also noted.
Calculation for Midazolam and Diazepam dosage:
Preparation: 0.5 mg/ml
Dosage: 0.5 mg/20g mouse
18
Dosage needed (mg):
0.5 mg = x in mg
20g weight of mouse
x = weight of mouse (g) x 0.5 mg
20g
x = 30g x 0.5 mg = 0.75 mg
20g
Volume to be injected (ml):
0.5 mg x X mg
1 ml Y ml
Y= 0.75 mg x 1 ml = 1.5 ml
0.5 mg
EXPECTED RESULT FOR DIAZEPAM:
Diazepam binds to specific subunit on GABA receptor at site that is distinct from
endogenous GABA molecule. It is a (+) allosteric modulator of GABA which causes
inhibitory effects due to hyperpolarization of postsynaptic membrane
Diazepam is expected to have a longer latency because it is less lipid soluble. It
has a pKa of 3 that contributes to the slower onset of action. Diazepam is also
exp[ected to have a longer duration of action because it is 99% bound to proteins and
the presence of its three active meatabolites which are, desmethyldiazepam,
temazepam and oxazepam.
EXPECTED RESULT FOR MIDAZOLAM:
Midazolam has shorter latency since it is water soluble due to the
benzodiazepine ring that is open at pH under 4. When midazolam is injected and the pH
is at 7.4, the benzodiazepine ring closes and it becomes much more lipid soluble,
facilitating its rapid uptake into nerve tissue and readily crosses the blood brain barrier.
Midazolam is expected to have a faster onset of action having a pKa of 6.15 therefore
predominantly unionized (>90%) at physiological pH and accounts for its rapid onset of action.
Compare to Diazepam, Midazolam has only one metabolite that is active á-hydroxymidazolam.
Midazolam also has a shorter half-life that is 1.94 hours and is less protein-bound which is
responsible for its shorter duration of action.
19
ACTUAL RESULTS:
DIAZEPAM
MIDAZOLAM
Section
Latency
Duration
Latency
Duration
A4
55 s
5.5 min
8.87 min
2.42 min
6.42 min
2.38 min
No loss in righting
reflex
2 min
15 min
No loss in righting
reflex
No loss in righting
reflex
3 min
17 min
No loss in righting
reflex
No loss in righting
reflex
18 s
29 min
9.53 min
14.38 min
2.55 min
20 min
4.1 min
14.35 min
13.58 min
1.7 min
25 min
40.58 min
B4
C4
D4
No loss in righting
reflex
No loss in righting
1.42 hr
23.58 min
1.4 hr
reflex
Values are measured in (s)
seconds; seconds of large values are converted to
(min) minutes/(hr) hours
DATA ANALYSIS: LATENCY
Drug
Mean
Standard
deviation
No. of
sample
D
223
sec
296
6
M
775
sec
542
6
LATENCY
Degree of
freedom
(n-1)
T
score
11
1.6135
p = 0.05
2.20 > 1.6135
Therefore, there is no significant difference in the latency of Diazepam and Midazolam
DATA ANALYSIS: DURATION
Drug
Mean
(sec)
Standard
deviation
No. of
sample
D
882
595
6
M
1581
1890
6
Degree of
freedom
(n1+n2) - 2
T score
10
0.689
p = 0.05
2.23 > 0.689
20
Therefore, there is no significant difference in the duration of action of Diazepam and
Midazolam
DISCUSSION:
Diazepam, also known as methyl diazepinone or diacepin, is a benzodiazepine
central nervous system depressant and anxiolytic. The drug has muscle relaxant
properties and is indicated in organophosphate poisoning where convulsions or
pronounced muscle fasciculation are present to reduce the duration of convulsions that
might be expected to cause structural damage to the central nervous system. It has also
been used in combination with atropine in treating carbamate poisoning and in other
anxiety cases for sedation.
The main site of action of diazepam is at the gamma-aminobutyric acid (GABA)
receptor, which is a ligand-gated chloride ion channel and the major inhibitory
neurotransmitter in the central nervous system (CNS). Benzodiazepines including
diazepam alter GABA binding at the GABAA receptor in an allosteric fashion causing
inhibitory effects due to hyperpolarization of postsynaptic membrane.
The volume of distribution of diazepam has been estimated to be 0.95-2 L/kg.
The peak plasma level is within 30 minutes- 2 hours. Desmethyldiazepam is the major
metabolite of diazepam upon metabolism in the liver, which involves demethylation, and
3-hydroxylation followed by glucuronidation and temazepam and oxazepam serve as
the minor metabolites. Diazepam has a plasma clearance of 26-35 mL/min after a single
intravenous dose and the metabolites conjugated with glucuronide are excreted in urine.
The standard intravenous dose of diazepam used for convulsions is 10-20 mg i.v.
in adults and 300-400 µ g/kg i.v. in children, depending on the severity of symptoms and
repeated as necessary.The most frequent adverse effects of diazepam include
drowsiness, muscle weakness and ataxia. Dysarthria, dizziness, confusion, visual
disturbances, urinary retention or incontinence and amnesia may also accompany it.
Midazolam is a benzodiazepine sedative used as adjunct to general anesthesia.
It is a short-acting benzodiazepine with sedative-general anesthetic properties. It has
peak plasma levels of 45 minutes and maximum effect is achieved between 30-60 min.
Time of recovery is usually within 2 hr up to 6 hrs. The plasma-protein binding of
midazolam is 97%. It is rapidly metabolized in the liver to inactive compounds and is
excreted through the urine.
Midazolam is generally indicated for preoperative sedation, anxiolysis, and
amnesia. It is administered in children to help alleviate anxiety before a diagnostic or
therapeutic procedure or before anesthesia induction. Midazolam is contraindicated in
cases of hypersensitivity to benzodiapenes and in acute narrow-angle glaucoma.
21
For adults, the dosage of Midazolam that should be administered is 0.07-0.08
mg/kg IM 1 hr before surgery and 0.1-0.15 mg/kg in children. The adverse effects of
Midazolam include cardiovascular and respiratory depression including hypotension,
cardiac arrest, decreased respiratory rate and tidal volume, apnea, variations in blood
pressure and pulse rate. It may also cause over-sedation, headache, drowsiness,
grogginess, confusion, retrograde amnesia, euphoria, nervousness, agitation, anxiety,
and restlessness in the CNS.
Midazolam, as compared to diazepam, has shorter latency because of the
benzodiazepine ring, which closes at pH 7.4 making it more lipid soluble, favoring its
rapid uptake into the nerve tissue and readily crosses the blood brain barrier. It also has
faster onset of action because it has a pKa of 6.15, therefore predominantly unionized
(>90%) at physiological pH. It has anionic fluorine, making benzodiazepine more
reactive to cationic site in the GABA receptor so it binds faster because of enhanced
affinity. Its duration of action is shorter because only one metabolite is active, named áhydroxymidazolam, it is rapidly metabolized via cytochrome P-450 enzymes and also it
has less protein binding. It has short half-life of 1.94 hours.
Diazepam, on the other hand, has longer latency because it is less lipid soluble,
has slower onset of action because its pKa is 3 and has longer duration of action
because it has more active metabolites than midazolam: desmethyldiazepam,
temazepam and oxazepam and has 99% protein binding. Its half-life is 43 hours.
Statistical analysis of the result revealed that there was no significant difference
in the latency of Diazepam and Midazolam. There was also no significant difference in
the duration of action of the two drugs. The inaccuracy of the results may be due to the
following:
1. Errors may have occurred in the calculation of the dosage.
2. The observers of each section may have varied definition of the righting reflex.
3. The weights of the mice may be out of range.
4. Errors in the administration of the drug.
REFERENCES:
T.C.
Marrs,
MD.
IPCS
Inchem.org.
http://www.inchem.org/documents/antidote/antidote/diazepam.htm#1.0.
Accessed: July 28,2006
Unnamed
author.
Health
Digest.org.
http://www.healthdigest.org/drug-
prescribing-database/midazolam-hydrochloride-1908.htm.
Accessed:
July
28,2006
22
FACTORS AFFECTING DRUG ACTION
Influence of Potentiation on Drug Action
Section D Subgroup 3
OBJECTIVES:
1. To observe the latency and duration of loss of the righting reflex upon
administration of Midazolam.
2. To observe the latency and duration of loss of the righting reflex upon
administration of Midazolam and Cimetidine.
3. To compare latency and duration of loss of the righting reflex between
Midazolam alone and Midazolam and Cimetidine using statistical analyses.
4. To review and observe the pharmacodynamic and pharmacokinetic principles
of Midazolam and Cimetidine in white mice.
INTRODUCTION
Drug interactions should always be remembered in drug administration. The
desired effect of one drug may be drastically changed by the presence of other drugs in
the system. In some cases, this results in decreased bioavailability of the desired drug.
However, it is also possible to increase a particular drug’s bioavailability in the presence
of another drug.
When there is an additive affect between drugs, synergism ensues.
Mathematically, this can be represented as 1 + 1 = 2. There are also cases when simple
synergism thus not occur, resulting in the increased effect of one drug, and this is called
potentiation. Mathematically, we can represent this as 1 + 1 = >2.
Midazolam was first synthesized in 1976 by Fryer and Walser. It is an
imidazobenzodiazepine
with
unique
properties
when
compared
with
other
benzodiazepines. It is water soluble in its acid formulation but is highly lipid soluble in
vivo. It also has a relatively rapid onset of action, high metabolic clearance and short
elimination half life (2-4 hours) and readily crosses the blood-brain barrier when
compared with other benzodiazepines. It has powerful anxiolytic, amnestic, hypnotic,
anticonvulsant, skeletal muscle relaxant and sedative properties.
Midazolam is used in the preoperative period including premedication,
anesthesia induction and maintenance, and sedation for diagnostic and therapeutic
procedures because of its sedative and amnestic property.
Cimetidine belongs to a class of medications called histamine H2-antagonists.
Histamine is a natural chemical that stimulates stomach cells to produce acid.
Histamine H2-antagonists or H2RA inhibit the action of histamine on the acid-producing
cells of the stomach and reduce stomach acid. It is the first drug used to block the acid
23
producing cell in the stomach. However, in the context of this experiment, Cimetidine’s
effect on the P450 system in the liver is very important.
The righting reflex is an automatic righting reaction that ensures that the head
and body are correctly oriented in relation to each other and with the environment. This
reflex is most integrated in the midbrain and the hindbrain. Loss of this reflex is defined
as the time at which the subject is unable to stand on its feet and maintain its balance.
In this experiment, we observed two properties of drug action: latency and
duration. Latency is the time it takes for the desired effects of a particular drug to be
observed in a subject. Duration is the length of time between the onset of the effect and
its reversal.
METHODOLOGY
Materials
A. Test Animals:
Mice (Same Sex and about the same weight)
-2 mice for Midazolam
-2 mice for Midazolam and Cimetidine
B. Instruments:
Animal weighing scale
Tuberculin syringe
Animal cages
Asbestos gloves
C. Drugs:
Cimetidine
- Preparation: 10mg / ml (1%)
- Dosage: 1 mg/20g mouse
Midazolam
- Preparation: 1 mg/ml (0.1%)
- dosage: 0.3mg/20g mouse
Procedure
Four rats were weighed to determine the dose of the drugs that will be
administered. Two rats were assigned to the Midazolam group (control) and the
remaining two were assigned to the Midazolam-Cimetidine group (experimental).
24
The dosages were computed using the weights of the rats, the recommended
dosage of the drugs and the preparation available. The computations for the weights
are showed at the end of this section.
One member of the group was assigned to note the loss of the righting reflex and
the time at which it is regained. Another member timed the latency and duration of the
desired response. This procedure was applied to both groups.
The latency and duration of response from the different sections were compiled
for further analysis using the independent t-test.
Calculation for Midazolam dosage:
Preparation: 1mg/ml (0.1%)
Dosage: 0.1 mg/20g mouse
a) Dosage of Midazolam needed (mg):
0.1 mg
X (mg)

20 g
weight of mouse
X
weight of mouse (g)  0.1mg
20 g
b) Volume of Midazolam to be injected (ml):
1 mg X mg

1 ml
Y ml
Y
X mg  1 ml
1 mg
Calculation for Cimetidine dosage:
Preparation: 10mg/ml (1%)
Dosage: 1mg/20g mouse
a) Dosage of Cimetidine needed (mg):
1 mg
X (mg)

20 g weight of mouse
X
weight of mouse (g)  1mg
20 g
25
b) Volume of Cimetidine to be injected (ml):
10 mg X mg

1 ml
Y ml
Y
X mg  1 ml
10 mg
Table 1. Summary of dosages
Subject
Weight
Midazolam (ml)
Midazolam and Cimetidine
Midazolam (ml)
Cimetidine (ml)
A
11.9
0.0595
B
16
0.08
C
15.7
0.0785
0.0785
D
10.2
0.051
0.051
Rationale for Intraperitoneal Injection:
If given orally, Midazolam is metabolized almost completely by cytochrome P4503A4 in the liver. Likewise, metabolism in the gut wall is also equal to that of the
liver. Intraperitoneal injection was used to ensure that the Midazolam will not be
metabolized in the liver and gut wall, and thus, lose it's efficacy before it enters the
systemic circulation. Intravenous injection could also be done; however, the said
method will require greater precision and care as the veins found in the tail where the IV
injection should be placed are prone to blanching and hemorrhage if done incorrectly.
On the other hand, Cimetidine decreases the P450 activity of the liver. The ideal
route of administration of Cimetidine is orally. However, its effects will only come after a
longer period of time as it has to be absorbed by the GIT first. Therefore, a peritoneal
injection would be ideal as it will allow a faster rate of absorption by the organ.
Apart from the rationale listed, another advantage of using the intraperitoneal
route will ensure that the correct dosage will be given as compared to force-feeding.
There will be less spillage of the drugs which will lead to inaccurate results.
RESULTS AND DISCUSSION
Theoretical background
Midazolam
Pharmacodynamics
Midazolam was first synthesized in 1976 by Fryer and Walser. It is an
imidazobenzodiazepine
with
unique
properties
when
compared
with
other
26
benzodiazepines. Benzodiazepines act on post-synaptic pathways thus increasing the
pre-synaptic inhibition. Structurally, it is described as a 1,4-benzodiazepine class with
imidazole rings in the 1,2-position and contains carboxamide group in the 7- membered
heterocyclic ring. A substituent is found in the 7th position which is a halogen or nitro
group which serves for the sedative-hypnotic activity.
Midazolam is a powerful anxiolytic, and has amnestic, hypnotic, anticonvulsant,
skeletal muscle relaxant and sedative properties. It has a rapid onset and a short
duration. Midazolam is preferred to other drugs (such as Diazepam) since it is rapid, the
induction of the drug is non-painful and lack of venous irritation.
Benzodiazepines and with the presence of GABAA receptor in the neuronal
membrane in the CNS would activate the chloride-ion channel and would therefore
increase chloride conductance and the increase of GABA levels would result to
anticonvulsant effects. There are two types of receptors found in the brain that has
different reactions upon the presence of benzodiazepines. Type 1 receptor would result
to anti-anxiety while with type 2, it would cause sedative actions.
Pharmacokinetics
Midazolam is water soluble in its acid formulation but is highly lipid soluble in
vivo. It also has a relatively rapid onset of action, high metabolic clearance and short
elimination half life (2-4 hours) and it readily crosses the blood-brain barrier when
compared with other benzodiazepines.
It is water soluble because the benzodiazepine ring is open at pH below 4 but
when it is injected and the pH is 7.4, the benzodiazepine ring closes. This makes it
much more lipid soluble thus facilitating its rapid uptake into the nerve tissue, because
of this and having a pKa of 6.15 and is predominantly unionized at physiological pH
causes this drugs’ rapid onset of action and its high protein binding in the blood. It has a
half life of 1.9 hours and peak plasma concentrations are reached within 1 hour.
Liver Metabolism
The metabolism of Midazolam in humans is principally due to metabolic
biotransformation catalyzed by CYP3A isoforms namely, CYP 3A3, 3A4 and 3A5,
27
subfamilies of P450 enzymes. Most of the administered dose would be excreted as a
conjugated alpha-hydroxy metabolite through hydroxylation. Other metabolites would be
excreted through the urine in large amounts.
Indications and Usage
Midazolam is used in the preoperative period including premedication,
anesthesia induction and maintenance, and sedation for diagnostic and therapeutic
procedures because of its sedative and amnestic property.
Adverse Effects
When administered the drug produces certain side effects such as hypnosis, amnesia,
and anti-anxiety effects.
Contraindications
It is contraindicated for patients with hypersensitivity, acute narrow angle
glaucoma, shock, hypotension, head injury and drug or alcohol use.
Cimetidine
Phamacodynamics
Cimetidine belongs to a class of medications called histamine H2-antagonists.
Histamine is a natural chemical that stimulates stomach cells to produce acid.
Histamine H2-antagonists or H2RA inhibit the action of histamine on the acid-producing
cells of the stomach and reduce stomach acid. It is the first drug used to block the acid
producing cell in the stomach.
Cimetidine is chemically related to histamine. Its empirical formula is C10H16N6S
and it contains an imidazole ring.
Pharmacokinetics
Cimetidine has a half-life of about two hours. It can be given orally,
intramuscularly or intravenously but if given orally, Cimetidine is absorbed rapidly. Since
this drug reduces stomach acid, it may reduce the absorption of drugs that are best
28
absorbed in acidic conditions and this may also increase the blood levels of several
drugs by reducing their elimination by the liver. The kidney excretes the metabolized
and unmetabolized forms through filtration and renal tubular secretion and most of the
drug is excreted unchanged in the urine.
Liver Metabolism
There is evidence suggesting that this drug may act as an inactivator of
cytochrome P450 enzymes after metabolic activation. Clinically, Cimetidine therapy
impairs the clearance of various drugs metabolized by CYP2D6 and is also known to
reversibly inhibit CYP2D6 in vitro.
Cytochrome P450 enzymes is the one responsible for phase I metabolism of
many drugs.
Indications and Usage
Cimetidine is used for the short-term treatment of active duodenal ulcer, active
gastric ulcers, and maintenance therapy for duodenal ulcer patients at reduced dosage
after healing of active ulcer, short-term treatment of active benign gastric ulcer, erosive
gastroesophageal reflux disease (GERD), pathological hypersecretory conditions,
heartburn and the prevention of gastrointestinal bleeding.
Adverse Effects
The side effects for this drug is said to be and generally reversible once the
medication is stopped.
Minor side effects include constipation, diarrhea, fatigue, headache, insomnia,
muscle pain, nausea, and vomiting. Major side effects include confusion and
hallucinations, enlargement of the breasts, impotence, decreased white blood cell
counts.
Other side effects include irregular heartbeat, impotence, rash, visual
changes, allergic reactions, and hepatitis.
Contraindications
It is contraindicated for patients known to have hypersensitivity to the drug.
Potentiation: Cimetidine and Midazolam
As mentioned, the metabolism of Midazolam in humans is principally due to
metabolic biotransformation catalyzed by cytochrome enzymes in the liver. Cimetidine
inhibits cytochrome P450 because of the interaction of P450 heme iron through one of
the nitrogen atoms of the imidazole nucleus, thus when a drug is co-administered with
29
Cimetidine in-vivo, this would reduce the metabolism of the drugs that are metabolized
through this pathway.
Results and Interpretation of Statistical Analyses
Table 2. Results of Midazolam and Midazolam-Cimetidine administration on the latency
and duration of loss of righting reflex. There were eight subjects for the Midazolam
group, and only seven for the Midazolam-Cimetidine group.
Section
Midazolam
Midazolam and Cimetidine
Latency
Duration
Latency
Duration
(sec)
(sec)
(sec)
(sec)
A
311
111
115
338
385
422
271
128
B
1209
720
1160
85
C
600
120
1070
26
780
190
960
235
D
42
720.9
147
1800.1
75
840.5
88
1682.6
Table 3. Descriptive analyses on latency and duration of response from the experiment.
For the experiment of section D, the loss of righting reflex was observed to be the
time when the subjects are unable to keep all their feet on the ground – dragging of feet
and imbalance was observed and recorded.
Results from the four sections did not show a definite trend with regards to either
latency or duration of loss of righting reflex. All subjects in the Midazolam-Cimetidne
group showed an increase in the latency of response, except for those form section A. A
more variable set of results is seen with regards to duration of the response, with the
results from section D exhibiting the largest difference between the set-ups. The results
of latency and duration of response are summarized in Table 2.
However, according to Table 3, there is an increase in the duration of response
in the Midazolam-Cimetidine group, while the latency for both set-ups remained
practically the same. The mean latency and duration for the Midazolam group was 486
sec and 446 sec, respectively. The mean latency and duration for the MidazolamCimetidine group was 544 sec and 614 sec, respectively.
Statistical analysis using Independent T-test, with p=0.05 and 95% CI.
30
Alpha = 0.05
Df
= 12
Null hypothesis: There is no difference between the Midazolam and Midazolam +
Cimetidine group
Alternative hypothesis: There is a difference
CRITICAL VALUE: 2.18
T-test score for latency: - 0.038 (p value: >0.05)
T-test score for duration: - 0.523 (p value: >0.05)
Since both t-scores are >0.05, the null hypothesis is accepted.
Although there was an increase in the duration of response, statistical analysis
shows that this was insignificant. Results of the experiment do no conclusively show
that there is any effect of Cimetidine on the drug action of Midazolam. No significant
difference was observed in the latency of the response.
The result regarding the effect of potentiation on the duration of response does
not agree with the theoretical data. It was expected that there will be a significant
increase in the duration of drug response in the Midazolam-Cimetidine group, given that
Cimetidine increases the bioavailability of Midazolam.
Latency was not expected to be affected because this dependent primarily on the
interaction of Midazolam with its receptors, not on the effects that can be attributed to
Cimetidine.
DISCUSSION
Drug interactions may sometimes result in potentiation, wherein the effect on one
drug is increased. Potentiation in this experiment, is theoretically due to the effect of
Cimetidine on Midazolam metabolism. With the former decreasing the activity of the
cytochrome P-450 system, the bioavailability of the latter is expected to increase.
Increase in duration of response is expected to follow.
In sedative effects of Midazolam are attributed to its interaction with the Type II
receptors in the cerebral cortex. The drug readily crosses the blood brain barrier and
readily interacts with the receptor. The effects are therefore rapid in onset but the
31
duration is relatively short because of rapid metabolism via the cytochrome P-450
enzymes.
In the Midazolam-Cimetidine set-up, the increase in duration agrees with the
theoretical results. The explanation for this mechanism has already been mentioned in
the theoretical background of the experiment.
Theoretically, there should be no significant difference in the latency of
Midazolam in the two set-ups. The effect of Cimetidine lies on the metabolism of the
drug, not on the speed by which the Midazolam interacts with its receptors.
As mentioned, statistical analysis of the results revealed that there was no
significant difference of potentiaton on the duration of loss of the righting reflex. The
error of the experiment may lie in the following:
1. The weights of the mice may not be in the same range. For the section D
experiment, there was a range of 16 g – 10.2 g. The difference in the weights
may have contributed to the pharmacokinetic properties of the drugs that were
used.
2. There could have subcutaneous instead of intraperitoneal administration of the
drugs.
3. Although the subjectivity of observation was eliminated by using only one
observer, the observer’s definition of the righting reflex may have varied between
the sections.
4. Errors in dosage calculations may have occurred and resulted in erroneous
observation and recording of the loss of righting reflex.
RECOMMENDATIONS
1. The definition of the “loss of righting reflex” should be properly discussed with the
assigned observers. Observation of diminished activities such as limping or
slowed movements may not be the response needed, but may easily be
misinterpreted as such.
2. The weight range of the subjects in all the sections should be within a certain
range to decrease pharmacokinetic effects on the drugs.
3. The students should be adequately familiar with drug dosage calculations in
order to properly carry out the experiment.
32
FACTORS AFFECTING DRUG ACTION
Influence of Drug 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 determine the effect of administration of morphine and naloxone on the duration
of ail erection in mice
3. To determine the effect of administration of morphine and nalbuphine on the
duration of tail erection of mice
4. To determine the difference in the duration of tail erection among nos. 1, 2 and 3.
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
33
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.
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.
Naloxone hydrochloride, a narcotic antagonist, is a synthetic congener of
oxymorphone. In structure it differs from oxymorphone in that the methyl group on the
nitrogen atom is replaced by an allyl group. It has the following molecular formula
C19H21NO4•HCl with a molecular weight of 363.84. The chemical name for naloxone
hydrochloride is: (-)-17-Allyl-4, 5-epoxy-3,14-dihydroxymorphinan-6-one hydrochloride
Naloxone
34
Naloxone hydrochloride occurs as a white to slightly off-white powder, and is
soluble in water, in dilute acids, and in strong alkali; slightly soluble in alcohol;
practically insoluble in ether and in chloroform. Naloxone Hydrochloride Injection is a
sterile solution intended for intramuscular, subcutaneous or intravenous use.
Morphine is derived from a substance known as thebaine and has an extremely
high affinity for μ-opioid receptors in the CNS. Naloxone is used to oppose the effects of
opioid overdose, an example is morphine overdose. It is a μ-opioid receptor competitive
antagonist, and its fast inhibition of those receptors often create quick onset of
withdrawal symptoms. Naloxone also has an antagonist action, though with a much less
affinity for at κ- and δ-opioid receptors. Structurally, Naloxone can be illustrated as a
substituted oxymorphone.
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.
35
MATERIALS
1. Forty (40) mice of the same sex and about the same weight
(20 for morphine alone, 12 for morphine + naloxone, 8 for morphine +
nalbuphine)
2. animal weighing scale
3. tuberculin syringe
4. animal cages
5. Asbestos gloves
6. Stopwatches
7. Drugs
- Morphine (10mg/mL or 1%)
- Naloxone (0.4mg/mL or 0.04%)
- Nalbuphine (0.5mg/mL or 0.05%)
METHODOLOGY
Forty male white mice of similar weight were used in the experiment. They were
divided into 3 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 naloxone 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,
naloxone was given. Duration of tail erection was also observed and recorded.
Groub C was also given morphine. But when tail erection was exhibited,
nalbuphine was given. Duration of tail erection was also observed and recorded.
ANOVA was used for the statistical analysis of the different data gathered.
Significance of the data was also tested. This is done through the use of t-test,
specifically on the data gathered for the duration of tail erection in mice (Group A vs.
Group B and Group A vs. Group C).
RESULTS AND DISCUSSION
Duration of Tail Erection in Seconds
Morphine Alone
Morphine + Naloxone
Morphine + Nalbuphine
Mouse
5400s
Mouse
65s
Mouse
2304s
1
1
1
4320s
110s
2150s
2
2
2
4740s
85s
2057s
3
3
3
4586s
140s
2000s
4
4
4
7870s
183s
2259s
5
5
5
5775s
105s
2226s
6
6
6
36
2912s
194s
7
2242s
7
7
2394s
151s
8
2222s
8
8
2009s
160s
9
9
1839s
204s
10
10
1584s
178s
11
11
2602s
255s
12
12
2062s
13
1916s
14
1648s
15
885s
16
954s
17
920s
18
868s
19
1013s
20
Mean
(X)
Between
Groups
Within
Groups
TOTAL
2814.85s
160.83s
2182.5s
Sum of
Squares
53671419.58
df
2
Mean of
Squares
26835709.79
74113028.2
37
2003054.82
127784447.8
39
F Ratio
F Critical
13.40
3.26
P value (morphine + Naloxone) p>0.0005
P value (morphine + Nalbuphine) p> 0.2
Morphine
Alone
Morphine with
Naloxone
Morphine
Alone
Morphine with
Nalbuphine
Number of
subjects
20
Mean
Variance
2814.85
3895152.134
Standard
Deviation
1973.61
12
160.83
3801.61
61.66
Number of
subjects
20
Mean
Variance
2814.85
3895152.134
Standard
Deviation
1973.61
8
2182.5
11102.86
105.37
37
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. The mu receptors was acted upon by morphine and
was shown to have a durational effect of 2814.85s as seen in table 1. On the other
hand, naloxone, which is a narcotic antagonist, blocks the mu receptors, decreasing
morphine binding, thus limiting or inhibiting morphine’s action. Data showed that indeed
there was a large decrease of tail erection duration as recorded (only 160.83s).
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 (2182.5s).
CONCLUSION
Based on the data, there is a statistically significant difference on the duration of
tail erection in mice administered with morphine alone, morphine and naloxone, and
morphine and nalbuphine.
According to t-test values between Group A and B, there is a significant
difference between the duration of tail erection in mice administered with morphine
alone and morphine with naloxone. Group A had significantly longer duration of tail
erection as compared to Group B.
There is no significant difference between the duration of tail erection in mice on
Groups A and C. this may be due to the limitation of the experiment such as erroneous
computation of dosage and different subject number for each group set up.
REFERENCES:
www.emsb.qc.ca/laurenhill/science/morphine.html
Basic and Clinical Pharmacology. 9th edition. 2004 Katzung. Mc Graw Hill, lange
medical
Publications.
www.infoplease.com/dictionary/nalbuphine
Laboratory Experiment (manual). Department of Pharmacology, Faculty of
Medicine and Surgery.
SY 2006-2007
38
DETERMINATION OF LD50
OBJECTIVES:
General

To determine the lethal dose 50 of Lidocaine HCl
Specific

To determine the influence of dose on response of the animal by plotting the
number of deaths against the dose used by the different groups.
INTRODUCTION:
LD stands for "Lethal Dose". LD50 is the amount of a material, given all at once,
which causes the death of 50% (one half) of a group of test animals. The LD50 is one
way to measure the short-term poisoning potential (acute toxicity) of a material.
Toxicologists can use many kinds of animals but most often testing is done with rats
and mice. It is usually expressed as the amount of chemical administered (e.g.,
milligrams) per 100 grams (for smaller animals) or per kilogram (for bigger test subjects)
of the body weight of the test animal. The LD50 can be found for any route of entry or
administration but dermal and oral methods are most common.
Chemicals can have a wide range of effects on our health. Depending on how
the chemical will be used, many kinds of toxicity tests may be required. Since different
chemicals cause different toxic effects, comparing the toxicity of one with another is
hard. We could measure the amount of a chemical that causes kidney damage, for
example, but not all chemicals will damage the kidney. We could say that nerve damage
is observed when 10 grams of chemical A is administered, and kidney damage is
observed when 10 grams of chemical B is administered. However, this information does
not tell us if A or B is more toxic because we do not know which damage is more critical
or harmful. Therefore, to compare the toxic potency or intensity of different chemicals,
researchers must measure the same effect. One way is to carry out lethality testing (the
39
LD50 tests) by measuring how much of a chemical is required to cause death. This type
of test is also referred to as a "quantal" test because it measures an effect that "occurs"
or "does not occur".
In general, the smaller the LD50 value, the more toxic the chemical is. The
opposite is also true: the larger the LD50 value, the lower the toxicity. The LD50 gives a
measure of the immediate or acute toxicity of a chemical in the strain, sex, and age
group of a particular animal species being tested. Changing any of these variables (e.g.,
type animal or age) could result in finding a different LD50 value. The LD50 test was
neither designed nor intended to give information on long-term exposure effects of a
chemical. Once you have an LD50 value, it can be compared to other values by using a
toxicity scale.
If the immediate toxicity is similar in all of the different animals tested, the degree
of immediate toxicity will probably be similar for humans. When the LD50 values are
different for various animal species, one has to make approximations and assumptions
when estimating the probable lethal dose for man. Tables 1 and 2 have a column for
estimated lethal doses in man. Special calculations are used when translating animal
LD50 values to possible lethal dose values for humans. Safety factors of 10,000 or 1000
are usually included in such calculations to allow for the variability between individuals
and how they react to a chemical, and for the uncertainties of experiment test results.
The LD50 can be used:

As an aid in developing emergency procedures in case of a major spill or
accident.

To help develop guidelines for the use of appropriate safety clothing and
equipment. For example, if the dermal LD50 value for a chemical is rated as extremely
toxic, it is important to protect the skin with clothing, gloves (etc.) made of the right
40
chemical-resistant material before handling. Alternatively, if a chemical has an
inhalation LC50 value which indicates that it is relatively harmless, respiratory protective
equipment may not be necessary (as long as the oxygen concentration in the air is in
the normal range - around 18%).

For the development of transportation regulations.

As an aid in establishing occupational exposure limits.
METHODOLOGY:
Materials
D. Test Animals:
a. Mice
i.
80 Female Mice for the whole batch
1. Only 20 for Section D as the mice were divided into four
sections
E. Instruments:
a. Animal weighing scale
b. 80 Tuberculin syringes
c. Animal cages
d. Asbestos gloves
F. Drugs:

Lidocaine HCl
i.
Preparation: 0.1% and 1% (freshly prepared)
Procedure
*Due to time constraints, the labor and the 80 mice had to be divided among the four (4)
sections of batch 2009, assigning 20 mice for sections D, B, C and A. Since this is the
41
case, the only factors that could be held constant was the drug used and the gender of
the mice. Factors noted not held constant were the experimenters and the dose.
1. The volume of each dose for each mouse was computed and shown to the
facilitator for approval.
2. The drug was given intraperitoneally by one member of each section to minimize
technique variation.
3. Mice were observed for one hour and the number of deaths noted.
4. Percentage of deaths per dose-group was computed using a correction factor of
±0.25.
5. Percentages were then converted to probit to get the dose response curve.
Procedure
Twenty female albino mice were weighed individually using the animal weighing
scale to determine if the mice approximately had the same weight. This was done to
standardize the experiment and minimize error caused by different body mass. Sixty
mice were randomly divided into six groups of ten and assigned to be injected with
1mg/100g, 2 mg/100g, 8 mg/100g, 16 mg/100g, and 32mg/100g doses of Lidocaine.
The remaining twenty mice were assigned to be injected with 4mg/100g of lidocaine.
For Section D, the group was tasked to inject ten mice with 1mg/100g dose and another
ten mice with a 2 mg/100g dose of Lidocaine.
Table 1: Summary of assignments and their doses
Subsection Lidocaine
Dose
#
assigned
preparation (mg/100g) mice
D1
0.1%
1
10
D1
0.1%
2
10
B1
0.1%
4
20
of
42
C1
1%
8
10
C1
1%
16
10
A1
1%
32
10
A1
1%
64
10
To compute for the volume of each dose per mouse since it is dependent on their
body mass, the following equations were used:
Equation 1: Equation for deriving the volume needed for 2mg/100g Dose of
Lidocaine
Equation 2: Equation for deriving the volume needed for 2mg/100g Dose of
Lidocaine
Drug dosage calculations were verified by the facilitator. The correct dosages
were obtained and placed in the syringes. The drug was then injected intrapertioneally.
Only one member of the group was tasked to inject the mice in order to minimize
technique variation which may lead to errors.
Lidocaine, a local anesthetic, although efficiently absorbed in the GIT, is 90%
metabolized by the liver by CYP1A2 and CYP3A4. As a result, the oral administration of
43
the drug would make it lose its efficacy. Likewise, spillage can occur is the mice were
force-fed (with or without the gavage tube). Intraperitoneal injection is ideal as it allows
the drug to be absorbed into the systemic circulation without having to pass through the
liver immediately. Venous injection could also be administered, however, this may be
difficult as the veins in the tail of the mice are fragile and may blanch easily. Also, the
tendency to clot is greater.
The mice were then observed for one hour and the number of deaths were
counted. The percentages of deaths per dose-groups were calculated with a correction
factor of ±0.25. A plot was then made with the number of deaths against the dose used
by the different groups. Then, another curve was plotted by converting to the probit in
order to get the dose response curve.
RESULTS AND DISCUSSION:
Theoretical background:
Lidocaine is the 1st amino acid type local anesthetic to be developed. It was
discovered by Nils Lofgren & Benet Lundquist in 1943 & was first marketed in 1948 as
an anesthetic & anti arrhythmic drug. It is usually available in the form of Lidocaine HCl
& has a chemical formula of C14H23ClN2O2. It can be bought in different preparations
which include injected local anesthetics, dermal patches & nasal instillations or sprays.
Pharmacodynamics:
Lidocaine exerts its effects thru its ability to block both activated & inactivated Na
Channels with rapid kinetics. As an anesthetic drug, lidocaine alters depolarization in
neurons by blocking the fast Na channels in the cell membrane. Blockage would then
prevent depolarization which would lead to a decrease in the synthesis & transmission
of action potential bringing about its anesthetic effects. On the other hand, Blocking of
the Na channels in the inactivated state has greater effects on cells with long action
potentials such as your Purkinje & ventricular cells & is thus responsible for the drug’s
anti arrhythmic effects. Classified as a 1b Antiarrythmic agent, lidocaine blocks the Na
44
channels of cardiac action potential which would then decrease automaticity of heart
contractility by decreasing the slope of phase 0 of depolarization.
Pharmacokinetics:
Lidocaine has a more rapid onset of actions & longer duration of action than
amino ester type local anesthetics such as procaine. Approximately 90% is metabolized
in the liver by CYP1A2, monoethylglycinexylidide &glycinexylidide & only about 3% of
orally administered lidocaine reaches the plasma. For this reason, lidocaine must be
given parenterally. Half life of lidocaine is very short, lasting for only about 1 to 2 hours.
Which is why, a loading dose of 150 – 200 mg administered over about 15 min should
be followed by a maintenance infusion of 2 to 4 mg/min in order to reach therapeutic
plasma levels. In some cases, presence of alpha1-acid glycoprotein, an acute phase
reactant protein that binds lidocaine decreases the amount of free drug available. When
this happens higher dosages of lidocaine are required before it can exert its effects.
There are some instances when half life can be prolonged. In patients with congestive
heart failure both volume of distribution & total body clearance may be decreased which
would lead to an increase in half life. However, since these 2 factors counteract each
other, only minimal change (approximately 136 min) can be seen. Increase in half life is
more markedly increased in patients with liver disease whose plasma clearance is
greatly reduced while volume of distribution is increased. Half life in these patients may
increase 3 folds or with an average amount of 343 min. Thus usual loading dose can be
given but maintenance dose should be decreased in patients with liver disease. Steady
state concentration requires a longer time for these patients as compared to those who
are normal. Some drugs could also reduce clearance by decreasing liver blood flow &
so increases toxicity of lidocaine.
Toxicity:
When lidocaine is used as a local anesthetic & if administered correctly, adverse
drug reactions are rare. Most of the adverse effects of lidocaine arise from wrong
administration techniques which could result to systemic exposure. Systemic exposure
to a great amount of lidocaine will initially manifest as problems in the CNS which would
45
include nervousness, tingling sensations, tinnitus, blurred vision & seizures followed by
episodes of depression. Further increase in systemic exposure to lidocaine would bring
about signs & symptoms arising from the cardiovascular system which includes
hypotension, bradycardia, muscle twitching, asystole & cardiac arrest. This is why even
though lidocaine is the agent of choice in treating ventricular tachycardia it should not
be used routinely because prophylactic use of lidocaine may increase mortality by
increasing incidence of asystole.
Results
A. Tabulation of Results
Subsection Lidocaine
Dose
#
of # of deaths in
Percentage
assigned
preparation (mg/100g) mice
1 hr
D1
0.1%
1
10
0
2.5%
D1
0.1%
2
10
0
2.5%
B1
0.1%
4
20
0
2.5%
C1
1%
8
10
0
2.5%
C1
1%
16
10
8
80%
A1
1%
32
10
5
50%
A1
1%
64
10
9
97.5%
**correction factor of ±0.25
46
B. DoseResponse
curve
Effects
Dose Response Curve
10
9
8
7
6
5
4
3
2
1
0
1
2
4
8
16
32
64
Dose
C. Probit Curve
A.
B.
47

green line shows LD 50
The results for the performed experiment did not show a specific trend of increasing
toxicity of the drug with an increase in dosage. As shown in the tabulation of results,
there were a lesser number of deaths for the rats administered with a dosage of 32
mg/1oo g than for those who were administered 16 mg/100 g. There was also a
discrepancy in terms of the lethal dose. A lethal dose of 16.9 mg/100g was attained
instead of the expected lethal dose of 8 mg /100 g
Discussion:
In the manufacturing of new drugs, it is not enough for the drugs to be proven
effective, drug companies must also make sure that the drugs they produce have an
adequate amount of safety. One of the parameters to ensure this safety would be to
check for the drugs LD 50 which is defined as the amount of drug given that would
48
produce death to 50% of the tested population. It can therefore be used to set
parameters on prescription of certain substances in terms of administration & dosages.
For Lidocaine HCl, which is the drug used in this experiment the normal dosage
that is deemed to be safe for mice is approximately 0.27 mg/100g. Dosages higher than
this would produce either CNS or cardiovascular manifestations. Since one of the
functions of lidocaine is to treat arrhythmia, increasing its dosage could lead to asystole
which could then lead to cardiac arrest & to eventual death of the subject.
It was expected in this experiment that there would be a direct relationship
between the dose of the drug & the LD50. It was also expected that the LD50 of
lidocaine would be 8 mg/100g. However this was not achieved. Instead an LD50 of
16.9mg/100g was attained. These errors could have been brought about by factors
such as improper calculation of the dosages, improper administration of the drug or
internal factors from the subjects as discussed in the theoretical background.
CONCLUSION:
The experiment results show that a concentration of 16.9 mg/100 g is lethal to
50% of the experimental animals and is greater than that of the standard and expected
LD50 at 8 mg/100 g.
49