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The University of Zambia
School of veterinary medicine
Department of paraclinical studies
Name: Musalo Brian
Computer #: 10008047
Course code: VMP-4500
Lab: Drug interaction and effects of liver on drugs
Attention: Dr. Muzandu
Date: 17/02/14
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Title: Drug interaction in mice
Aim: to investigate the interaction of drugs and the effects of liver function on drug action in
animals (mouse).
INTRODUCTION
Drug interaction is the modification of the action of one drug by another. There are three kinds of
mechanisms: pharmaceutical, pharmacodynamics and pharmacokinetics. Pharmaceutica l
interaction occur by chemical reaction or physical interaction when drugs that influence the same
physiological function ( for example drugs that influence the state of alertness or blood pressure);
the result of adding a second such drug during treatment with another may be to increase the
effect of the first(for example alcohol increases the sleepiness caused by benzodiazepines).
Drug interaction is very important because, whereas judicious use of more than one drug at a time
can greatly benefit patients, adverse interactions are not uncommon, and may be catastrophic, yet
are often avoidable. Multiple drug use (ploypharmacy) is extremely common, so the potential for
drug interaction is enormous. One study showed that on average 14 drugs were prescribed to
medical in-patients per admission (one patient received 36 different drugs). The problem is likely
to get worse, for several reasons such as that many drugs are not curative, but rather ameliorate
chronic conditions (e.g. arthritis) and it’s too easy to enter an iatrogenic spiral in which a drug
results in an adverse effect that is countered by the introduction of another drug, and so on.
Therefore hospital admission provides an opportunity to review all the medications that any patient
is receiving, to ensure that the overall regimen is rational (James M.R, 2008).
Pharmaceutical interactions: inactivation can occur when the drug such as heparin with
gentamicin are mixed. Drugs may also interact in the lumen of the gut for example tetracycline
with iron and colestyramine with digoxin. Pharmacodynamic interactions: these are very common
and mostly have a simple mechanism consisting of the summation or opposition of the effects of
drugs with, respectively similar or opposing actions. Since this type of interaction depends
broadly on the effect of the drug, rather than on its specific chemical structure, such interactions
are non-specific. Pharmacokinetics interaction: The gastrointestinal absorption of drugs may be
affected by concurrent use of other agents that (1) have a large surface area upon which the drug
can be adsorbed, (2) bind or chelate, (3) alter gastric pH, (4) alter gastrointestinal motility, or (5)
affect transport proteins such as P-glycoprotein. One must distinguish between effects on
absorption rate and effects on extent of absorption. A reduction in only the absorption rate of a
drug is seldom clinically important, whereas a reduction in the extent of absorption will be
clinically important if it results in sub-therapeutic serum levels.
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The mechanisms by which drug interactions alter drug distribution include (1) competition for
plasma protein binding, (2) displacement from tissue binding sites, and (3) alterations in local
tissue barriers, e.g., P-glycoprotein inhibition in the blood-brain barrier. Although competition
for plasma protein binding can increase the free concentration (and thus the effect) of the
displaced drug in plasma, the increase will be transient owing to a compensatory increase in drug
disposition. The clinical importance of protein binding displacement has been overemphasized;
current evidence suggests that such interactions are unlikely to result in adverse effects.
Displacement from tissue binding sites would tend to transiently increase the blood concentration
of the displaced drug.
The usefulness of drug interaction is very important and involves increased effect, drugs can be
used in combination to enhance their effectiveness. Disease is often caused by complex processes
and drugs influence their different components of the disease mechanism and may have an
additive effect such as combination of antimicrobial drugs, are used to prevent the selection of
drug–resistant organisms. Tuberculosis is the best example whose successful treatment requires
this approach. Minimize side effects, there are many situations where doses of two drugs may be
used and are better tolerated, and are more effective than single doses. Also blocks accurately
an unwanted/toxic effect: drugs can be used to block an undesired or toxic effect, as for example
when anesthetics are being used a cholinesterase is used to reverse the neuromuscular blockade,
or when antidotes such as naloxone are used to treat opioid overdose. The use of vitamin or fresh
plasma to reverse the effects of warfarin poisoning is another example (Ktuzung (2003).
Hypnotics are drugs, which can produce a state of CNS depression resembling normal sleep after
administration of a sufficient dose. At one time, the most likely used hypnotics were the barbiturates but nowadays safer drugs such as benzodiazepines are preferred. However, the barbiturate hypnotics are particularly suitable for illustrating certain pharmacokinetic and
Pharmacodynamic mechanisms. The barbiturates can be classified in pharmacokinetic terms as: ultra-short acting, short acting, intermediate acting and long acting. Most barbiturates are
metabolized to a greater or lesser extent in the liver, although some e.g. baritone are excreted virtually unchanged in the urine. The duration of action tends to be closely correlated with the lipid solubility of the barbiturate in question.
In this practical, thiopentone and Pentobarbitone are selected as examples of ultra-short and intermediate acting barbiturates and their respective duration of action are measured.
Administering simultaneously another CNS depressant, ethyl alcohol, can modify the duration of action of Pentobarbitone. Phenobarbitone is a long-acting barbiturate. It is one of the most potent liver microsomal enzyme inducers and may be a carcinogenic promoter. It is partially metabolized by hydroxylation (to hydroxyphenobarbitone) and then conjugated w ith glucuronic a c id for excretion in urine. Excretion of unmetabolized drug is facilitated by alkalization of urine. (The barbiturates are weak organic acids, and the drug will be ionized in alkaline urine thus reducing tubular reabsorption.). It is used as a sedative; anti-epileptic drug for long term control – one of the safest in cats; It may increase appetite in cats; some veterinarians may use it in chronic refractory skin conditions to
help reduce self-trauma (Brander G.C, 1977).
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Pentobarbital (US English) or Pentobarbitone (UK English) is a short-acting barbiturate.
Pentobarbital can occur as both a free acid and as a salts of elements such as sodium and calcium. The free acid is only slightly soluble in water and ethanol. In high doses, pentobarbital
causes death by respiratory arrest. In the United States, the drug has been used for executions
of humans. Sodium thiopental, also known as Sodium Pentothal, thiopental, is a rapid-onset
short-acting barbiturate general anesthetic. Sodium thiopental is a core medicine in the World Health Organization's "Essential Drugs List", which is a list of minimum medical needs for a
basic healthcare system. Carbon tetrachloride is an inorganic compound with the formula
CCl4. It was formerly widely used in fire extinguishers, as a precursor to refrigerants, and as a
cleaning agent. It is a colorless liquid with a "sweet" smell that can be detected at low levels.
MATERIAL
observation cage
stop watch
1 ml syringe
needles
animals: 5 mice for each group (male mice)
Small animal electronic balance
Drugs: Pentobarbitone, thiopentone and carbon tetrachloride.
PROCEDURE
The class was divided into 10 working groups and each group was assigned 5 mice. All the mice
were weighed and their weights were recorded and the drug doses for each mouse was calculated
based on weight and concentration of the drug. All the mice were pre-weighed.
Group A1, injected only the calculated doses of 80mg/kg Thiopentone and immediately the
stop watches were switched on and the observations were made and recorded. Here the mice
were provided with food and water until the day of the experiment. In group A2, the mice were
starved for 48 hours but later water was given to them. Then the calculated doses of 80mg/kg
Thiopentone were injected and immediately the stop watches were switched on and
observations were made and recorded.
In group B1, mice were injected with calculated doses of 80mg/kg of Phenobarbitone and
immediately the stop watches were switched on and the observations were made and recorded.
Here the mice were provided with food and water until the day of the experiment.
In group B2, the mice were starved for 48 hours and then were given water later. They were
injected with calculated doses of 80mg/kg Phenobarbitone. Stop watches were then switched
on immediately and observations were then made and recorded.
In group C1, there was normal treatment, normal food and water given. The mice were injected
only the calculated doses of 80mg/kg Thiopentone and immediately the stop watches were
switched on and the observations were made and recorded.
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In group C2, the mice were pre-treated with 0.1% (w/v) Pentobarbitone in drinking water 5
days before the experiment day. They were also on normal feeding regime. Then immedia te ly
after this they were injected with calculated doses of Thiopentone. Stop watches were switched
on immediately, observations were made and recorded.
In group D1, the mice were on normal feeding regime. These were injected with calculated
doses of 80mg/kg Pentobarbitone. The stop watches were switched on immediate ly,
observations were made and recorded.
In group D2, the mice were pre-treated with 0.1% Pentobarbitone in drinking water 5 days
before the experiment day. They were on normal feeding regime. These were injected with
calculated doses of 80mg/kg Pentobarbitone. Stop watches were switched on immediately and
observations were made and recorded.
In group E1, the mice were on normal feeding and water regime. . These were injected with
calculated doses of 80mg/kg Pentobarbitone. The stop watches were switched on immediate ly,
observations were made and recorded.
In group E2, the mice were injected with 10% carbon tetrachloride 5 days prior to the
experiment. They were on normal feeding and water regime. They were injected with
calculated doses of 80mg/kg of Pentobarbitone. Stop watches were switched on and
observations were made which were then recorded.
RESULTS
Group A1
Mouse
#
weight Thiopentone
(ml)
t 0 t 1 t 2 t 3 X (sec) Y (sec) Z (sec)
1 30.3 0.49 14:56 14:57 14:57 18:00 60 60 10980
2 27.7 0.44 14:59 15:00 15:00 Died 60 60 -
3 32.9 0.53 15:01 15:02 15:03 18:45 60 120 13320
4 32.8 0.52 15:03 15:04 15:05 24:15 60 120 33000
5 26.8 0.43 15:04 15:06 15:07 17:30 120 180 8580
Averages 72 108 16470
Group A2
Mouse
#
weight Thiopentone (ml)
+ 48hrs starving
t 0 t 1 t 2 t 3 X (sec) Y (sec) Z (sec)
1 26.2 0.42 15:09 15:15 15:25 17:10 360 960 8700
2 27.5 0.44 15:12 15:19 15:27 23:30 420 900 28980
3 22.8 0.36 15:18 15:23 15:28 18:30 300 600 10920
4 24.4 0.39 16:05 16:09 16:15 01:45 240 600 30600
5 22.9 0.37 15:26 15:32 15:40 17:24 360 360 8640
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Averages 366 684 17568
Group B1
Mouse
#
weight Pentobarbitone
(ml)
t 0 t 1 t 2 t 3 X (sec) Y (sec) Z (sec)
1 25.01 0.33 14:37 14:42 14:45 02:45 300 480 3600
2 27.50 0.37 14:4
5
14:48 14:52 19:35 180 420 19380
3 25.20 0.33 14:4
2
14:48 14:54 19:32 360 720 19080
4 23.09 0.32 14:4
6
14:51 14:56 20:04 300 600 20880
5 28.13 0.38 14:4
7
14:52 14:58 17:30 300 660 11520
Averages 288 576 14892
Group B2
Mouse
#
weight Pentobarbitone
(ml)
t 0 t 1 t 2 t 3 X (sec) Y(sec) Z (sec)
1 26.4 0.53 14:35 14:40 14:43 17:50 300 480 11220
2 23.0 0.46 14:45 14:51 14:52 17:55 360 420 10980
3 27.0 0.54 14:48 14:52 14:54 18:00 240 360 13560
4 24.4 0.49 14:52 14:58 15:02 19:05 360 600 14580
5 17.0 0.34 14:56 15:01 15:04 19:30 300 480 15960
Averages 312 468 13260
Group C1
Mouse
#
weight Thiopentone
(ml)
t 0 t 1 t 2 t 3 X (sec)
Y (sec) Z (sec)
1 28.2 0.34 14:52 14:53 14:5
4
17;10 60 120 10560
2 25.3 0.34 14:53 14:54 14:5
9
17:30 60 360 11460
3 30.9 0.41 15:00 15:01 15:0
2
17:20 60 120 8280
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4 24.0 0.32 14:55 Died - - - - -
5 24.2 0.32 14:57 14:58 14:5
4
16:40 60 120 8460
Averages 60 180 9690
Group C2
Mouse
#
weight Thiopentone
(ml)
t 0 t 1 t 2 t 3 X (sec) Y (sec) Z (sec)
1 28 0.448 12:25 12:35 12:4
2
- 600 1020 -
2 30 0.48 12:38 12:39 12:4
2
- 60 180 -
3 28.2 0.34 - - - - - - -
4 25.3 0.34 - - - - - - -
5 30.9 0.41 - - - - - - -
Average: 330 600
Group D1
Mouse
#
weight Pentobarbitone
(ml) t 0 t 1 t 2 t 3 X (sec) Y (sec) z(sec)
1 28.9 0.241 15:00 15:02 15:03 17:15 120 180 7920
2 29.0 0.24 14:55 14:57 14:59 16:49 120 240 6600
3 35.4 0.30 14:53 14:54 14:55 16:40 60 120 5700
4 27.3 0.23 15:04 15:07 15:11 17:42 180 420 9060
5 17.3 0.14 15:06 15:08 15:09 18:31 120 180 12120
Average: 120 228 8280
Group D2
Mouse
#
weight Pentobarbitone
(ml)
t 0 t 1 t 2 t 3 X (sec) Y (sec) Z (sec)
1 27.3 0.546 12:24 12:45 12:50 12:57 1260 1560 420
2 - - - - - - - - -
3 - - - - - - - - -
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4 - - - - - - - - -
5 - - - - - - - - -
Average: 1260 1560 420
Group E1
Mouse
#
weight Pentobarbitone
(ml)
t 0 t 1 t 2 t 3 X (sec) Y (sec) Z (sec)
1 27.4 0.37 14:49 14:51 14;52 19:04 120 180 15120
2 34.7 0.46 14:51 14:53 14:54 18:59 120 180 14700
3 25.9 0.35 14:54 14:55 14:56 19:05 60 120 14940
4 32.9 0.44 14:57 14:59 14:59 19:35 120 120 16560
5 29.3 0.39 14:59 15:00 15:01 18:55 60 120 14040
Average: 96 144 15072 15072
Group E2
Mouse
#
weight Pentobarbitone
(ml) t 0 t 1 t 2 t 3 X (sec) Y (sec) Z (sec)
1 25.2 0.504 15:00 15:03 15:04 22:50 180 240 27960
2 25.0 0.5 15:06 15:08 15:09 23:50 120 180 31260
3 28.0 0.56 15:11 15:13 15:15 23:09 120 240 28440
4 26.5 0.53 15:16 15:18 15:20 23:19 120 240 28740
5 23.5 0.47 15:17 15:20 15:26 23:22 180 540 28560
6 24.6 0.492 15:23 15:24 15:26 23:30 60 180 29040
Average: 130 270 29000
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Data Analysis
Group X sec Y sec Z sec Additional
drug dose
time of
injection
of ( drug )
( drug )
Dose: Mg/kg
A1 72 108 16470 Nil Nil 80mg/kg
A2 336 684 17568 starved 48 hrs.
before 80mg/kg
B1 288 576 21372 Nil Nil 80mg/kg
B2 312 468 13260 Starved 48 hrs.
before 80mg/kg
C1 60 180 9690 Nil Nil 50mg/kg
C2 330 600 - 0.1%
Pentobarbitone
in water
5 days
before 80mg/kg
D1 120 228 8280 Nil Nil 50mg/kg
D2 1260 1560 420 0.1%
Pentobarbitone
in water
10 minutes
before 80mg/kg
E1 96 144 15072 Nil Nil 80mg/kg
E2 130 144 29000 10% CCl4
injection
5 days
before 80mg/kg
DISCUSSION
Drug interaction occurs when two or more drugs interact in such a way that the effectiveness or
toxicity of one or more of the drugs is altered. Although not all drug interactions are clinica l ly
important to be alert for those that are. A knowledge of the main types of drugs (thiopentone &
Pentobarbitone) will act as a useful alert. Drugs most likely to be involved in interaction are those
with a narrow safety margin between the therapeutic and toxic dose, those requiring careful dosage
control and those that are either induced or inhibit liver microsomal enzymes.
The drug interaction and the effect of the liver on the drugs provided which were Pentobarbito ne,
Carbon tetrachloride and Thiopentone. Pentobarbitone and Thiopentone are barbiturates and are
metabolized in the liver and they also differ in their duration of action. Pentobarbitone is a short
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acting barbiturate whereas Thiopentone is an ultra-short acting barbiturate. Carbon tetrachloride is
an inorganic compound and it is as well metabolized in the liver. Barbiturates are in two groups,
the oxybarbiturates such as Pentobarbitone and Thiobarbiturates like Thiopentone. These two
groups of barbiturates differ in the way they are metabolized and excreted. Induction of general
anesthesia in mice can be achieved by a variety of drugs and techniques. The most commonly used
anesthetics in mice include the injectable agents such as Pentobarbital. In mice, injectable
anesthetics can best be administered via IP, IM, and IV routes.
In group A1, the mice were on normal treatment thus they were provided with food and water until
the day of the experiment. They were injected with calculated doses of 80mg/kg Thiopentone. The
mice took about 72 seconds to the onset of ataxia; they lost conscious for about 108 seconds and
slept for about 16470 seconds. In A2, the mice were starved for 48 hours and then treated with
calculated doses of 80mg/kg of Thiopentone and the mice slept for about 17568 seconds. In group
A2 the mice slept for a longer time as compared to those in A1, this can be attributed to the fact
that Thiopentone being a Thiobarbiturates, its duration of action tends to be closely correlated with
its lipid solubility. In A2 were the mice were starved for 48 hours, the fat was broken down to
provide energy for the mice thereby reducing the fat content in the tissue, hence making the mice
sleep for a longer time when subjected to Thiopentone treatment which tends to be redistributed
in fat tissues for its excretion (termination of action) as compared to those in A1 which were on
normal treatment regime (provided with food and water).
Groups B1 and B2, the mice were injected with calculated doses of 80mg/kg of Pentobarbitone,
the only difference being that the mice in B1 were on normal feeding regime whereas in B2 they
were starved for 48 hours. The mice were subjected to the treatment with Pentobarbitone.
Pentobarbitone is a short acting barbiturate and it undergoes first-pass metabolism in the liver and
possibly the intestines; it causes cardiorespiratory depression as well as hypotension. In B1 the
mice slept longer than the ones in B2 which were starved. The duration of action of Pentobarbitone
depends on metabolism. In the starved mice there was low metabolism thereby reducing the effect
of Pentobarbitone leading to its fast termination of action.
In group C1 the mice slept for 9690 seconds after being injected with calculated doses of 80mg/kg
Thiopentone. These mice were on normal feeding regime. In C2, only two mice were worked on
and these were injected with calculated doses of 80mg/kg Thiopentone though the time they woke
wasn’t noted.
In D1 the mice were on normal feeding regime, they slept for 8280 seconds after being injected
with calculated doses of 80mg/kg of Pentobarbitone. In D2, the mice were pretreated with 0.1%
Pentobarbitone in water before the experiment. Only one mouse was injected with calculated dose
of 80mg/kg Pentobarbitone, the other mice died during the pretreatment process. In D2 the mouse
slept for a short time and then woke, this was because when the mice were treated with 0.1%
Pentobarbitone, the microsomal hepatic enzyme, Cytochrome P450 was produced in high amounts
hence when calculated dose of 80mg/kg Pentobarbitone during the experiment was injected it was
metabolized quickly due to high concentration of cytochrome P450 enzyme, therefore fast
termination of action leading to the mouse sleeping for a short time.
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In E1, the mice were on normal feeding regime thus they were provided with food and water until
the day of the experiment and they were injected with calculated doses of 80mg/kg Pentobarbitone .
They slept for 15072 seconds. In E2, the mice were pre-treated with 10% carbon tetrachloride 5
days prior to the experiment and then they were injected with calculated doses of 80mg/kg of
Pentobarbitone. The mice slept for 29000 seconds. Carbon tetrachloride has a damaging effect on
the liver. The mice were pretreated with 10% carbon tetrachloride 5 days prior to the experiment.
Here the liver was damaged hence when the mice were treated with 80mg/kg Pentobarbitone; the
duration of the drug was prolonged leading to the mice sleeping for a very long time.
Carbon tetrachloride is a clear liquid with a sweet smell that can be detected at low levels. Carbon
tetrachloride metabolism is primarily in the liver, although it may also occur in other tissues. In
the rats and mice, cytochrome P450 (CYP) 2 E1 is primarily responsible for the bioactivation of
carbon tetrachloride.
The sex of mice influences the pharmacokinetics and metabolism of anesthetics probably due to
differences in plasma corticosteroids, sexual hormones, or hepatic enzymes (Hildebrandt et al.
2008). Male mice were used because females could have been in different physiological status that
could have affected the results of the experiment and some of the drugs used were ecbolic (cause
uterine muscle contractions) that would have induced abortion (Alexander, 1978).
Laboratory mice especially the males exhibit specific anatomic and physiologic peculiarities that
influence the effects of anesthetic drugs. Due to their small body size, drug metabolism and
excretion are extremely fast, reducing the half-life of injectable drugs and rendering the duration
of anesthesia a more critical factor compared with larger species. Moreover, the elevated body
surface area of mice promotes heat loss and hypothermia, while their reduced glycogen reserve
predisposes them to hypoglycemia. In addition, their high oxygen consumption rate reduces the
survival rate for hypoxemia. In fact, irreversible central nervous system damage occurs only a few
seconds after respiratory arrest in mice (Abou-Madi 2006).
During this practical it is very important to take note and ensure the following precautions for a
safe and successful practical The dosage of the drug must be calculated according to the weight of the individual mouse
When dealing with LIVE specimens, do not cause unnecessary pain or disturbance to the animals
Wear double layers of cotton gloves or avoid touching the mice if you are unfamiliar with the handling procedures
Be extremely careful during manipulation of the injection needles
Report all cases of injury or death.
CONCLUSION
The interaction of drugs (Pentobarbitone, carbon tetrachloride and Thiopentone) and the effect of
the liver on the given drugs were confirmed. The experiment showed that in drug interaction in
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animals (mice), duration of action of a drug depends on time of administration as well as dose rate
of the drug.
REFERENCES
Abou-Madi N. 2006. Anesthesia and Analgesia of Small Mammals: Recent Advances in
Veterinary Anesthesia and Analgesia: Companion Animals. In: Gleed RD, Ludders JW, eds.
Ithaca NY: International Veterinary Information Service (www.ivis.org). pp 1-9.
Alexander. F. (1978), An Introduction To Veterinary Pharmacology, 3rd Edition, Churchill
Livingstone, London
Brander G.C & push D.M, (1977), veterinary applied pharmacology & therapeutics, 3rd
edition, Bellaire Tindal, London.
James M.R, Lionel D.L, Timothy GK.M & Albert F (2008), Clinical pharmacology and
therapeutics, 5th edition, Hodder Arnold print, United Kingdom.
Ktuzung (2003), Basic and clinical pharmacology, 9th edition, McGraw hill, London.
Roach S.S et.al (2003), Introduction to clinical pharmacology, 7th edition
