Transcript LD 50 : Median lethal dose

THE MEASUREMENT OF LD50
Definition
• An LD50 represents the individual
dose required to kill 50 percent of a
population of test animals. It is an
index determination of medicine and
poison’s virulence. The lower the
LD50 dose, the more toxic the
pesticide.
LD50: Median lethal dose
• LD50 (median lethal dose):
– The dose of a drug that
produces death in 50% of the
animal population tested
Drug variability and toxicity
assessment
• ED50: Effective dose for 50% of
subjects
• LD50: Lethal dose for 50% of subjects
• The therapeutic index
TI = LD50 / ED50
No drug is 100% safe
Definition
• Historically, the LD50 was
determined with a high degree of
precision and was used to compare
toxicities of compounds relative that
high precision is not necessary to
compare toxities. Therefore, the
median lethal dose is now estimated
from the smallest number of animals
possible.
Definition
• Toxicity test can be divided into
three parts: acute toxicity testing,
chronic toxicity testing, and special
testing .
Definition
• Acute toxicity testing: single dosage
everyday, to observe its toxic
reaction after 7 days and 14 days.
• Acute toxicity test: that is lethal in
approximately 50% of animals.
Determine maximum toleterated
closes. Usually two species, two
routes, single close (compare with
therapeutic dose).
• Chronic toxicity test: 1 to 2 years.
Required when drug is intended to be used
in humans for prolonged periods. Usually
run concurrently with clinical trial. Goals
of chronic tests are to show which organs
are susceptible to drug toxicity. Clinical
chemistry physiologic signs, autopsy
studies, hematology, histology, electron
microscopy studies. Identify target organ
of toxicity.
Experimental purpose
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To grasp the basic metered method
of LD50 and the suitable condition
of the Karbar's method.
To be familiar with the general
group method for animals.
To comprehend the deduction
procedure of the Karbar's method.
Experimental method
• Karbar's method:
• There are many methods in determining
LD50 such as Bliss, sequential method,
grouping method ect. The most classic
and precise method is Bliss method
which is used in this experiment.But it
needs to use computer and corresponding
software. It is more complicated,so we
often use Karbar's method.
Experimental condition
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The dose must be increased with
geometric proportion series. (The
logarithm of Dose with arithmetic
series)
The number of every group must be
equal.
The response should satisfy the
Gaussian distribution roughly.
Experimental animals
•mice
Experimental drugs
• the different concentration of
Dipterex; 2, 4,6-trinitrophenol
Dipterex
It can irreversibly inhibits AChE
Mechanism of intoxication
 Organophosphates are“ irre-versible ”
anticholinesterase drugs. Their phosphor
atom combines with AChE by covalent bond
and forms phosphated AChE that is uneasy
to be hydrolyzed.
 The activity of AChE diminishes markedly
and the concentration of ACh increases.
 Administering as soon as possible in order to
prevent enzyme from “aging”.
Experimental apparatus
• mice cage, animal’s equi-armbalance,
1ml injection syring, and electronic
calculator
Experimental procedure
How to choose and divide the
experimental animals:
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Principle of choice: We should choose
the animals sensitive to the trial agents.
Considering about source, economic value,
convenient handling to the animals, we
often choose mice for our experiment.
Principle of group: The number of every
group should be more than the number of
groups. We divide the animals into 7
groups in this experiment, and there are
10 animals in each group.
Group
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How to group animals Principal: We
divide these animals into different
groups according to their weight this
time, and then group them in random.
Their weight of each group must be
almost the same, and the different of
the sum of weight in each group must be
less than 5g
How to group students: Divide the
students into 10 groups in order to
decrease errors which may be caused by
handling of different classmates.
Dosage
• Administration route: intraperitoneal
injection method.
• The concrete method as follows: raise
mouse’s tail root with the right hand, slip
to cervical part from back to the head
with left hand, knead its scalp with thumb
and forefinger, turn over it and fix its tail,
hold the injection with the right hand,
prick to left intraperitoneal (trumpet) and
pumpback to make sure that there is not
the blood.
• We should thrust and pluck quickly when
inject.
Observe the death number and
calculate the death rate
– observing time: one hour
– fill the death rate and data in the
table
Calculation formula should be
applied by these conditions:
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When the death rate（P n）of the
minimum dosage equals to 0％ and the
death rate（P m）of the maximal dosage
equals to 100％, we use the basic formulas.
When the P n less than 20% or the P m
more than 80%, we use the correction
formula.
When the P n more than 20% or the P m
less than 80%, we can not use any formula.
Basic formula
（ P n = 0% and P m = 100％ ）
LogLD50=XK-d (∑p-0.5)
quorum XK——death rate 100％logarithmic
dose
d ——grouping interval
∑p——the sum of all groups
Corrected formula
（ P n<20% or P m>80％ ）
3  Pm  Pn
LogLD50= XK –d (∑p )
4
-1
LD50＝Log LogLD50
95％ confidence level
LD50±4.5S Log LD50 ×LD50
• S Log LD50 ＝d
• S Log LD50 ——the standard error of
Log LD50
• p
——the every group’s death rate
• n
——the animals number of every
group
Attention
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All of us should give drugs at the same
time.
Dipterex can volatilize easily, so we
should be attention to cover bottle at
any time, and don’t touch it with your
hand directly.
Use the right way of intraperitoneal
injection to the mice in order to avoid
being griped by the mice.
Questions
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How to group the mice and the
students?
What’s the symptom of the mice
when they are injected with
metrifonate?
What’s the meaning of the 95％
confidence level?
How to prepare the drugs?