: THE MEASUREMENT OF LD50 Alok kr vishwakarma Mahendra singh Bipin katiyar Amit verma M.pharm(pharmaceutics) Psit,kanpur Definition : 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: LD 50 : Median lethal dose LD 50 (median lethal dose): The dose of a drug that produces death in 50% of the animal population tested Drug variability and toxicity assessment: Drug variability and toxicity assessment ED 50 : Effective dose for 50% of subjects LD 50 : Lethal dose for 50% of subjects The therapeutic index TI = LD 50 / ED 50 No drug is 100% safe Definition: 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 : Definition Toxicity test can be divided into three parts: acute toxicity testing, chronic toxicity testing, and special testing . Definition : 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 : Experimental purpose 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 : 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 : Experimental condition 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 : Experimental animals mice Experimental drugs : Experimental drugs the different concentration of Dipterex; 2, 4,6-trinitrophenol : Dipterex It can irreversibly inhibits AChE Slide 17: 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 : Experimental apparatus mice cage, animal’s equi-armbalance, 1ml injection syring, and electronic calculator Experimental procedure: Experimental procedure How to choose and divide the experimental animals:: How to choose and divide the experimental animals: 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 : Group 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 : 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 : 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: : Calculation formula should be applied by these conditions: 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 : Basic formula （ P n = 0% and P m = 100 ％ ） LogLD50=X K -d (∑p-0.5) quorum X K ——death rate 100 ％ logarithmic dose d ——grouping interval ∑p——the sum of all groups Corrected formula : Corrected formula （ P n<20% or P m>80 ％ ） LogLD50= X K –d (∑p - ) LD50 ＝ Log -1 LogLD50 95％ confidence level LD50±4.5S Log LD50 ×LD50: 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 : Attention 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 : Questions 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?