藥理實(shí)驗(yàn)報(bào)告1

LD50determinationofProcaine

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（ChinaPharmaceuticalUniversity，Nanjing210009）

AbstractThehalflethaldoseisoneoftheindacaatortoevaluatethedegreeofacutetoxicityofdrug.InthisexperimentwedeterminedtheLD50ofProcaine,wecalculateditbyimprovedKurbermethodandBlissmethod.TheresultsoftheexperimentshowsthattheofProcaineLD50=156.9mg/kg,andthe95%confidenceintervalofLD50is139.4mg/kg~176.6mg/kg.Therefore,Procaineisamedium-toxicity(3-degree)drug.

KeywordsProcaine,LD50,Kurbermethod,Blissmethod

1.IntroductionThelethaldoseisusuallyusedtoevaluatethedegreeofacutetoxicityofthedrug.Halflethaldoseisoneoftheindicatorsbecauseitisthehighestsensitivitypointonthedose-reactioncurve.Acutetoxicity(lethaldose,LD50)canbeviewedas‘cumulativepotential’tocausevariousacuteeffectsanddeathinanimals.Complexendpointssuchasacutetoxicityareamenabletopredictiononlytoalimitedextent,becauseitdependsonamultitudeofvariablesthatareinfluencedbyabroadvarietyofmechanisms.Procaineisalocalanesthetic.Localanesthetics,suchasprocaine,lidocaine,andcocaine,havebeendemonstratedtoprotectneuronaltissueagainsthypoxicdamage.[1]Procainehasbeenusedformorethan90yearsathomeandabroad.[2]ThedeterminationofLD50isarecurrentpharmacology,toxicologyimportantwork.ThecalculationmethodisgenerallybelievedtoBliss'searlyproposedalgorithmarethemostpreciseandaccurate.Butbecauseofitscomplicated,time-consumingmanualcalculationmethod,usingacomputerprogramforthepromotion.[3]

2MaterialandMethods

2.1Material

Animalrequired:44NIHmice,maleandfemale(halfandhalf),weighing14–26g,wereusedthroughoutthedurationofthestudies.Theroomtemperaturewascontrolledbetween18and22°C.

Equipmentrequired:mousecage,balance,andsyringe

Solutionrequired:1%Procaine(BatchNumber:FF120901;10ml×5Ampoules;ShanghaiXudonghaipuCo.;10ml:0.1g)，carbazoticacidformarkingthemouse.

2.2Methods

①.Separatethemalefromthefemale,andweighthemrespectively.Putthemiceofthesameweightclass(14.0~15.9g)intothesamemousecage.

②.Markthesamesignalonallthemiceinthesamecage.

③.Randomly,dividethemiceinto5groupsof10miceeachwithsimilarsexandweightintheturnsoftheweightclass(frombigtosmallorininverse).

④.Injectintraperitoneally1%procainetothemiceineachgroupwiththedoseof130mg/kg,153mg/kg,180mg/kg,212mg/kg,249mg/kgrespectivelyusing1:0.85astheratiobetweenadjacentgroups.Numberthedeadineachgroupwithin30min.

⑤.CalculateLD50and95%confidencelimitbyimprovedandBlissmethod.

3.ResultsandDiscussion

Ourgrouptookthepartthatwiththedoseof249mg/kginthewholeexperiment.Thephenomenonisasbelow:

Mousenumber

Mouseweight

（g）

Mouse

gender

（♂&♀）

Timeofdruggiving

（min：s）

Mousesymptomafterdruggiving

Deadtime

（min：s）

Timeofdying

（min：s）

1

25

♂

10：03

Hurrybreathing→Randomwalking→Drifting→Convulsions→Death

13：55

03：52

2

22

♂

08：20

Rigid→Shortnessofbreath→Convulsions→Jump→Hindlimbconvulsions→Death

11：19

02：59

3

17

♀

04：20

Randomwalking→Shortnessofbreath→Convulsions→Death

06：39

02：19

4

18

♂

01：18

Bradykinesia→Shortnessofbreath→Tailturnred→Convulsions→Death

06：00

04：42

5

21

♀

03：08

Shortnessofbreath→Convulsions→Tailturnred→Death

04：55

01：47

6

15

♂

07：18

→Bradykinesia→Shortnessofbreath→Thetwitching→→Convulsions→Hindlimbconvulsions→Tailturnred→Death

10：39

03：21

7

15

♂

14：59

Randomwalk→Shortnessofbreath→Tailturnred→Bradykinesia→Convulsions→Drifting→Jumpseveraltimes→Hindlimbconvulsions→Death

22：54

07：53

8

16

♀

11：14

Bradykinesia→Writhing→Convulsions→Shortnessofbreath→Death

14：06

02：52

9

16

♀

00：00

Shortnessofbreath→Drifting→Bradykinesia→Convulsions→Death

01：30

01：30

3.1ImprovedKurbermethodbySun

TocalculatetheLD50ofProcainebyImprovedKurbermethodweneedthedataincludemicedeathnumber,dosage,micenumberineachgroup.AndthenwecangetthedeathrateP,andLD50withits95%confidenceinterval.

Table1.TheexperimentalresultsofProcaineintraperitonealinjectioninmice

Group

numberofrat

dose(mg/kg)

Logarithmdose

NumberofDeath

Deathrates(P)

P2

1

2

3

4

5

9

9

8

9

9

249

212

180

153

130

2.40

2.33

2.26

2.18

2.11

9

8

6

3

2

1

0.889

0.750

0.333

0.222

1

0.79

0.56

0.11

0.04

P—deathrateineachgroup(expressedwithdecimal)

P2—thesquareofP

Table2.ProcessofProcaineintraperitonealinjectioninmiceresults

∑P

ΣP2

Pm

Pn

Xm

i

3.184

2.513

1

0.222

2.40

0.07

∑p—thetotaladditionofdeathrateineverygroup(p1+p2+p3…..)

∑p2—thesquareofthetotaladditionofdeathrateineverygroup(p12+p22+p32+……)

Pm—thedeathrateingroupwithmaximumdose

Pn—thedeathrateingroupwithminimumdose

Xm—thelogarithmofhighestdose

i—theminusoflogarithmdosesinnearinggroups

LD50=lg-1[Xm-i(Σp-(3-Pm-Pn)/4)]=159.78mg/kg

SlgLD50=i[(ΣP-ΣP2)/(n-1)]?=0.0204

95%confidenceintervalofLD50:lg-1(2.20±0.040)=145.73mg/kg~175.19mg/kg

3.2Blissmethod

UsingLD50CALCversion2.0softwaretoprocesstheexperimentaldataoftheBlissmethod,processingresultsareasfollows:

LD50=156.9mg/kg

95%confidenceintervalofLD50:139.4mg/kg~176.6mg/kg

3.3Discussion

DeterminationofthetoxicityofthedrugsuseddatamayalsoincludeLD90[4],ED50,LD1,LD10,LD5,ED95,ED99andsoon,totheoverallanalysisofdrugtoxicity,toguidethedosage,andthesecurityassessmentofdrug.Becausetheresourcesofmiceislimited,thisexperimentdoesnotmeettherequirementsof10miceineachgroup,themiddledosegroupofmiceisonly8,theremainingdosegroupmicefor9.Thisdefectwillgiveourexperimentresult.

Forthejudgmentofmicetodeath,therespirationandheartbeat,shouldbecarefullyobserved,therearemiceintermittenttremorisnotdeath.Asthehighdosegroupoftheexperiment,miceshowedsymptomsappearsoonaftergavedrug,mainlyforthefirstlyexcited,andthenactivityweakened,rathairerect,hindlegspedaling.Neardeath,miceasweakness,trembling,vitalsignsgraduallydisappear.

ThecurveofDeathRate-Doseistransformedtoanothercurvewithfeaturesofthesymmetryoftheleftandrightandthepointofsymmetryin50%deathratebyusinglogarithmtransformeddoseasabscissa.Atthepoint,theslopeismaximumandthechangeisobvious.Thatistosaythatthereactionoftheanimaltothedrugdoseismostsensitive,anddoseisthemostaccurateanderrorisminimum.SowechooseLD50toevaluatethedegreeofacutetoxicityofthedrug.

Thedefectsintheexperiment:

1、Theweightdifference,14~26g.

2、Theamountofmiceisnotenough,thisexperimentcancostalotofanimals.

3,Thecleangradecannotreachthestandardofmice(SPF).

4,Thetemperatureofenvironmental,humidityandotherfactorsdidnotgetundercontrol.

5,Manualoperationisnotstandard,suchasintraperitonealinjectionoperationmaybenotstandard,thiscaninfluencetheexperimentresult.

4.Conclusion

TheresultsshowedthattheLD50and95%confidenceintervalofLD50arealittledifferentbydifferentmethod.AsknownthatBlissmethodisthemostrigorousmethodmathematicallyamongmanycalculationmethodsofLD50andisrecommendedinthemethodofexaminationandapprovalofnewdrugs,wetaketheresultofBlissmethod.AsLD50=