B6
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Experiment No. 3
LD 50 1
OBJECTIVES • To determine the LD50 of Lidocaine in mice To determine the toxic effects of Lidocaine
2
Lethal Dose • refers to the dose (dose/Kg of body weight) at which a given percentage of subjects will die • LD values for humans are generally estimated by extrapolating results from testing on animals or on human cell cultures • depends on the species of animal and the route of administration
3
LD 50 • commonly used indicator of lethality • the dose at which 50% of the subjects die • important in determining the therapeutic index and margin of safety of a given substance
4
Acut e Toxi city • describes the adverse effects of a substance which result either from a single exposure or from multiple exposures in a short space of time (usually less than 24 hours)
5
LI DOC AINE Drug Information
•common local anesthetic and anti-arrythmic drug •weak base with a pKa of 8-9 •exists in either the neutral or cationic form 6
PHA RM ACOKI NE TI CS • undergoes extensive 1st pass metabolism in the liver I.V. route • as a local anesthetic: injection into the dermis for faster absorption • distribution: the brain, liver, kidney, and heart (rapid); GI and Muscles (slow) • metabolized by CYT P450 in the liver 7
PHAR MACO DYN AM ICS • 1o MOA: blockade of Na+ channels In the peripheral nerves: prevents the generation of an AP In the heart (PF and Ventricular Cells): slows depolarization at phase 4 and enhances K+ efflux at phase 3 shorten the duration of the AP 8
9
Toxi city (C NS)
•
sleepiness/light- headedness
•
visual and auditory disturbances
•
restlessness
•
circumoral and tongue numbness and metallic taste
•
nystagmus and muscular twitching
•
overt- tonic clonic convulsions 10
Toxici ty (Cardi ovascul ar) •
effect is on cardiac and smooth muscle membranes and partly on ANS
•
can block Ca channels SYSTEMIC HYPOTENSION
11
Toxici ty
(Hem atol ogi c)
• accumulation of metabolite o-toluidine, an oxidizing agent capable of converting hemoglobin to methemoglobin • may appear cyanotic and the blood “chocolate colored”
12
MATERIALS An imals : • 80 Mice (same sex and about the same weight)
Materials: • • • •
animal weighing scale tuberculin syringe animal cage asbestos gloves 13
In tervention: • Lidocaine HCl • Preparations: 0.1%, 1%, 2%
(freshly prepared)
14
ME THODS 80 Mice
• pre-weighed
10 Mice
128 mg / 100g
10 Mice
64 mg / 100g
10 Mice
32 mg / 100g
10 Mice
16 mg / 100g
10 Mice
8 mg / 100g
10 Mice
4 mg / 100g
10 Mice
2 mg / 100g
10 Mice
1 mg / 100g
15
Determ in ation of t he d osage to be a dmin istere d 16 mg = 100g
X_ (dosage in mg to be administered) 20 g (assumed wt of the mouse)
X = 3.2 mg 3.2 mg =
1% = 10 mg 1 ml
X
10 mg 1 ml
X = 0.32 ml (for the 20 g mouse) 16
• Inject lidocaine intraperitoneally • Count the number of deaths 1 hour after injection • Compute for the percentage of deaths for each dose • Determine the LD-50 using the PROBIT METHOD
17
RE SU LT S Section
Dose
D6 D6 c/o Pharma C6 B6/C6 B6/A6 A6 c/o Pharma
1mg / 100g 2mg / 100g 4mg / 100g 8mg / 100g 16mg / 100g 32mg / 100g 64mg / 100g 128mg / 100g
Log Dose No. of mice No. of death Percentage Probit 0.00 0.30 0.60 0.90 1.20 1.51 1.81 2.11
10 10 10 10 10 10 10 10
0 0 1 3 8 10 10 10
2.50% 2.50% 10% 30% 80% 97.50% 97.50% 97.50%
3.035 3.035 3.72 4.48 5.84 6.97 6.97 6.97
A correction factor of 2.5% and 97.5% was used for results showing 0% and 100%, respectively.
18
PR OB IT • created by Chester Ittner Bliss • a statistical tool that converts the percentage killed into a “probability unit” • equal to 0 for 0.0001 and 10 for 0.9999
19
Pr ob it Me th od • convert percentage of death to probit ex. 32% mortality = 4.53 % 0 10 20 30
0 3.72 4.16 4.48
1 2.67 3.77 4.19 4.5
2 2.95 3.82 4.23 4.53
3 3.12 3.87 4.26 4.56
4 3.25 3.92 4.29 4.59
5 3.36 3.96 4.33 4.61
• take the log of the doses used 20
Section
Dose
D6 D6 c/o Pharma C6 B6/C6 B6/A6 A6 c/o Pharma
1mg / 100g 2mg / 100g 4mg / 100g 8mg / 100g 16mg / 100g 32mg / 100g 64mg / 100g 128mg / 100g
Log Dose No. of mice No. of death Percentage Probit 0.00 0.30 0.60 0.90 1.20 1.51 1.81 2.11
10 10 10 10 10 10 10 10
0 0 1 3 8 10 10 10
2.50% 2.50% 10% 30% 80% 97.50% 97.50% 97.50%
3.035 3.035 3.72 4.48 5.84 6.97 6.97 6.97
21
99 P 99 E R C 9090 E 80 80 N 70 T 70 6060 5050 4040 3030 20 20 1010
PROBIT
LD 50
0
.1 .2
.3 .4 .5 .6 .7.8 .9 1 1.5 2 LOG DOSE
3
4 5
6 7
22
COORDINATES: (log of the concentration used, probit)
PLOT THE COORDINATES LOG DOSE AT 50% = .86 ANTI-LOG= 7.24 mg/100g (LD50) Theoretical LD50 = 8 mg/100g
23
Lin ea r Re gr es sion
24
Lin ea r Re gr es sion slope, m= 57.32470697 y-int, b= -8.247072324 Using:
if x = 0; y = -8.247
y = mx + b if y = 0; x = 0.144 (not LD50 -for best fit line)
to compute LD50 : if y = 50%, compute for x which is your log dose
x = 1.016
log-1 x = 10.37 mg/100g (LD50)
25
Re sults • restless prior to convulsions (running around the cage) • overt tonic clonic convulsions
26
conc lus ion Value of LD50 using different methods: •
Actual LD50 : 8 mg / 100 g
•
Probit method : 7.24 mg / 100 g
•
Linear Regression : 10.37 mg / 100g
Toxic effects of Lidocaine: •
CNS
•
Cardiovascular
•
Hematologic
27
28
29
30
Number of deaths vs. Dose 12 Number of deaths
10 8 6 4 2 0 -2 0
20
40
60
80
100
120
140
Dose (mg/100ml)
31
Percentage of Death vs. Log of Dose
Percentage of Death
120.00% 100.00% 80.00% 60.00% 40.00% 20.00% 0.00% 0.00
0.50
1.00
1.50
2.00
2.50
Log of Dose
32
Probit vs. Log of Dose 8
Probit
6 4 2 0 0.00
0.50
1.00
1.50
2.00
2.50
Log of Dose
33
34
Other characte ris ti cs ✎ Use dependent- the more the channels are opened, the greater the block becomes ✎ Voltage and time dependent- higher affinity for activated and inactivated channels ✎ Usually in the form of salts ✔Buffering effect after administration ✔Repeated administration may lead to tachyphylaxis
35
Antiarrythmic churva • Class 2- sympatholytic; reduce B-adrenergic activity in the heart • Class 3- prolong APD; blocks potassium current • Class 4- blocks cardiac calcium current; slows conduction in SA and AV nodes ■
Class1 (A, B, C) ✦ 1A- prolong the action potential duration (APD) and dissociate from the channel with intermediate kinetics ✦ 1C- minimal effect on APD and dissociate from the channel with slow kinetics
36
37
FAST RESPONSE 1 0 4
SLOW RESPONSE
2 0
3
4
3
4 38
39
Acute toxicity describes the adverse effects of a substance which result either from a single exposure[1] or from multiple exposures in a short space of time (usually less than 24 hours).[2] To be described as acute toxicity, the adverse effects should occur within 14 days of the administration of the substance.[2] Acute toxicity is distinguished from chronic toxicity, which describes the adverse health effects from repeated exposures, often at lower levels, to a substance over a longer time period (months or years). It is obviously unethical to test for acute (or chronic) toxicity in humans. However, some information can be gained from investigating accidental human exposures (e.g. factory accidents). Otherwise, most acute toxicity data comes from animal testing or, more recently, in vitro testing methods and inference from data on similar substances.
40
Abstract Background and objective: Clinical studies suggest that lidocaine may induce irreversible neurological damage after spinal application in human beings. The mechanisms underlying the possible cytotoxic action of lidocaine have only been suggested from animal studies. This study aimed to investigate if lidocaine exhibited cytotoxic action in a human model widely used for the study of neuronal apoptosis. This is important to know as it may help one to judge on possible neurotoxic risks imposed by the spinal application of lidocaine. Methods: The concentration- and time-dependent effects of lidocaine on retinoic acid-differentiated human neuroblastoma SH-SY5Y cells were quantified by trypan blue staining, the release of lactate dehydrogenase, immunocytochemistry and flow cytometry. 41
Results: The local anaesthetic caused a significant increase in the number of cells staining positive for trypan blue, a significant increase of LDH release into the incubation medium, and a significant increase of 7AAD and annexin V binding. Lidocaine induced apoptosis already at 3 mm. At a concentration of 10 mmol 47% of the cells and at 30 mmol 98% of the cell population was necrotic. Both necrosis and apoptosis were time-dependent. Conclusions: The results demonstrate that lidocaine exhibited neurotoxic effects in a human model established for the study of drug-induced neuronal apoptosis. The results were consistent with the neurotoxic clinical effects of lidocaine. These effects may be produced by more than a single mechanism. (Accepted October 2001) 42
■ ■
■ ■ ■ ■ ■
Phase 0-depol, Slow response Sa nd av nodal tissue, low RMP, depndent on ECF ca Automaticity Fast response –purkinje STEEP O-NA spike, high rmp, Na dependent K AND CL CA AN2 P2 IN ISCHEMIA , RAPID NA IS INHIBITED 43
ED 50 ✎ Uses: ✔To compare potency of drugs ✔To obtain an index of selectivity of a drug’s action (via comparison) ✔To determine a drug’s therapeutic index
44
ED 50 ✎ Median Effective Dose ✎ The amount of drug that produces a desired effect on 50% of the population ✎ Usually the effect referred to is the beneficial effect but it can also stand for the harmful effect of the drug
45
LD 50 1
OBJECTIVES • To determine the LD50 of Lidocaine in mice To determine the toxic effects of Lidocaine
2
Lethal Dose • refers to the dose (dose/Kg of body weight) at which a given percentage of subjects will die • LD values for humans are generally estimated by extrapolating results from testing on animals or on human cell cultures • depends on the species of animal and the route of administration
3
LD 50 • commonly used indicator of lethality • the dose at which 50% of the subjects die • important in determining the therapeutic index and margin of safety of a given substance
4
Acut e Toxi city • describes the adverse effects of a substance which result either from a single exposure or from multiple exposures in a short space of time (usually less than 24 hours)
5
LI DOC AINE Drug Information
•common local anesthetic and anti-arrythmic drug •weak base with a pKa of 8-9 •exists in either the neutral or cationic form 6
PHA RM ACOKI NE TI CS • undergoes extensive 1st pass metabolism in the liver I.V. route • as a local anesthetic: injection into the dermis for faster absorption • distribution: the brain, liver, kidney, and heart (rapid); GI and Muscles (slow) • metabolized by CYT P450 in the liver 7
PHAR MACO DYN AM ICS • 1o MOA: blockade of Na+ channels In the peripheral nerves: prevents the generation of an AP In the heart (PF and Ventricular Cells): slows depolarization at phase 4 and enhances K+ efflux at phase 3 shorten the duration of the AP 8
9
Toxi city (C NS)
•
sleepiness/light- headedness
•
visual and auditory disturbances
•
restlessness
•
circumoral and tongue numbness and metallic taste
•
nystagmus and muscular twitching
•
overt- tonic clonic convulsions 10
Toxici ty (Cardi ovascul ar) •
effect is on cardiac and smooth muscle membranes and partly on ANS
•
can block Ca channels SYSTEMIC HYPOTENSION
11
Toxici ty
(Hem atol ogi c)
• accumulation of metabolite o-toluidine, an oxidizing agent capable of converting hemoglobin to methemoglobin • may appear cyanotic and the blood “chocolate colored”
12
MATERIALS An imals : • 80 Mice (same sex and about the same weight)
Materials: • • • •
animal weighing scale tuberculin syringe animal cage asbestos gloves 13
In tervention: • Lidocaine HCl • Preparations: 0.1%, 1%, 2%
(freshly prepared)
14
ME THODS 80 Mice
• pre-weighed
10 Mice
128 mg / 100g
10 Mice
64 mg / 100g
10 Mice
32 mg / 100g
10 Mice
16 mg / 100g
10 Mice
8 mg / 100g
10 Mice
4 mg / 100g
10 Mice
2 mg / 100g
10 Mice
1 mg / 100g
15
Determ in ation of t he d osage to be a dmin istere d 16 mg = 100g
X_ (dosage in mg to be administered) 20 g (assumed wt of the mouse)
X = 3.2 mg 3.2 mg =
1% = 10 mg 1 ml
X
10 mg 1 ml
X = 0.32 ml (for the 20 g mouse) 16
• Inject lidocaine intraperitoneally • Count the number of deaths 1 hour after injection • Compute for the percentage of deaths for each dose • Determine the LD-50 using the PROBIT METHOD
17
RE SU LT S Section
Dose
D6 D6 c/o Pharma C6 B6/C6 B6/A6 A6 c/o Pharma
1mg / 100g 2mg / 100g 4mg / 100g 8mg / 100g 16mg / 100g 32mg / 100g 64mg / 100g 128mg / 100g
Log Dose No. of mice No. of death Percentage Probit 0.00 0.30 0.60 0.90 1.20 1.51 1.81 2.11
10 10 10 10 10 10 10 10
0 0 1 3 8 10 10 10
2.50% 2.50% 10% 30% 80% 97.50% 97.50% 97.50%
3.035 3.035 3.72 4.48 5.84 6.97 6.97 6.97
A correction factor of 2.5% and 97.5% was used for results showing 0% and 100%, respectively.
18
PR OB IT • created by Chester Ittner Bliss • a statistical tool that converts the percentage killed into a “probability unit” • equal to 0 for 0.0001 and 10 for 0.9999
19
Pr ob it Me th od • convert percentage of death to probit ex. 32% mortality = 4.53 % 0 10 20 30
0 3.72 4.16 4.48
1 2.67 3.77 4.19 4.5
2 2.95 3.82 4.23 4.53
3 3.12 3.87 4.26 4.56
4 3.25 3.92 4.29 4.59
5 3.36 3.96 4.33 4.61
• take the log of the doses used 20
Section
Dose
D6 D6 c/o Pharma C6 B6/C6 B6/A6 A6 c/o Pharma
1mg / 100g 2mg / 100g 4mg / 100g 8mg / 100g 16mg / 100g 32mg / 100g 64mg / 100g 128mg / 100g
Log Dose No. of mice No. of death Percentage Probit 0.00 0.30 0.60 0.90 1.20 1.51 1.81 2.11
10 10 10 10 10 10 10 10
0 0 1 3 8 10 10 10
2.50% 2.50% 10% 30% 80% 97.50% 97.50% 97.50%
3.035 3.035 3.72 4.48 5.84 6.97 6.97 6.97
21
99 P 99 E R C 9090 E 80 80 N 70 T 70 6060 5050 4040 3030 20 20 1010
PROBIT
LD 50
0
.1 .2
.3 .4 .5 .6 .7.8 .9 1 1.5 2 LOG DOSE
3
4 5
6 7
22
COORDINATES: (log of the concentration used, probit)
PLOT THE COORDINATES LOG DOSE AT 50% = .86 ANTI-LOG= 7.24 mg/100g (LD50) Theoretical LD50 = 8 mg/100g
23
Lin ea r Re gr es sion
24
Lin ea r Re gr es sion slope, m= 57.32470697 y-int, b= -8.247072324 Using:
if x = 0; y = -8.247
y = mx + b if y = 0; x = 0.144 (not LD50 -for best fit line)
to compute LD50 : if y = 50%, compute for x which is your log dose
x = 1.016
log-1 x = 10.37 mg/100g (LD50)
25
Re sults • restless prior to convulsions (running around the cage) • overt tonic clonic convulsions
26
conc lus ion Value of LD50 using different methods: •
Actual LD50 : 8 mg / 100 g
•
Probit method : 7.24 mg / 100 g
•
Linear Regression : 10.37 mg / 100g
Toxic effects of Lidocaine: •
CNS
•
Cardiovascular
•
Hematologic
27
28
29
30
Number of deaths vs. Dose 12 Number of deaths
10 8 6 4 2 0 -2 0
20
40
60
80
100
120
140
Dose (mg/100ml)
31
Percentage of Death vs. Log of Dose
Percentage of Death
120.00% 100.00% 80.00% 60.00% 40.00% 20.00% 0.00% 0.00
0.50
1.00
1.50
2.00
2.50
Log of Dose
32
Probit vs. Log of Dose 8
Probit
6 4 2 0 0.00
0.50
1.00
1.50
2.00
2.50
Log of Dose
33
34
Other characte ris ti cs ✎ Use dependent- the more the channels are opened, the greater the block becomes ✎ Voltage and time dependent- higher affinity for activated and inactivated channels ✎ Usually in the form of salts ✔Buffering effect after administration ✔Repeated administration may lead to tachyphylaxis
35
Antiarrythmic churva • Class 2- sympatholytic; reduce B-adrenergic activity in the heart • Class 3- prolong APD; blocks potassium current • Class 4- blocks cardiac calcium current; slows conduction in SA and AV nodes ■
Class1 (A, B, C) ✦ 1A- prolong the action potential duration (APD) and dissociate from the channel with intermediate kinetics ✦ 1C- minimal effect on APD and dissociate from the channel with slow kinetics
36
37
FAST RESPONSE 1 0 4
SLOW RESPONSE
2 0
3
4
3
4 38
39
Acute toxicity describes the adverse effects of a substance which result either from a single exposure[1] or from multiple exposures in a short space of time (usually less than 24 hours).[2] To be described as acute toxicity, the adverse effects should occur within 14 days of the administration of the substance.[2] Acute toxicity is distinguished from chronic toxicity, which describes the adverse health effects from repeated exposures, often at lower levels, to a substance over a longer time period (months or years). It is obviously unethical to test for acute (or chronic) toxicity in humans. However, some information can be gained from investigating accidental human exposures (e.g. factory accidents). Otherwise, most acute toxicity data comes from animal testing or, more recently, in vitro testing methods and inference from data on similar substances.
40
Abstract Background and objective: Clinical studies suggest that lidocaine may induce irreversible neurological damage after spinal application in human beings. The mechanisms underlying the possible cytotoxic action of lidocaine have only been suggested from animal studies. This study aimed to investigate if lidocaine exhibited cytotoxic action in a human model widely used for the study of neuronal apoptosis. This is important to know as it may help one to judge on possible neurotoxic risks imposed by the spinal application of lidocaine. Methods: The concentration- and time-dependent effects of lidocaine on retinoic acid-differentiated human neuroblastoma SH-SY5Y cells were quantified by trypan blue staining, the release of lactate dehydrogenase, immunocytochemistry and flow cytometry. 41
Results: The local anaesthetic caused a significant increase in the number of cells staining positive for trypan blue, a significant increase of LDH release into the incubation medium, and a significant increase of 7AAD and annexin V binding. Lidocaine induced apoptosis already at 3 mm. At a concentration of 10 mmol 47% of the cells and at 30 mmol 98% of the cell population was necrotic. Both necrosis and apoptosis were time-dependent. Conclusions: The results demonstrate that lidocaine exhibited neurotoxic effects in a human model established for the study of drug-induced neuronal apoptosis. The results were consistent with the neurotoxic clinical effects of lidocaine. These effects may be produced by more than a single mechanism. (Accepted October 2001) 42
■ ■
■ ■ ■ ■ ■
Phase 0-depol, Slow response Sa nd av nodal tissue, low RMP, depndent on ECF ca Automaticity Fast response –purkinje STEEP O-NA spike, high rmp, Na dependent K AND CL CA AN2 P2 IN ISCHEMIA , RAPID NA IS INHIBITED 43
ED 50 ✎ Uses: ✔To compare potency of drugs ✔To obtain an index of selectivity of a drug’s action (via comparison) ✔To determine a drug’s therapeutic index
44
ED 50 ✎ Median Effective Dose ✎ The amount of drug that produces a desired effect on 50% of the population ✎ Usually the effect referred to is the beneficial effect but it can also stand for the harmful effect of the drug
45
