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Animal Models for PK/PD
Studies
William A. Craig, M.D.
University of Wisconsin
USA
Animal Versus In-Vitro Models
 Infection of specific body sites
 Interaction of multiple host factors
 Growth environment less favorable than
broth
 Faster drug elimination than in humans, but
can still simulate human pharmacokinetics
by inducing renal impairment or computercontrolled drug administration
Use of Animal Models in PK/PD Evaluation
of Anti-Infective Agents
• Describing the time-course of antimicrobial
activity at sites of infection
- pattern of killing (concentration or timedependent)
- presence or absence of persistent effects
Neutropenic Mouse Thigh-Infection Model
1. Neutropenia induced by 2
injections of cyclophosphamide
on days -4 and -1
4. Thighs removed,
homogenized, serially
diluted and plated for
CFU determinations
2. Bacteria injected into
thighs on day 0 (106-7)
3. Treatment (usually given SQ)
started 2 hr after infection and
continued for 1-5 days
Time Course of Antibacterial Activity of
Tobramycin and Ticarcillin Against
Pseudomonas aeruginosa
Log CFU per Thigh
8.0
7.5
Ticarcillin
Tobramycin
7.0
6.5
6.0
5.5
5.0
4.5
0
2
4
6
0
2
4
Time (hours)
4 mg/kg
12 mg/kg
20 mg/kg
Craig and Gudmundsson 1996
300 mg/kg
800 mg/kg
2400 mg/kg
Effect of Increasing Concentrations
on Killing of Pneumococci
in Thighs of Neutropenic Mice
Telithromycin
Log10 CFU per Thigh
8
Azithromycin
Clarithromycin
6
4
40 mg/kg
10 mg/kg
2.5 mg/kg
18.8 mg/kg
4.7 mg/kg
1.17 mg/kg
2
0
2
4
CFU = colony-forming unit.
6
0
2
4
40 mg/kg
10 mg/kg
2.5 mg/kg
6
0
2
4
6
Time (hours)
Craig WA, et al. 40th ICAAC Toronto, Ontario, September 17-20, 2000.
Use of Animal and In Vitro Models in PK/PD
Evaluation of Anti-Infective Agents
• Describing the time-course of antimicrobial
activity at sites of infection
• Identifying PK/PD indices correlating with
efficacy (Peak/MIC, AUC/MIC, Time>MIC)
- dose-fractionation studies to reduce
inter-dependence among the
various indices
Log10 CFU/Thigh at 24 Hrs
Relationship Between PK/PD Indices and
Efficacy for Ceftazidime against Klebsiella
pneumoniae in a Murine Pneumonia Model
10
9
8
7
6
5
4
3
2
10
100
1000
24-Hr AUC/MIC
1
10
100 1000
Peak/MIC
0
25
50
75
100
Time Above MIC
Log10 CFU/Thigh at 24 Hrs
Correlation of PK/PD Indices with Efficacy of
Levofloxacin against Streptococcus
pneumoniae in Thighs of Neutropenic Mice
10
8
6
4
2
0
10
100
1000
24-Hr AUC/MIC
1
10
100
Peak/MIC
1000 0
25
50
75
100
Time Above MIC
Use of Animal and In Vitro Models in PK/PD
Evaluation of Anti-Infective Agents
• Describing the time-course of antimicrobial
activity at sites of infection
• Identifying PK/PD indices correlating with
efficacy (Peak/MIC, AUC/MIC, Time>MIC)
• Determining magnitudes of the PK/PD indice
required for efficacy and identifying factors
that affect the magnitude
- cfu changes (short durations of therapy)
vs survival (longer courses)
- multiple factors can alter magnitudes
- predicting efficacy in humans
PK/PD Magnitude Variables
•
•
•
•
•
•
•
•
•
•
•
Animal
Antibiotic Class
Protein binding
Organism/strain
Presence of resistance mechanism(s)
Immune status (normal vs neutropenic)
Infection site
Location of organisms (intracellular vs extracellular)
Kinetics and shape of concentration-time curve
Duration of therapy
Time survival is determined
Half-Lives in Mice and Humans
Drug
Penicillin G
Imipenem
Cefazolin
Amikacin
Ciprofloxacin
Erythromycin
Minocycline
Half-life in Minutes
Mice
Humans
5
30
8
60
15
108
17
150
32
240
35
180
120
1080
Efficacy of Once-Daily Dosing of Amikacin
against K. pneumoniae (MIC=0.5 mg/L) in
Neutropenic Mice with Murine and Human
Pharmacokinetics
Dose (mg/kg)
Peak (mg/L)
T1/2 (min)
AUC
T>MIC (hr)
PAE (hr)
Efficacy
Murine
15
16
17
14
1.9
4.1
NO
Human
15
46
104
128
11.7
12.3
YES
Static Dose (mg/kg/24 hrs)
Impact of Dosing Interval on Static Dose
for Amikacin against K. pneumoniae and E. coli
in Mice with Normal and Impaired Renal Function
1000
K. pneumoniae
E. coli
Normal
T1/2=17 min
100
K. pneumoniae
E. coli
Renal Impaired
T1/2=104 min
10
1
3
6
12
24
Dosing Interval (Hours)
Reddington and Craig, JAC 1995
24-Hr AUC/MIC with Total and Free Drug for
the Static Dose of Different Fluoroquinolones
with S. pneumoniae ATCC 10813
24-Hr AUC/MIC
160
120
Total
Free
80
40
0
Gati
Sita
Moxi Gemi Garen Levo Cipro
Andes & Craig 40th and 41st ICAAC, 2000 and 2001
Time Above MIC for Total and Free Drug for
the Static Dose of Different Cephalosporins
with Klebsiella pneumoniae ATCC 43816
70
60
50
40
Total
Free
30
20
10
0
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C
C
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Craig Infect Dis Clin N Amer 2003
Time Above MIC Required for a Static
Effect with 4 Cephalosporins
Time Above MIC (% of Dosing Interval)
Drug
GNB
S. pneumoniae
S.aureus
Ceftazidime
36 (27-42)
39 (35-42)
22 (19-24)
Cefpirome
35 (29-40)
37 (33-39)
22 (20-25)
Cefotaxime
38 (36-40)
38 (36-40)
24 (20-28)
Ceftriaxone
38 (34-42)
39 (37-41)
24 (21-27)
Craig Diagn Microbiol Infect Dis 22:89, 1995
T>MIC for Cephalosporins, Penicillins and
Carbapenems with Susceptible and NonSusceptible Strains of S. pneumoniae
T>MIC (%)
50
40
30
Cephalosporins
Penicillins
Carbapenems
20
10
0
0.008 0.03
0.12
0.5
2
MIC (mg/L)
8
Magnitude of PK/PD Indice for Free Drug
Required for Static Dose of Gemifloxacin
Against S. pneumoniae
in Thighs of Neutropenic Mice
Drug
Susceptible
Gyrase, PAR C or E
Efflux
MIC
Mean AUC/MIC
0.015
28.3
0.03-0.5
31.3
0.12-0.25
5.8
Craig & Andes 2005 ECCMID
Change in Log10 CFU/Thigh
over 24 Hours
In Vivo Activity of Moxifloxacin
against S. pneumoniae and K. pneumoniae
in Thighs of Normal and Neutropenic Mice
3
S. pneumoniae
2
K. pneumoniae
1
0
-1
-2
-3
-4
-5
1.17
4.69
18.8
75
0.58 2.34 9.38 37.5 150
Dose (mg/kg/12 hrs)
600
Log10 CFU/Thigh or Lung
after 24 Hrs of Therapy
Activity of Vancomycin against
S. pneumoniae in Lungs and Thighs of
Neutropenic Mice
2
0
-2
Thigh
Lung
Starting CFU
-4
0.38
0.75
1.5
3
6
Dose (mg/kg/12 hrs)
12
Survival/Mortality Studies
• Mortality in control animals 80-100% by
end of therapy
• Animals treated for at least 48 hrs
• Mortality assessed within 24 hrs of end
of therapy
• Pharmacokinetics included so PK/PD
parameters could be estimated
T>MIC for ß-Lactams Versus Mortality
in Animal Models: Literature Review
Mortality (%)
100
Cephalosporins
Penicillins
80
60
40
20
0
0
20
40
60
80
100
Time Above MIC (% of Interval)
Craig Antimicrobial Pharmacodynamics in Theory and Practive 2002
Bacterial Eradication (%)
Relationship of T>MIC with Bacterial
Eradication in Acute Otitis Media
100
80
60
40
PSSP
PISP-PRSP
H. flu
Best Fit
20
0
0
20
40
60
80
Time Above MIC (%)
Craig Infect Dis Clin N Amer 2003
100
Relationships Between Mortality and 24-Hr
AUC/MIC for Fluoroquinolones in Animal Models
Mortality (%)
100
80
60
40
20
0
2.5
10
25
100 250
24-Hr AUC/MIC
Andes, Craig Int J Antimicrob Agents, 2002
1000
Intracellular Concentrations of
Fluoroquinolones vs Intracellular Pathogens
• Tissue homogenate concentrations from
multiple tissues are 2 to 8-fold higher than
plasma levels
• A variety of efficacy studies using survival as
the end point have been conducted with
fluoroquinolnes against both extracellular
and intracellular pathogens.
• The primary intracellular pathogens studied
were M. tuberculosis, C. psittaci, L.
pneumophila, and L. moncytogenes.
Relationship Between 24 Hr AUC/MIC and Mortality
for Intracellular and Extracellular Pathogens in Various
Infection Models for Fluoroquinolones
100
Percent Mortality
80
60
Intracellular
Extracellular
40
20
0
3
10
30
100
24 Hr AUC/MIC
300
1000
Use of Animal and In Vitro Models in PK/PD
Evaluation of Anti-Infective Agents
• Describing the time-course of antimicrobial
activity at sites of infection
• Identifying PK/PD indices correlating with
efficacy (Peak/MIC, AUC/MIC, Time>MIC)
• Determining magnitudes of the PK/PD indice
required for efficacy and identifying factors
that affect the magnitude
• Identifying magnitudes of the PK/PD indice
promoting and suppressing the emergence
of resistance
Relationship of AUC/MIC to Enhancement
and Suppression of Resistant P. aeruginosa
in a Murine Thigh Infection
157/1
52/1
J Clin Invest 2003;112:275-285 &
Nature Rev Microbiology 2004;2:289-300
Conclusions
• Animal and in vitro models have been very useful
for determining the PK/PD target for efficacy
(PK/PD indice and appropriate magnitude required
for bacteriologic cure and survival
• Additional studies are identifying the PK/PD targets
that enhance and suppress the emergence of
resistance
• Despite the variety of techniques and models, there
is marked consistency in the PK/PD data from
these in these animal model studies