Post on 17-Dec-2015
Prevention of Emergence of Resistance:
A Pharmacodynamic Solution
G.L. Drusano, M.D.
Professor and Director
Division of Clinical Pharmacology
Clinical Research Institute
Albany Medical College &
New York State Department of Health
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• Resistance to antimicrobial agents often occur as a function of single point mutations
• Other mechanisms include spread of plasmids with multiple resistance determinants
• Horizontal transmission also confuses the issue
• Examples of a point mutation providing drug resistance are stable derepression of AMP C beta lactamases for 3rd generation cephalosporins and target mutations or pump upregulation for fluoroquinolones
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• As these occur at a frequency of 1/108 or less frequently, infection site populations exceed this frequency, often by multiple logs
• Consequently, such total populations do not behave as a single, sensitive population, but as a mixture of two populations of differing drug susceptibility
• This raises an important question:
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
Can a drug exposure be identified that will prevent the resistant subpopulation from
taking over the total population?
The Team
N. L. Jumbe, A. Louie, W. Liu,V. Tam, T. Fazili, R. Leary, C. Lowry, M.H. Miller and
G. L. Drusano
S. pneumoniae outcome studies
P. aeruginosa outcome studies
Rf in vitro Rfin vivo MIC (g/mL) MBC (g/mL)
2.35x10-6 2.2x10-6 0.8 1.6
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• Clearly, Pseudomonas and Pneumococcus differ in their response
• Pneumococcus has no inoculum effect; Pseudomonas has a major inoculum effect
• The explanation probably rests in the mutational frequency to resistance
• Pseudomonas has a high frequency, while Pneumococcus has a frequency that is not measurable at the bacterial densities used in these experiments with this fluoroquinolone
Peripheral (thigh)Compartment (Cp)
Central Blood Compartment (Cc)
IPinjection
kcp kpc
+ Bacteria(XT/R)
f(c)
dCc= kaCa+kpcCp-kcpCc-keCc
dt
ke
dXS=KGS x XS x L - fKS(CcH ) x XS
dtdXR= KGR x XR x L- fKR(Cc
H ) x XR
dt
Kmax CcH
C H
50+CcH
f(CcH)=
Y1=XT=XS+XR
Y2=XR
[3]
[4]
[5]
[6]
[7]
, =K and = S,R
[1]
L = (1-(XS+XR)/POPMAX)
[8]
dCp = kcpCc - kpc Cp
dt
[2]
KmaxGS
0.117
KmaxGR
0.163
KmaxKS
94.01
KmaxKR
12.16
HKS
6.26
HKR
2.37
C50KS
123.5
C50KR
129.8
KmaxG -maximum growth rate (hr-1) in the presence of drug
KmaxK -maximum kill rate (hr-1)
C50K -drug concentration (g/mL) to decrease kill rate by half
HK -rate of concentration dependent kill
Popmax -maximal population size
Mean Parameter Estimates of the Model.
Popmax = 3.6 x 1010
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• All regimens were simultaneously fit in a large population model
• The displayed graph is the predicted-observed plot for the total population after the Maximum A-posteriori Probability (MAP) Bayesian step
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• All regimens were simultaneously fit in a large population model
• The displayed graph is the predicted-observed plot for the resistant population after the Maximum A-posteriori Probability (MAP) Bayesian step
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• In this experiment, a dose was selected to generate an exposure that would prevent emergence of resistance
• As this was at the limit of detection, the measured population sometimes had “less than assay detectable” for the colony count
• These were plotted at the detection limit
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• We were able to determine how the overall (sensitive plus resistant) population responds to pressure from this fluoroquinolone
• More importantly, we were able to model the resistant subpopulation and choose a dose based on simulation to suppress the resistant mutants
• The prospective validation demonstrated that the doses chosen to encourage and suppress the resistant mutants did, indeed, work
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• Now, for Pneumococcus
• We were unable to recover resistant mutants with levofloxacin as the selecting pressure in the mouse thigh model
• However, we then examined ciprofloxacin as the selecting agent
• Now, selecting mutants was straightforward
Study Design: Mouse Thigh Infection Model- Ciprofloxacin Studies [50mg/kg BID ~
AUC/MIC 100:1]
Begin therapy
Sacrifice, harvest,homogenize muscle
-2 hr 0 hr1. Microbial eradication
2. Selection of resistance
Infect
24 hr
BID
+ 2xMIC Cipro - Drug + 4xMIC Cipro + 3xMIC Levo
Drug #58 RC2
Cipro/±Reserpine 0.6/0.6 3.5/1.0
Levo/±Reserpine 0.6/0.6 0.6/0.6
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• Strain 58, the RC2 and RC4 mutants were sequenced through Gyr A, Gyr B, Par C & Par E.
• The entire open reading frames were sequenced.
• No differences were seen between parent and the RC2 daughter strain.
• This, coupled with the decrement in ciprofloxacin MIC with reserpine exposure (3.5 mg/L 1.0 mg/L), implies RC2 is a pump mutant.
• For RC4, a mutation was found in parC (aa 79, sertyr) and this strain also decreased its MIC with addition of reserpine.
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• We have examined other new fluoroquinolones in this system or in our hollow fiber pharmacodynamic system
• All resemble levofloxacin and do not allow emergence of resistance for wild type isolates
• Why is ciprofloxacin different?
• Likely because it is the most hydrophilic drug and is most efficiently pumped by the PMRA pump
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• Are there other factors that can alter the probability of emergence of resistance?
• The most likely is duration of therapy
• Fluoroquinolones induce an SOS response
• This resembles a “hypermutator phenotype”
• Therapy intensity and therapy duration should influence the probability of having the resistant population becoming ascendant
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• Hollow fiber System allows simulation of human PK in vitro
• Useful for dose ranging and schedule dependency determinations
• Allows examination of different classes (beta lactams, fluroquinolones, etc.)
The original hollow fiber system was used by Blaser & Zinner
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• A 10 day hollow fiber experiment was performed for MSSA and MRSA (CS) for 6 regimens
• The time to complete replacement of the population with resistant organisms was recorded
• CART was employed to look for a breakpoint in the exposure
• > 200/1 AUC/MIC ratio was identified
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• A stratified Kaplan-Meier analysis was performed with this breakpoint
• The breakpoint was significant (Mantel test p = 0.0007); Tarone-Ware and Breslow Gahan tests were also significant
• To prevent resistance, hit hard (> 200 AUC/MIC) and stop early (< 7 days)
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• The intensity of therapy and the duration of therapy have an impact upon the probability of emergence of resistance
• Short duration therapy trials should examine an endpoint of resistance frequency
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
Placebo
0
2
4
6
8
10
12
0 6 12 18 24 30 36 42 48Time (h)
Total
ToyamaresistantCiproresistant
Tam et al ICAAC 2001
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
Cipro (AUC/MIC 65.6)
0
1
2
3
4
5
6
7
8
9
10
0 6 12 18 24 30 36 42 48
Time (h)
Total
ToyamaresistantCiproresistant
Tam et al ICAAC 2001
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
T600 (AUC/MIC 3.2)
0
2
4
6
8
10
0 6 12 18 24 30 36 42 48Time (h)
Log1
0 CFU
/mL
Total
Toyamaresistant
Ciproresistant
Tam et al ICAAC 2001
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
T1800 (AUC/MIC 10.4)
0
1
2
3
4
5
6
7
8
9
10
0 6 12 18 24 30 36 42 48 Time (h)
Log1
0 CF
U/m
L
Total
ToyamaresistantCiproresistant
Tam et al ICAAC 2001
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
T13500 (AUC/MIC 88.6)
0
1
2
3
4
5
6
7
8
9
0 6 12 18 24 30 36 42 48
Time (h)
Total
ToyamaresistantCiproresistant
Tam et al ICAAC 2001
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
T18000 (AUC/MIC 108.3)
0
1
2
3
4
5
6
7
8
9
0 6 12 18 24 30 36 42 48
Time (h)
Total
ToyamaresistantCiproresistant
Tam et al ICAAC 2001
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
T36000 (AUC/MIC 200.8)
0
1
2
3
4
5
6
7
8
9
0 6 12 18 24 30 36 42 48
Time (h)
Total
ToyamaresistantCiproresistant
Tam et al ICAAC 2001
Central Compartment (Cc)Infusion + Bacteria
(XT/R)
f(c)
dCc=Infusion-(SCl/V)xCc
dt
SCl
dXS=KGS x XS x L - fKS(CcH ) x XS
dtdXR= KGR x XR x L- fKR(Cc
H ) x XR
dt
Kmax CcH
C H 50 +Cc
H
f(CcH)=
Y1=XT=XS+XR, IC(1)=2.4x108
Y2=XR , IC(2)= 30
[2]
[3]
[4]
[5]
[6]
, =K and = S,R
[1]
L = (1-X/POPMAX)
[7]
KmaxGS
0.745
KmaxGR
0.614
KmaxKS
27.85
KmaxKR
31.72
HKS
2.24
HKR
3.50
C50KS
16.94
C50KR
107.0
KmaxG -maximum growth rate (hr-1) in the presence of drug
KmaxK -maximum kill rate (hr-1)
C50K -drug concentration (g/mL) to decrease kill rate by half
HK -rate of concentration dependent kill
Popmax -maximal population size
Mean Parameter Estimates of the Bacterial Growth/Kill Model.
Popmax = 3.3 x 1010
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• All regimens were simultaneously fit in a large population model
• The displayed graph is the predicted-observed plot for the drug concentrations after the Maximum A-posteriori Probability (MAP) Bayesian step
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• All regimens were simultaneously fit in a large population model
• The displayed graph is the predicted-observed plot for the total bacterial counts after the Maximum A-posteriori Probability (MAP) Bayesian step
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• All regimens were simultaneously fit in a large population model
• The displayed graph is the predicted-observed plot for the resistant bacterial counts after the Maximum A-posteriori Probability (MAP) Bayesian step
Prevention of Emergence of Resistance:
A Pharmacodynamic Solution
•‘Inverted-U’ Phenomenon
– Resistant sub-populationis are initially amplified & then decline with increasing drug exposure 0
1
2
3
4
5
6
0 1 2 3 4 5 6 7
Therapeutic Intensity
Lo
g10
CF
U/m
L
ResistantSub-Population
Prevention of Emergence of Resistance:
A Pharmacodynamic Solution P. aeruginosa - Prevention of Amplification of
Resistant Subpopulation• The amplification of the
resistant sub-population is a function of the AUC/MIC ratio
• The response curve is an inverted “U”.
• The AUC/MIC ratio for resistant organism stasis is circa 187/1
Tam et al ICAAC 2001
Prevention of Emergence of Resistance:
A Pharmacodynamic Solution P. aeruginosa - Prevention of Amplification of
Resistant Subpopulation
Placebo
0
5
10
15
0 12 24 36 48 60 72 Time (h)
Lo
g10 C
FU
/mL
Total
Resistant
AUC/MIC 136.7
0
2
4
6
8
10
0 12 24 36 48 60 72 Time (h)
Lo
g10 C
FU
/mL
Total
Resistant
AUC/MIC 199.7
0
2
4
6
8
10
0 12 24 36 48 60 72 Time (h)
Lo
g10 C
FU
/mL
Total
Resistant
AUC/MIC 165.8
0
2
4
6
8
10
0 12 24 36 48 60 72 Time (h)
Lo
g10 C
FU
/mL
Total
Resistant
Prospective Validation
Prevention of Emergence of Resistance:
A Pharmacodynamic Solution • This was the same strain as employed in the mouse
model, but a different fluoroquinolone
• The mouse model contained granulocytes, while the hollow fiber system does not
• The total drug target for the mouse model was 157 which is a free drug target of 110
• The hollow fiber system target is 187 (1.7 fold )
• Craig found that targets increase by 1.5 -2.0 fold when granulocytes are removed
• These results are concordant with this finding
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
Multiple Bacterial Populations Do Make a Difference!
• In Vitro pharmacodynamic model investigations frequently only examine the total bacterial population
• The presence of a small pre-existent population more resistant to the selecting drug pressure has major implications, particularly as the bacterial population size increases to (near) clinical infection size
0
2
4
6
8
10
0 50 100 150 200
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
P aeruginosa
Lo
g10
CF
U/m
L
Daily AUC/MIC
Breakpoint = 187
0
2
4
6
8
10
0 50 100 150 200
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
K. pneumoniae
Lo
g10
CF
U/m
L
Daily AUC/MIC
Breakpoint = 93
0
1
2
3
4
5
6
0 20 40 60 80 100 120 140 160
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
MSSA
Lo
g10
CF
U/m
L
Daily AUC/MIC
Breakpoint = 66
0
2
4
6
8
0 20 40 60 80 100 120 140 160
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
MRSA-CS
Lo
g10
CF
U/m
L
Daily AUC/MIC
Breakpoint = 143
0
2
4
6
8
10
12
0 100 200 300 400 500
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
MRSA-CR
Lo
g10
CF
U/m
L
Daily AUC/MIC
Breakpoint = 484
Prevention of Emergence of Resistance: A Pharmacodynamic Solution
• Some drug exposures allow amplification of the resistant subpopulations
• Exposures can be identified that will prevent this amplification and, functionally suppress emergence of resistance