# Presentation: 316973

## 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

*S. pneumoniae* outcome studies

## P. aeruginosa outcome studies

*P. aeruginosa* outcome studies

**R****f *****in vitro*****  ****	****R****f*****in 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

## Slide 10

## +

**Compartment (C****p****)**

**Central Blood **
**Compartment (C****c****)**

**IP**
**injection**

**k****cp**

**k****pc**

**+**

**Bacteria**
**(X****T/R****)**

**f(c)**

_**dC**_**c****= k****a****C****a****+k****pc****Cp-k****cp****C****c****-k****e****C****c**
** ****dt**

**k****e**

_**dX**_**S****=K****GS ****x**** ****X****S  ****x**** L - f****KS****(C****c****H**** ********) ****x**** X****S**
** ****dt**

_**dX**_**R****= K****GR ****x**** ****X****R ****x ****L- f****KR****(C****c****H**** ********) ****x ****X****R**
** ****dt**

**K****max**** ******** ********C****c****H**** ******
**          **
** ****C ****H ********50********+C****c****H ******

**f************(C****c****H********)=**

**Y****1****=X****T****=X****S****+X****R**
**Y****2****=X****R**

**[3]**

**[4]**

**[5]**

**[6]**

**[7]**

- , ******=K and** ****** = S,R**

**[1]**

**L = (1-(X****S****+****X****R****)/POPMAX)**

**[8]**

_**dC**_**p ****= k****cp****C****c**** - k****pc**** C****p**
**dt**

**[2]**

## Mean Parameter Estimates of the Model. 

**0.117**
**K****maxGR**
**0.163**

**K****maxKS**
**94.01**
**K****maxKR**
**12.16**

**H****KS**
**6.26**
**H****KR**
**2.37**

**C****50KS**
**123.5**
**C****50KR**
**129.8**

**K****maxG**	  -maximum growth rate (hr-1) in the presence of drug
**K****maxK****	****   **-maximum kill rate (hr-1)
**C****50K****	****   **-drug concentration (g/mL) to decrease kill rate by half
**H****K****	****   **-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

## Slide 16

## 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 hr**

**1. Microbial eradication**

**2. Selection of resistance**

**Infect**

**24 hr**

**BID**

**+ 2xMIC Cipro**

**+ 4xMIC Cipro**

**+ 3xMIC Levo**

## Prevention of Emergence of Resistance: A Pharmacodynamic Solution

**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, sertyr) 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

- Tam et al ICAAC 2001

## Prevention of Emergence of Resistance: A Pharmacodynamic Solution

- Tam et al ICAAC 2001

## Prevention of Emergence of Resistance: A Pharmacodynamic Solution

- Tam et al ICAAC 2001

## Prevention of Emergence of Resistance: A Pharmacodynamic Solution

- Tam et al ICAAC 2001

## Prevention of Emergence of Resistance: A Pharmacodynamic Solution

- Tam et al ICAAC 2001

## Prevention of Emergence of Resistance: A Pharmacodynamic Solution

- Tam et al ICAAC 2001

## Prevention of Emergence of Resistance: A Pharmacodynamic Solution

- Tam et al ICAAC 2001

## +

**Compartment (C****c****)**

**Infusion**

**+**

**Bacteria**
**(X****T/R****)**

**f(c)**

_**dC**_**c****=Infusion-(SCl/V)xC****c**
** ****dt**

**SCl**

_**dX**_**S****=K****GS ****x**** ****X****S  ****x**** L - f****KS****(C****c****H**** ********) ****x**** X****S**
** ****dt**

_**dX**_**R****= K****GR ****x**** ****X****R ****x ****L- f****KR****(C****c****H**** ********) ****x ****X****R**
** ****dt**

**K****max**** ******** ********C****c****H**** ******
**          **
** ****C ****H ******** ****50**** ********+C****c****H ******

**f************(C****c****H********)=**

**Y****1****=X****T****=X****S****+X****R****, IC(1)=2.4x10****8**
**Y****2****=X****R ****, IC(2)= 30**

**[2]**

**[3]**

**[4]**

**[5]**

**[6]**

- , ******=K and** ****** = S,R**

**[1]**

**L = (1-X********/POPMAX)**

**[7]**

## Mean Parameter Estimates of the Bacterial Growth/Kill Model. 

**0.745**
**K****maxGR**
**0.614**

**K****maxKS**
**27.85**
**K****maxKR**
**31.72**

**H****KS**
**2.24**
**H****KR**
**3.50**

**C****50KS**
**16.94**
**C****50KR**
**107.0**

**K****maxG**	  -maximum growth rate (hr-1) in the presence of drug
**K****maxK****	****   **-maximum kill rate (hr-1)
**C****50K****	****   **-drug concentration (g/mL) to decrease kill rate by half
**H****K****	****   **-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

***Therapeutic Intensity***

***Log10 CFU/mL***

***Resistant******Sub-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

- 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

## Prevention of Emergence of Resistance: A Pharmacodynamic SolutionP aeruginosa

- Prevention of Emergence of Resistance: A Pharmacodynamic Solution*P aeruginosa*

***Log10 CFU/mL***

***Daily AUC/MIC***

**Breakpoint = 187**

## Prevention of Emergence of Resistance: A Pharmacodynamic Solution K. pneumoniae

- Prevention of Emergence of Resistance: A Pharmacodynamic Solution* **K. pneumoniae*

***Log10 CFU/mL***

***Daily AUC/MIC***

**Breakpoint = 93**

## Prevention of Emergence of Resistance: A Pharmacodynamic Solution MSSA

- Prevention of Emergence of Resistance: A Pharmacodynamic Solution* **MSSA*

***Log10 CFU/mL***

***Daily AUC/MIC***

**Breakpoint = 66**

## Prevention of Emergence of Resistance: A Pharmacodynamic Solution MRSA-CS

- Prevention of Emergence of Resistance: A Pharmacodynamic Solution* **MRSA-CS*

***Log10 CFU/mL***

***Daily AUC/MIC***

**Breakpoint = 143**

## Prevention of Emergence of Resistance: A Pharmacodynamic Solution MRSA-CR

- Prevention of Emergence of Resistance: A Pharmacodynamic Solution* **MRSA-CR*

***Log10 CFU/mL***

***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

## Slide 52