1. 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 & NYSDOH

2. Prevention of Emergence of Resistance: A Pharmacodynamic Solution Currently, the therapeutic armamentarium is amazingly limited for many Gram-negative pathogens Discovery programs do not promise any relief for at least 5 years We must learn to use available drugs more intelligently to preserve the susceptibility of the infecting flora to current agents

3. Prevention of Emergence of Resistance: A Pharmacodynamic Solution

4. Many organisms have resistance mechanisms that occur as a function of single point mutations Examples are stable derepression of type I beta lactamases for 3 rd generation cephalosporins and target mutations or pump upregulation for fluoroquinolones As these occur at a frequency of 1/10 8 or greater, infection site populations exceed this frequency, often by multiple logs

5. Prevention of Emergence of Resistance: A Pharmacodynamic Solution 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:

6. 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?

7. The Team N. L. Jumbe, A. Louie, W. Liu,V. Tam, T. Fazili, R. Leary, C. Lowry, M.H. Miller and G. L. Drusano

8. Prevention of Emergence of Resistance: A Pharmacodynamic Solution Let Us Compare and Contrast the Pharmacodynamics of Levofloxacin for Streptococcus pneumoniae and Pseudomonas aeruginosa in a Mouse Thigh Infection Model

9. S. pneumoniae outcome studies

10. P. aeruginosa outcome studies Rf in vitro Rf in vivo MIC (  g/mL) MBC (  g/mL) 2.35x10 -6 2.2x10 -6 0.8 1.6

11. 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 Levofloxacin

14. Peripheral (thigh) 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 keke 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]

15. K maxGS 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 10 10

16. 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

17. 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

18. Prevention of Emergence of Resistance: A Pharmacodynamic Solution

19. We wished to evaluate the model prospectively Models, to be useful, need to predict the future We simulated a dose not previously studied that would encourage selection of resistance The study was carried out for 48, not 24 hours The model predicted the change in the resistant mutant population well

21. 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

22. Prevention of Emergence of Resistance: A Pharmacodynamic Solution We were able to determine how the overall (sensitive plus resistant) population responds to pressure from Levofloxacin 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

23. 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

24. 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 - Drug + 4xMIC Cipro + 3xMIC Levo

25. 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

26. Strain 58, the RC2 and RC4 mutants were sequenced through Gyr A, Gyr B, Par C & Par E. The sequences examined were: GyrA (ORF 822 aa) aa 4- 229; Gyr B (ORF 648 aa) aa 346-579; ParC (ORF 823 aa) aa 1-178; ParE (ORF 647 aa) aa 359-561. 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.

27. Begin therapy Sacrifice, harvest, homogenize muscle -2 hr 0 hr + 2xMIC Cipro - Drug Infect 24 hr BID + 4xMIC Cipro Begin therapy Sacrifice, harvest, homogenize muscle -2 hr 0 hr + 3xMIC Levo - Drug Infect 24 hr BID + 3xMIC Cipro #58-RC2 #58-WT + 3xMIC Levo Study Design: Second Passage of First Stage Ciprofloxacin Resistant S. pneumoniae

28. Total Counts

29. Cipro Resistance

30. Levo Resistance * = no colonies detected in any sample. Sample size  4 animals

31. Prevention of Emergence of Resistance: A Pharmacodynamic Solution What next? We are currently examining the RC2 mutant in the mouse thigh model In preliminary data, exposures to levofloxacin that would kill the wild-type isolate did not kill the mutant, even though the MIC has not changed This finding has been recreated with another later generation fluoroquinolone in a hollow fiber model This implies that, counter to the output of Resistance 2000, sometimes newer drugs preserve the sensitivity of the flora better than older drugs

32. 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

33. 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

34. 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)

35. Prevention of Emergence of Resistance: A Pharmacodynamic Solution CONCLUSIONS Probability of emergence of resistance is impacted upon by the intensity of therapy and by the duration of therapy Short duration therapy trials should examine an endpoint of resistance frequency As importantly, doses should be chosen to provide resistance counterselection exposures for a large fraction of the population. An example follows:

36. Prevention of Emergence of Resistance: A Pharmacodynamic Solution Target Attainment

38. Prevention of Emergence of Resistance: A Pharmacodynamic Solution While this example is for microbiological outcome, a similar analysis could (and should!) be performed for a prevention of resistance target Such a dose choice, coupled with short duration therapy will yield the highest probability of a good clinical and microbiological outcome and the lowest probability of the resistant subpopulation taking over the whole population