1. Mutant Prevention Concentration and the Selection Window Hypothesis Karl Drlica, Xilin Zhao, and Tao Lu Public Health Research Institute Newark, NJ

2. Selecting a treatment plan for a particular patient Individual patient issues Probability of “cure” without serious side effects Public health issues Probability for avoiding enriching a resistant bacterial subpopulation

3. wild type single mutant double mutant frequency ~ 10 -7 frequency ~ 10 -14 (number of bacteria during infection: < 10 10 ) Blocking Growth of Single Mutants Forces Cells to Have a Double Mutation to Overcome Drug attack by drug

4. Introduction to MPC Mutant Selection Window Dosing above the MPC Closing the Window Combination Therapy Comparison with Traditional PK/PD Application to S. pneumoniae Application to S. aureus Other antimicrobial-pathogen combinations

5. Introduction to MPC: Fluoroquinolone studies with mycobacteria

6. Quinolone + DNA Gyrase Cell death (c)(d) (a) (b)Gyrase mutations Cell lysis SDS Cm Blocks DNA replication and cell growth (MIC) Intracellular Consequences of Fluoroquinolone Treatment

7. O OH F N O N HN O H3C H3C H5C2 H5C2 Fluoroquinolone Structure O OH F N O N HN H5C2 H5C2 C-8-H compound C-8-methoxy compound

8. Bacteriostatic Activity with M. bovis BCG Fluoroquinolone C-8-moiety ID 50 (  g/ml) gyrA + gyrA r ciprofloxacinH0.15 6.1 PD161148OMe0.05 0.61 PD160793H0.08 7.0

9. [Fluoroquinolone] (  g/ml) 10 -2 10 -1 1 10 10 2 10 3 0.010.11 10 Survival (%) C-8-OMe C-8-H 110100 Bactericidal Activity of Fluoroquinolones with M. bovis BCG AB Incubation time: 6 days wild type gyrA mutant

10. non-gyrA 0 20 40 60 80 100 Percent recovered A91V D95A D95Y D95G D95N D95H D95GG89CD95HD95GG89C 10 -2 1 0.010.1110 [Fluoroquinolone] (  g/ml) Fraction of cells recovered 10 -4 10 -8 10 -6 A B D C ABDC Mycobacterium smegmatis Effect of Fluoroquinolone Concentration on Mutant Recovery

11. Mutant Selection Window

12. [Fluoroquinolone] ( µ g/ml) 10 -9 10 -7 10 -5 10 -3 10 -1 0.010.1110 Fraction of cells recovered MIC 99 MPC MIC 99 Mutant Prevention Concentration (MPC) M. bovis BCG C-8-H C-8-OMe

13. Drug Concentration (log 10 ) MIC MPC Mutant Selection Window C-8-methoxy C-8-hydrogen Fraction of colonies recovered (log 10 ) A Mutant Selection Window Time post-administration MIC MPC Serum or tissue drug concentration Mutant Selection Window C ma x B

14. [Moxifloxacin] (  g/ml) MIC (  g/ml) at 24 h before treatment at 48 h at 72 h Selection Window Demonstrated by Dynamic, in vitro Model Data from Firsov et al. Organism: Staphylococcus aereus Dosing ProtocolAnalysis of recovered cells A MPC MIC 10 1 0.1 B MPC MIC 10 1 0.1 C MPC MIC 10 1 0.1 Time (h) 72482496120 0.3 0.2 0.1 0 0.3 0.2 0.1 0 0.3 0.2 0.1 0

15. Selective Pressure Sele ctive Pres MIC Time Concentration Traditional Explanation for Enrichment of Mutants

16. Mutants are not selected at concentrations below MIC

17. Time post-administration Serum or tissue drug concentration Dose above MPC Strategies for Restricting the Development of Resistance MPC MIC Narrow the window MPC~MIC 2-drug therapy MPC=MIC

18. Dosing Above the Window

19. Antibiotic MPC C max MPC/C max Rifampicin >80 9.5 >8 Streptomycin >320 34 >9 Isoniazid 20 7.62.6 Ethionamide 35 201.8 Ethambutol 50 68.3 Capreomycin 160 334.8 Kanamycin >800 21 >38 Cycloserine 70 35 2 Fluoroquinolones Ciprofloxacin 8.0 4.41.8 Levofloxacin 7.5 5.71.3 Sparfloxacin 2.5 1.41.6 Moxifloxacin 2.5 4.5 0.55 Gatifloxacin 1.5 3.7 0.41 Time post-administration Serum or tissue drug con. Dose above MPC Relationship of Pharmacokinetics and MPC in M. tuberculosis

20. Closing the Window

21. Narrowing the Selection Window with S. aureus MPC MIC (99) MPC/MIC (99 ) (  g/ml) 0.60.05 12 1.70.05 34 4 0.3 13 0.45 0.08 6 O OH F N O O H 3 C N N O OH F N O N N O F N O N HN O H 3 C O OH F N O N HN Narrow the window Time post- administration Serum or tissue drug con.

22. Problems with Combination Therapy

23. Serum or tissue drug concentration Time post-administration Drug 1 Drug 2 open closed MIC #1 #2#3 Mutant Selection Window Closing The Mutant Selection Window

24. HIV + TB patients (drug-susceptible) Treat with INH, Rif, Pz, Em (2 months; DOT) INH/Rifapentine (1/wk, 4 months; DOT) INH/Rif (2/wk, 4 months; DOT) 5/30 relapse 3/31 relapse 4/5 Rif-resistant 0/3 Rif-resistant Treatment Protocol for a Dual-drug Failure Source: A. Vernon et al. Lancet 353: 1843-1847 (1999)

25. Pharmacodynamic Comparison of Rifampicin and Isoniazid 1 10 100 1000 0102030 Plasma drug concentration (fold of MIC) Time post-administration (hr) MIC Isoniazid Rifampicin M. tuberculosis

26. Pharmacokinetic mismatch between rifapentine and isoniazid 1 10 100 1000 010203040506070 Plasma drug concentration (fold of MIC) Time post-administration (hr) MIC Rifapentine Isoniazid M. tuberculosis

27. 0.1 1 10 100 500 02468101214 Time post-administration (hr) Serum drug concentration (fold of MIC) MIC INH Rifampicin Pyrazinamide Normalized pharmacokinetic profiles of Rifater (M. tuberculosis)