1. Pharmacodynamics of Fluoroquinolones G.L. Drusano, M.D. Professor and Director Division of Clinical Pharmacology Clinical Research Institute Albany Medical College & New York State Department of Health

2. Pharmacodynamics of Fluoroquinolones

3. What Pharmacodynamic covariate is linked to outcome? Why? What does this have to do with emergence of resistance? What are the factors that amplify resistant mutant populations?

4. Pharmacodynamics of Fluoroquinolones Let us examine fluoroquinolones and determine the pharmacodynamically- linked variable

5. 24681012 White Blood Cell Count After I.P. Cyclophosphamide (100 mg/kg on Day 0 and 75 mg/kg on Day 4 ) 3 3 Cyclophosphamide Bacterial Challenge End of Experiment Drusano GL, et al. Antimicrob Agents Chemother. 1993;37:483-490.

6. Lomefloxacin: Pharmacokinetics and Pharmacodynamics in Septic, Neutropenic Rats Mean Peak Mean AUC Time > MIC Concentration per 24 h Dosage (mg/L) (mg  h/L) (h) 20 mg/kg q6h4.757.216.8 40 mg/kg q12h6.963.614.0 80 mg/kg q24h20.864.39.6 20 mg/kg q6h4.757.216.8 40 mg/kg q12h6.963.614.0 80 mg/kg q24h20.864.39.6 For all determinations, N=3. Drusano GL, et al. Antimicrob Agents Chemother. 1993;37:483-490.

7. Lomefloxacin Therapy for Pseudomonas Sepsis in Neutropenic Rats: Effect of Dose Fractionation (N=50/Group) Drusano GL, et al. Antimicrob Agents Chemother. 1993;37:483-490.

8. Lomefloxacin Pharmacodynamics: Survivorship After Challenge (N=20/Group) Drusano GL, et al. Antimicrob Agents Chemother. 1993;37:483-490.

9. Pharmacodynamics of Fluoroquinolones Sometimes AUC/MIC Ratio is the pharmacodynamically-linked covariate, but sometimes it is Peak/MIC Ratio. Peak/MIC probably occurs when there are mixtures of sensitive and less-sensitive bacterial populations (more about this anon)

10. PK/PD Paramters with Fluoroquinolones 24-hr AUC/MIC (incorrectly referred to as AUIC) is the parameter that best predicts activity of fluoroquinolones. 24-hr AUC/MIC (using free drug levels) for static dose range from 25-50 for most organisms in neutropenic mice W.A. Craig, M.D.

11. 24-Hr AUC/MIC for Static Doses of Gatifloxacin, Sitafloxacin and Gemifloxacin Against 6 Strains of Streptococcus pneumoniae W.A. Craig, M.D. Protein Binding Does Matter! - G.L. Drusano, M.D.

12. Impact of Neutrophils on Activity of Fluoroquinolones in Mice  Based on studies with 2 organisms (K. pneumoniae and S. pneumoniae) that grow well in normal mice  Neutrophils reduced static doses by 20-40% for K. pneumoniae both in thigh- and lung-infection models  Neutrophils reduced static doses by 75-85% for S. pneumoniae in thigh-infection model W.A. Craig, M.D.

13. Pharmacodynamics of Fluoroquinolones  Magnitude of 24-Hr AUC/MIC in serum required for 90-100% survival in animal infection models varies from about 25 in immunocompentent animals for Streptococcus pneumoniae to about 100 in immunocompromised animals for gram- negative bacilli  24-Hr AUC/MIC values of 25 and 100 are equivalent to averaging one and four times the MIC over a 24-hr period W.A. Craig, M.D.

14. Relationship Between 24 Hr AUC/MIC and Mortality for Fluoroquinolones in Immunocompromised Animal Models W.A. Craig, M.D.

15. Relationship Between 24 Hr AUC/MIC and Mortality for Fluoroquinolones against Streptococcus pneumoniae in Immunocompetent Animals W.A. Craig, M.D.

16. Pharmacodynamics of Fluoroquinolones Now that we know what to do for mice and rats, what about clinical data?

17. Pharmacodynamics of Fluoroquinolones Percent of Patients Remaining Culture-positive Days of therapy AUC/MIC <125 AUC/MIC 125-250 AUC/MIC >250 100 75 50 25 0 02468101214 Forrest et al

18. Pharmacodynamics of Fluoroquinolones It should be appreciated that this was a retrospective analysis Further, the number of patients with Streptococcus pneumoniae infections was ZERO!

19. Levofloxacin Clinical Outcome: Probability of a Successful Outcome Patient n=134; 7 Patients Failed McFadden’s Rho 2 = 0.337 Breakpoint = 12.2 Pulmonary Infections (n=87) (500 mg qd) Skin and Soft Tissue Infections (n=25) (500 mg qd)

20. Levofloxacin Clinical Outcome: Probability of Successful Therapy Patient n=134; 7 Patients Failed McFadden’s Rho 2 = 0.192 Breakpoint = 50.25

21. Pharmacodynamics of Fluoroquinolones This was the first prospective, multicenter study for the delineation of the linked pharmacodynamic variable for fluoroquinolones There was a mix of Gram-negative isolates as well as Streptococcus pneumoniae However, the difference in identified breakpoints makes one wonder about the issue of mixed populations of sensitive and less-sensitive pathogens

22. Pharmacodynamics of Fluoroquinolones Ambrose PG et al. AAC 2001;45:2793-2797

23. Pharmacodynamics of Fluoroquinolones Clearly, Streptococcus pneumoniae differs from other organisms For other pathogens, an AUC/MIC ratio around 100 will produce acceptable response rates For S pneumoniae, an AUC/MIC ratio of 20-40 is an appropriate exposure target

24. Pharmacodynamics of Fluoroquinolones What about emergence of resistance?

25. Suppression of Emergence of Resistance: A Pharmacodynamic Solution Resistance to antimicrobial agents often occurs 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 3 rd generation cephalosporins and target mutations or pump upregulation for fluoroquinolones

26. Suppression of Emergence of Resistance: A Pharmacodynamic Solution As these occur at a frequency of circa 1/10 8, 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:

27. Suppression 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?

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

29. S. pneumoniae outcome studies

30. 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

31. Suppression 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

33. 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 [4] [5] [6] [7] [8],  =K and  = S,R [2] L = (1- (X R + X S ) /POPMAX) [9] dC p = k cp C c - k pc C p dt [3] dC a = -k a C a dt [1]

34. 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

35. Suppression 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

36. Suppression 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

37. Suppression of Emergence of Resistance: A Pharmacodynamic Solution

39. 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

40. Suppression 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

41. Suppression 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

42. 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

43. Drug #58 RC2 Cipro/ ±Reserpine 0.6/0.6 3.5/1.0 Levo/ ±Reserpine 0.6/0.6 0.6/0.6 Suppression of Emergence of Resistance: A Pharmacodynamic Solution

44. 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.

45. Suppression 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

46. Suppression 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, fluoroquinolones, etc.) The original hollow fiber system was used by Blaser, Dudley & Zinner

47. Suppression 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

48. Suppression of Emergence of Resistance: A Pharmacodynamic Solution Pseudomonas aeruginosa Tam et al ICAAC 2001

49. Suppression of Emergence of Resistance: A Pharmacodynamic Solution Tam et al ICAAC 2001

50. Suppression of Emergence of Resistance: A Pharmacodynamic Solution Tam et al ICAAC 2001

51. Suppression of Emergence of Resistance: A Pharmacodynamic Solution Tam et al ICAAC 2001

52. Suppression of Emergence of Resistance: A Pharmacodynamic Solution Tam et al ICAAC 2001

53. Suppression of Emergence of Resistance: A Pharmacodynamic Solution Tam et al ICAAC 2001

54. Suppression of Emergence of Resistance: A Pharmacodynamic Solution Tam et al ICAAC 2001

55. Central 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]

56. K maxGS 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 10 10

57. Suppression 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

58. Suppression 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

59. Suppression 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

60. Suppression 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

61. Suppression of Emergence of Resistance: A Pharmacodynamic Solution P. aeruginosa - Prevention of Amplification of Resistant Subpopulation Prospective Validation

62. Suppression 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

63. Suppression 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

64. Suppression of Emergence of Resistance: A Pharmacodynamic Solution Emergence of Resistance and the Flight of Time’s Arrow (Is the duration of therapy important?)

65. Suppression 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

66. Suppression 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)

67. Suppression 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

68. Suppression of Emergence of Resistance: A Pharmacodynamic Solution Targets for Fluoroquinolone Therapy: Does One Size Fit All? (A Question Answered in the Affirmative by a Group in the Western Part of New York State - See an AUC/MIC Ratio of 125)

69. Suppression of Emergence of Resistance: A Pharmacodynamic Solution ‘Inverted-U’ Phenomenon –Resistant sub- populations are initially amplified & then decline with increasing drug exposure Therapeutic Intensity Log10 CFU/mL Resistant Sub-Population

70. Suppression of Emergence of Resistance: A Pharmacodynamic Solution P aeruginosa Log10 CFU/mL Daily AUC/MIC Breakpoint = 187

71. Suppression of Emergence of Resistance: A Pharmacodynamic Solution K. pneumoniae Log10 CFU/mL Daily AUC/MIC Breakpoint = 93

72. Suppression of Emergence of Resistance: A Pharmacodynamic Solution MSSA Log10 CFU/mL Daily AUC/MIC Breakpoint = 66

73. Suppression of Emergence of Resistance: A Pharmacodynamic Solution MRSA-CS Log10 CFU/mL Daily AUC/MIC Breakpoint = 143

74. Suppression of Emergence of Resistance: A Pharmacodynamic Solution MRSA-CR Log10 CFU/mL Daily AUC/MIC Breakpoint = 484

75. Suppression 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 The exposures differ by isolate We can do better in designing doses to not only optimize outcome, but also suppress emergence of resistance Attainment of target can be assessed through use of Monte Carlo simulation