1. 1 Developments in pK/pD: optimising efficacy & prevention of resistance A critical review of pK/pD in in vitro models Alasdair MacGowan Bristol Centre for Antimicrobial Research & Evaluation University of Bristol & North Bristol NHS Trust Southmead Hospital Bristol, UK

2. 2 OBJECTIVES OF STUDYING pK/pD IN IN VITRO MODELS tests of efficacy determination of dominant pK/pD parameter determination of the magnitude of the dominant pK/pD parameter emergence of resistance determination of dominant pK/pD parameter determination of the magnitude of the dominant pK/pD parameter

3. 3 TESTS OF EFFICACY human dosing or other activity of agent vs target pathogen  co resistance's activity of agent and comparator(s) vs target pathogen activity of agent v target pathogen with various MIC values of the agent activity of agent vs target pathogen with various mechanisms of resistance to the agent activity of agent vs different bacterial species of target pathogen

4. 4 ACTIVITY AGAINST TARGET PATHOGENS i.e. moxifloxacin 400mg 24hrly species MIC 50 strainlog reduction (mg/L) MIC viable count (mg/L) 24h S pneumoniae 0.06 0.08 5.6  0.4 H influenzae 0.03 0.06 6.5  0.2 M catarrhalis 0.06 0.08 5.1  1.2 S aureus 0.06 0.06 2.4  1.1 Group A strep 0.12 0.16 3.1  1.9 MacGowan, 1999; MacGowan et al, 1998; MacGowan et al, 1999a; MacGowan et al, 1999b

5. 5 ACTIVITY OF AGENT AND COMPARATOR AGAINST TARGET agents targetreference pathogen trova/cipro S aureus Firsov et al, 1999 trova/cipro S aureus O’Brien et al, 1999 levo/cipro S pneumoniae Lister & Saunders, 1999 moxi/grepa/clari S aureus Esposito et al, 2000 Gp A streptococci trova/gati/clina S pneumoniae Hershberger & Rybak, 2000 spar/levo/cipro moxi/levo/gati/cipro S pneumoniae Firsov et al, 2000 various

6. 6 ACTIVITY OF AGENTS AGAINST TARGET PATHOGENS WITH VARIOUS MICs - USUALLY INCREASING gemifloxacin 320mg 24hrly vs S. pneumoniae (MIC 50 0.015; MIC 90 0.03/6mg/L) Strains MIClog reduction in viable count (mg/L) 24h 48h 0.0165.3  0.3 5.3  0.3 0.063.4  1.4 6.2  0.1 0.104.1  0.5 5.7  0.2 0.160.6  1.4 3.9  0.9 0.240.2  0.4 0.5  0.2 MacGowan et al, (in press)

7. 7 ACTIVITY OF AGENTS AGAINST PATHOGENS WITH DIFFERENT MECHANISM OF RESISTANCE (1) S aureus with Nor A efflux pump S aureus (Nor A expression induced) log cfu/ml at 72h levofloxacon 4.8  0.4 levofloxacin + omeprazole 4.8  0.4 ciprofloxacin 9.5  0.3 ciprofloxacin + omeprazole 7.4  0.5 Aeschlimann et al, 1999

8. 8 ACTIVITY OF AGENTS AGAINST PATHOGEN WITH DIFFERENT MECHANISMS OF RESISTANCE (2) gemifloxacin 320mg od, for 72hr log reduction time to kill MIC count at 99.9% (mg/L) mechanism 72h (h) 0.06 none 5.3  0.3 26  2 efflux pump 5.8  0.3 15  12 gyr A mutation 1.2  0.3 > 72 par C mutation 1.2  0.3 > 72 0.12 efflux pump 3.8 21  8 gyr A mutation 0.4  0.4 > 72

9. 9 ACTIVITY AGAINST DIFFERENT TARGET PATHOGENS i.e. moxifloxacin 400mg od vs S pneumoniae or P aeruginosa both MIC 0.25mg/L S pneumoniaeP aeruginosa log reduction count @ 24h 3.4  1.2 0.1  0.2 @ 48h 4.2  1.0 0.7  0.5 @ 72h 4.8  0.5 0.1  0.6 time to kill 99.9% (h) 16  7 >72

10. 10 CONCLUSIONS ON TESTS OF EFFICACY - easy, descriptive, limited but useful information

11. 11 DETERMINATION OF DOMINANT pK/pD PARAMETER  dosing regimen employed (differentiation between AUC/MIC; C max /MIC; T > MIC)  end point chosen  analytic tools used  susceptibilities of target strains  effects of aggregation of data (i.e. species or mechanisms)

12. 12 PRODUCING VARIABILITY IN pK/pD PARAMETERS dose escalationMIC differences dose fractionation ranges in pD/pK parameters (AUC/MIC; Cm/MIC; T > MIC)

13. 13 DOSING REGIMENS doseconclusion ciprofloxacin Cm 5mg/LAUC/MIC related to ofloxacin 5mg/L, 8mg/L;outcome different t/ 12 Madaras-kelly et al, 1996a ofloxacin Cm 3.5-62mg/LAUC/MIC related ciprofloxacin Cm 1.1-20mg/Lto outcome plus othersMadaras-Kelly et al, 1996b gemifloxacinAUC/MIC or T>MIC 160mg bd; 320mg od;related to outcome 640mg 48hBowker et al, 2000

14. 14 GEMIFLOXACIN DOSE FRACTIONATION PLUS MIC RANGE 0.016 - 0.24mg/L Spearman rank Correlation (95% CI) AUC/MIC v Cm/MIC 0.77 (0.42 - 0.92) AUC/MIC v T > MIC 0.87 (0.60 - 0.96) Cm/MIC v T > MIC 0.42 (0.14 - 0.77)

15. 15 END POINTS - measures of antibacterial effect (ABE) measures in time (X axis) time to kill 90, 99, 99.9 etc time to maximum kill measures in viable count (Y axis) log kill at 12, 24, 36 etc h log kill after dose ( ~ ) maximum kill “integrated” measures (X and Y) slope of kill curve areas around the kill curve

16. 16 AREA MEASURES log cfu/ml control IEIE AUBC test time AAC: area above the curve AUBC: area under the bacterial (kill) curve AUBKC I E : intensity of effect (area between curves, (log C - log T) xt) AUBKC (test) /AUBKC (control) ratio AAC

17. 17 PERCENTAGE CO-EFFICIENTS OF VARIATION OF ANTIBACTERIAL EFFECT MEASURES moxifloxacin 69 simulations number of occasions measure measurable % CV (median; range) log change @ 12hr 15 32 (9 - 100) 24hr 13 55 (8 - 72) 36hr 10 25 (4 - 97) 48hr 12 43 (6 - 132) maximum log 12 17 (4 - 88) T99 17 25 (0 - 70) T99.9 17 27 (5 - 75) AUBKC 24 21 14 (1 - 65) AUBKC 48 21 14 (1 - 74

18. 18 EFFECT OF CHOSEN ANTIBACTERIAL EFFECT MEASURE ON DOMINANT pK/pD PARAMETER gemifloxacin160mg x 4 over 48 hr 320mg x 2 over 48 hr 640mg x 1 over 48 hr five strains of S pneumoniae (MIC 0.016; 0.06; 0.1; 0.16; 0.24 mg/L) pD parameters: AUC/MIC (72 - 1219) C max /MIC (3 - 131) T > MIC (38 - 100%)

19. 19 using E max model AUC/MIC related to AUBKC 48 best C max /MIC and T>MIC less well weighted least squares regression analysis AUC/MIC and T>MIC predictors of AUBKC 48 C max /MIC not predictive of AUBKC 48

20. 20 Cox proportional hazards regression - ABE: time to kill 99.9% inoculum univariate analysis AUC/MIC, Cm/MIC and T > MIC related to T99.9 multivariate model C max /MIC related to T99.9 Cm/MICrelative risk95% CI <5 1.0 5 - 9.9 7.7 2.2 - 27.2 10 - 29.9 11.6 4.3 - 31.8 >30 20.7 2.3 - 68.3

21. 21 WHY AUC/MIC AND T > MIC PREDICT AUBKC 48 log change count dosing regimen MIC (mg/L)160mg x 4320mg x 2640mg x 1 0.016maximum - 5.2 - 5.3 - 4.8 final - 4.8 - 5.3 - 4.8 0.06maximum - 4.5 - 6.2 - 6.2 final - 4.0 - 6.2 + 0.1 0.10maximum - 6.6 - 5.7 - 6.2 final - 5.2 - 3.9 0 0.16maximum - 2.8 - 3.9 - 4.6 final - 2.3 - 0.6 0 0.24maximum - 3.3 - 0.5 - 4.7 final - 0.8 + 0.3 + 0.3

22. 22 If exclude simulation with T > MIC of <70% then AUC/MIC predicts AUBKC 48 in multi variate analysis

23. 23 COX PROPORTIONAL HAZARDS REGRESSION ANALYSIS FOR T99.9 AND REGROWTH 81 experiments with gemifloxacin and moxifloxacin against S. pneumoniae In 59/81 experiment T99.9 achieved In 24/59 regrowth occurred AUC/MIC, Cm/MIC and T > MIC related to both measures T99.9 related to Cm/MIC especially > 5 Regrowth related to T > MIC AUC/MIC did not predict T99.9 or regrowth

24. 24 THEREFORE - T99.9 Cm/MIC slope  log cfu/ml AUBKC  AUC/MIC Regrowth I E  T > MIC

25. 25 ANTIBACTERIAL EFFECT MEASURES Strengths Weakness T99.9 easy to understand highly variable easy to measure not always related to pD suitable for time to parameters event analysis doesn’t capture regrowth data intensity of effect less variable depends on regrowth strong comparative T > MIC driven (inter quinolone predictor) database linear over a large linear over therapeutic AUC/MIC range AUC/MIC range AUC/MIC an intra quinolone predictor i.e. FQ specific

26. 26 ANTIBACTERIAL EFFECT MEASURES Strengths Weakness area under curve less variable size-counter intuitive AUC/MIC driven i.e. AUC/MIC  1/AUBKC sigmoid relationship to AUC/MIC

27. 27 CONCLUSION - DETERMINATION OF Pd PARAMETER end point matters

28. 28 DETERMINATION OF THE MAGNITUDE OF THE pK/pD PARAMETER AUC/MIC for optimal outcome v S. pneumoniae (1) AUC/MIC 30-55 sustained 4 log reduction with levofloxacin Lacey et al, 1999a AUC/MIC >45 sustained 5 log reduction with trovafloxacin and ofloxacin Lister & Saunders, 1999b AUC/MIC of 44 sustained 5 log reduction with levofloxacin Lister & Saunders,

29. 29 AUC/MIC AND S. PNEUMONIAE (2) AUC/MIC 50 = 60 for In AUBKC levofloxacin, ofloxacin ciprofloxacin Madaras - Kelly et al, 1997 AUC/MIC >250 for lowest AUBKC gemifloxacin Bowker et al, 2000 AUC/MIC >250 for lowest AUBKC moxifloxacin Bowker et al, 2001

30. 30 AUC/MIC and S. PNEUMONIAE (3) Moxifloxacin doseMICAUC/MICclearance (sustained 5 log reduction 400 OD0.08 375 Yes 400 BD0.25 320 Yes 800 OD0.25 320 Yes 400 OD0.12 167 No 400 OD0.25 160 No 400 OD1 30 No 400 BD4 20 No 800 OD4 20 No 400 OD2 15 No 400 OD4 10 No Gemifloxacin 320 OD0.016 625 Yes 320 OD0.06 166 No 320 OD 0.10 100 No 320 OD0.16 62 No 320 OD0.25 41 No

31. 31 WHY THE DIFFERENCE?  inoculum  growth phase  total organisms exposed

32. 32 EFFECT OF INOCULUM ON I E ; CIPROFLOXACIN AND TROVAFLOXACIN relative I E S aureusE coli 8 log 1.05 1.12 6 log0.98 (-7%) 0.92 (-18%) from Fig 4, Firsov et al, 1999

33. 33 IS THE AUC/MIC THE SAME FOR DIFFERENT SPECIES? MoxifloxacinAUBKC 48 (log cfu/ml) MIC (mg/L)Gram +Gram -  0.195  4433  15 0.1 - 0.5 170  6563  14 > 0.5 240  3099  21 Gram +; S pneumoniae, S aureus, GpA streptococci Gram -; H influenzae, M catarrhalis (MacGowan, 1999)

34. 34 IS THE AUC/MIC THE SAME FOR DIFFERENT SPECIES? (2) AUC 0-72 /MIC for 50% effect AUBKC 72 (log cfu/ul.h) S pneumoniae 136 P aeruginosa > 1000

35. 35 CONCLUSION ON MAGNITUDE OF pK/pD PARAMETER - not uniform across models or species - models need to be calibrated on the basis of good clinical studies

36. 36 EMERGENCE OF RESISTANCE - THE STORY SO FAR enoxacin ~ P. aeruginosa emergence of resistance observed, related to time of exposure, and total exposure (Cm/MIC) Blaser et al, 87 Ciprofloxacin ~ P. aeruginosa dose fractionation (1200 OD; 600BD; 400TDS) 1200 OD less resistance than 600 BD or 400 TDS Marchbanks et al, 1993

37. 37 Ciprofloxacin/ofloxacin ~ P. aeruginosa AUC/MIC or C max /MIC related to emergence of resistance (MIC  ) Madaras-Kelly et al, 1996

38. 38 End points MIC before and after exposure subculture onto recovery with increasing FQ concentrations (MIC x 0.5; MIC x 1; MIC x 2 etc) -absolute count on plates -heterogencity of population -area-under-curve - population-analysis- profile (AUC-PAP)

39. 39 Log cfu/ml 0Antibiotic concentration time 0 time 48 time 24 AUC - PAP o

40. 40 EMERGENCE OF RESISTANCE TO MOXIFLOXACIN WITH S. PNEUMONIAE & P. AERUGINOSA P. aeruginosa MIC 0.25mg/L log cfu/ml 0hr 24hr72hr conc in 200 400 400 800200 400 400 800 recovery OD OD BD ODOD OD BD OD plate 0 8.5 8.3 7.6 7.5 7.58.3 8.2 7.6 7.5 4 4.7 8.0 7.3 2.6 3.5 8.0 8.2 4.0 4.0 16 <2 ND 3.4 <2 <2 ND 8.3 3.2 3.5

41. 41 AUC 72 /MIC for 50% effect - S. pneumoniaeP. aeruginosa ABE: AUBKC 72 136 >1000 Resistance log cfu/ml MIC x 4 < 50 862 log cfu/ml MIC x 16 < 50 1186 PAP ~ AUC < 50 910

42. 42 Emergence of resistance depends on (i)species (P. aeruginosa > S pneumoniae) (ii)duration of exposure (long > short) (iii)exposure (large AUC/MIC > small) (iv)AUC/MIC  Cm/MIC (v)pD parameter magnitude different to effect

43. 43 Conclusions in vitro models have a vital role in pD assessments in vitro models are flexible tools to assess many pD questions, i.e. dosing in vitro models can be used for hypothesis testing and generation for best value they should compare with animal data but much important, human data