EMA Workshop Non-Clinical Models to Identify PK/PD Indices and PD Targets In Vitro

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1 EMA Workshop Non-Clinical Models to Identify PK/PD Indices and PD Targets In Vitro G.L. Drusano, M.D. Professor and Director Institute for Therapeutic Innovation University of Florida

2 In Vitro There are many in vitro systems for delineating the relationships between drug regimen intensity and in vitro outcomes of interest (bacterial cell kill and resistance suppression) I will limit myself to the system we employ in our laboratory the hollow fiber infection model first employed by Jurg Blaser and Steve Zinner and used extensively by Mike Dudley I have no experience with the other systems

3 In Vitro There are many advantages and disadvantages to both in vitro systems, as well as in vivo systems I will concentrate on in vitro systems Advantages: 1) any half-life can be simulated 2) any bacterial burden can be examined 3) any organism can be studied 4) Resistance emergence is straightforward to find and study 5) Other physiologic states can be induced and studied (e.g. Non-Replicative Persister Phenotype)

4 In Vitro Advantages (cont d): 6) The system can be employed at any stage of discovery/development Really? Even if I do not know the PK in man? Yes! Simply look at a small animal half-life for effect and then empirically dial in longer half-lives likely to be seen in man (e.g. 2, 4, 6, 8 hr half-lives) and ascertain the impact on the dynamic index We have done this before (AAC 2011;55: and AAC 2015;59: )

5 In Vitro What are the disadvantages? 1) THERE IS NO IMMUNE SYSTEM! 2) There is no physiology 3) you cannot look at issues such as tissue penetration and effect on outcome 4) cannot look directly at protein binding issues (we do employ free drug concentrationtime curves, but there are other issues)

6 In Vitro Cell Kill and Resistance Emergence

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8 Resistance Suppression in Pseudomonas aeruginosa The use of the hollow fiber model for studying antimicrobial regimens was described by Blaser and Zinner and employed extensively by Dudley

9 Resistance Suppression in Pseudomonas aeruginosa Tam V et al. Bacterial-population responses to drug selective pressure: Examination of garenoxacin s effect on Pseudomonas aeruginosa. J Infect Dis 2005;192:

10 Central + Bacteria Infusion Compartment (C c ) (X T/R ) SCl f(c) [1] dc c =Infusion-(SCl/V)xC c dt dx S =K GS x X S x L - f KS (C c H ξ ) x X S dt dx R = K GR x X R x L- f KR (C c H ξ ) x X R dt [2] [3] L = (1-(X R + X S )/POPMAX) [4] f ψξ (C c Hξ)= K max ξ C c H ξ C H ξ 50 ξ +C c H ξ, ψ=k and ξ = S,R [5] Y 1 =X T =X S +X R, IC(1)=1.01x10 8 Y 2 =X R, IC(2)= 58 [6] [7]

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12 Resistance Suppression in Pseudomonas aeruginosa 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 185/1 Resistant organisms at baseline Resistant Mutants (CFU/mL) AUC 0-24 :MIC Ratio All other data points represent resistant organism counts at 48 hours of therapy

13 Prospective Validation Experiment

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15 In Vitro Time to Resistance Continuous infusion of Avibactam (AUC = 8 x 24 = then called NXL104) worked and suppressed resistance for the duration of the experiment (D); AUC=192 Q 24 h (E) failed, as did AUC = 96 Q12 h (F) AUC = 64 Q 8 h (G) succeeded for the whole experiment, implying that for this agent Time > Threshold (or Cmin) drives β-lactamase inhibition Note in (F) that resistance did not emerge until after day 10 you must study long enough

16 In Vitro Impact of Therapy Duration

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23 Prospective Validation Experiment

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27 In Vitro Very Reproducible

28 In Vitro Looking at Agents in Combination

29 Mono-Rx Pseudomonas aeruginosa

30 In Vitro So, what s going on? Why the failure of mono-rx and why the success of combo-rx? AAC 2012; 56:

31 In Vitro So, what is going on? We looked at the stability of cefepime over time at different baseline inocula Inoculum and timedependent hydrolysis was seen Hypothesis: β-lactamase mediated problem Antimicrob Agents Chemother 2012;56:

32 In Vitro No resistance emergence!

33 In Vitro Success of Combination Therapy As a protein synthesis inhibitor, we hypothesize that the aminoglycoside shuts down the expression of the ampc β-lactamase AAC 2012; 56:

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35 In Vitro We have gone as long as 6 months; 1-2 months is standard for us in MTB studies

36 In Vitro - Conclusions This in vitro system is flexible, powerful and reproducible It allows study of differences in PK, organisms, bacterial burden and resistance emergence It allows linkage of measures of regimen intensity to effect (cell kill and resistance suppression) It allows experiments to be carried out for clinicallyrelevant durations All the data are straightforwardly able to be modeled fully parametrically to increase insight and allow design of validation experiments

37 In Vitro - Conclusions WHAT IS MISSING IS MODELING ALL THE OUTPUTS AND USING THE DATA TO PEFORM A PROSPECTIVE VALIDATION STUDY THIS WILL IMPROVE CONFIDENCE!

38 Thank You for Your Attention!

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