Preclinical Testing of Novel Agents in Tuberculosis Treatment Regimens

1. Issues in testing regimens containing multiple novel agentsI. Preclinical Testing Jacques Grosset Johns Hopkins University School of Medicine, Baltimore, MD

2. What is Preclinical Testing? • In the year 2005, the general objective of preclinical testing is to determine whether a drug active in vitro against Mycobacterium tuberculosis is likely to contribute to improved treatment of tuberculosis • Its specific objectives are to assess the toxic, pharmacokinetic (PK), and pharmacodynamic (PD) properties of a given drug .

3. In vitro assessmentof properties of the drug alone • MIC, lowest drug concentration that preventsthe growth of at least 99% of the inoculated bacilli (CFU) • MBC, lowest drug concentration that killsat least 99% of the inoculated bacilli (CFU) • EmaxC, Concentration of Maximal Effect, i.e., lowest drug concentration beyond which there is no additional killing

4. EmaxC MBC MIC Exp Expt 2,inoculum 5.41 log10cfu; Expt 3, inoculum 6.27 log10

5. EmaxC MBC MIC Inoculum 6.27 log10 cfu; CFU counts after 2 weeks of culture in 7H9+OADC

6. In vivo sequential assessment of properties of the drug alone • Toxicity • Basic PK assessment: bioavailability (SIT, SBT) at non-toxic doses • Basic PD assessment: dose-ranging activity (MED, MBD, EmaxD) • If basic PK & PD data are favorable (the higher the ratio of toxic dose/effective dose, the better), - Dose fractionation studies to establish the PD parameters most closely correlated with bactericidal activity (AUC/MIC, Cmax/MIC, Time>MIC )

7. Example of a dose fractionation studyJayaram et al, AAC (2003); 47:2118-2124 After 10mg/kg CFU counts in mouse lung CFU counts after 6 days of treatment RIF pharmacodynamic parameter

8. An example What should not be done…. (to jump to mouse experiments without solid data)

9. CMC 4wks after infection Vehicle Control many visible lesions

10. (after 4wkstreatment) INH 25mg/kg no visible lesions

11. Four unknown compounds X1 100mg/kg X2 100mg/kg X3 100mg/kg X4 100mg/kg Even in groups treated with the highest doses, all mice had nodular lung lesions similar to those observed in controls

12. Serum Inhibitory Titer of compound “x” po, single oral dosing; ip, single intraperitoneal dosing INH, 25mg/kg; Compound “x”, 100mg/kg +, culture positive; 0, culture negative on day 14 Conclusion: No active serum concentrations of “x”

13. What should be done ? Take no short cuts but apply sequential procedures • Screen MIC (5, 1.25, and 0.15µg/ml) by standard validated method • If, and only if, MIC is favorable (≤1.25 µg/ml), perform serum inhibitory test in the mouse • If, and only if, the titer of the serum is favorable (≥1/4), determine MED, MBD EmaxD in the mouse.

14. Dose-ranging activity of PA-824 MED MBD MED MED MBD 2 logs EmaxD≥200 After 4 weeks of daily (5/7) treatment in mice aerosol infected with 5x103 CFU

15. Properties of the drug alone Secondary in vivo assessment • Confirmation of “bactericidal” activity: - select drug- resistant mutants when given alone - prevent selection of INH-resistant mutants when combined with INH • Assessment of “sterilizing” activity: ability of the compound to kill bacilli that persist after 2 months of daily treatment with RHZ.

16. Initiation of treatment Death of untreated controls 6.02 5.83 5.53 3.94

17. Selection of drug-resistant mutants 1Stover et al, Nature (2000);405:962

18. CFU counts in the lungs of mice treated with PA-824 in the continuation phase No treatment Initial phase regimen: RHZ Continuation phase regimen: 3.94 2.48 1.92 0.60 0

19. 1) the impact of rifampin2) the impact of pyrazinamide Assessment of activity in combination therapy: Two past examples

20. Comparative bactericidal activity of INH + SM vs. INH + RIF in mice … as in humans(Tubercle 1967;48:11-26; Tubercle 1962;43: 201-67; Tubercle 1969; 50 (march suppl):12-21) Log10 cfu in lungs months

21. Failure and relapse rates after INH+SM and INH+RIF* *From Mitchison; and Grosset & Ji; in Gangadharam & Jenkins, Chapman & Hall, 1998 Conclusion: because the mouse model is a pessimistic model, results achieved in the mouse are likely achievable in humans

22. Log10 cfu in lungs Comparative bactericidal activity of INH + SM, INH + RIF, and INH + RIF + PZA in mice… as in humans(Grosset, Tubercle 1978: 59:287; EA/BMRC, Tubercle 1986;67:5) months 22

23. Failure and relapse rates after INH+RIF (HR) and INH+RIF+PZA (HRZ) From Mitchison; and Grosset & Ji; in Gangadharam & Jenkins, Chapman & Hall, 1998

24. Assessment of activity in combination regimens • Activity after incorporation into the first-line regimen (2RHZ/4RH) • as supplement • as substitution • Activity after incorporation into new regimens

25. Pre-requisites for combination experiments in murine model • Realistic appraisal of doses to be tested • Assurance of compatible pharmacokinetics • Selection of infection parameters and outcomes relevant to human disease

26. 1. Assessment after incorporation in the first-line regimen (2RHZ/4RH*) • Activity of moxifloxacin * R, rifampin; H, isoniazid; Z, pyrazinamide

27. Results of log10 CFU counts from lung homogenates in mice treated with MXF and standard regimen 2RHZ/4RH.

28. Conclusions 1. The addition of MXF did not significantly improve the sterilizing activity of RHZ. 2. The substitution of MXF for R or Z was detrimental to the activity of RHZ 3. But, the substitution of MXF for H provided a regimen with substantially improved sterilizing activity 4. Phase II clinical studies evaluating the RMZ regimen will soon be underway

29. 2. Assessment after incorporation in new regimens • Activity of PA-824 in the RMZ* regimen * R, rifampin; M, moxifloxacin, Z, pyrazinamide

31. Proportion of mice relapsing after 3 months of therapy * * 79% 78% 46% 3 2 1 *p<0.05 vs. RMZ

32. Conclusions 1. The addition of PA-824 to the RMZ regimen did not improve the sterilizing activity of RMZ. 2. R is more sterilizing than PA-824, and the substitution of Pa for M or Z was detrimental to the activity of RMZ 3. But, the substitution of Pa for R provided a regimen with sterilizing activity approaching that of RMZ 4. Such a “PaMZ” regimen, without R and H, has great potential for HIV-TB, MDR-TB, etc.

33. To conclude 1. To date, the mouse model of TB chemotherapy has provided results predictive of clinical outcomes. 2. However, it is essential that the model utilizes the: - appropriate mouse species - appropriate infection with M. tuberculosis - equipotent dose of drugs - appropriate time points to assess cure 3.Preclinical testing of a drug is the best way to determine whether there is a need for a clinical trial (Nardell & Rubin, AJRCCM 2005; 172: 1361-62)

34. Acknowledgements All of these studies could not have been performed without the support of: • TB Alliance - NIAID (NIAID-DAIDS N01 AI 40007,NIAID K08 AI 58993, NIAID-DAIDS R01 AI 43846).