Issues in testing regimens containing multiple novel agents

I. Preclinical Testing

Jacques Grosset

Johns Hopkins University School of Medicine, Baltimore, MD

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 .

In vitro assessment of properties of the drug alone

- MIC, lowest drug concentration that prevents the growth of at least 99% of the inoculated bacilli (CFU)

- MBC, lowest drug concentration that kills at least 99% of the inoculated bacilli (CFU)

- EmaxC, Concentration of Maximal Effect, i.e., lowest drug concentration beyond which there is no additional killing

Log10 kill of M. tuberculosis by INH in 7H9 broth

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0 0.01 0.03 0.06 0.12 0.25 0.5 1 2 4 8concentrations

log1

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Exp Expt 2, inoculum 5.41 log10cfu; Expt 3, inoculum 6.27 log10

EmaxC

MBC

MIC

Log10 kill of M. tuberculosis by RIF in 7H9 broth

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Inoculum 6.27 log10 cfu; CFU counts after 2 weeks of culture in 7H9+OADC

In vivo sequential assessment of properties of the drug alone

1. Toxicity

2. Basic PK assessment: bioavailability (SIT, SBT) at non-toxic doses

3. Basic PD assessment: dose-ranging activity (MED, MBD, EmaxD)

4. 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 )

Example of a dose fractionation studyJayaram et al, AAC (2003); 47:2118-2124

RIF pharmacodynamic parameter

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CFU counts after 6 days of treatment

After 10mg/kg

An example

What should not be done….

(to jump to mouse experiments without solid data)

Vehicle Control 4wks after infectionCMC

many visible lesions

INH 25mg/kg (after 4wks treatment)

no visible lesions

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

Serum Inhibitory Titer of compound “x”

Serum Control Dilutions of serum in 7H9 broth

0 +, + 1/2 1/16 1/32 1/64 1/128

INH po 0 0 +

INH ip 0 0 +

“x1” po + +

“x1” ip + +

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

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.

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Dose-ranging activity of PA-824

MEDMBD

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MED

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After 4 weeks of daily (5/7) treatment in mice aerosol infected with 5x103 CFU

EmaxD≥200

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.

CFU counts in the lungs of mice treated with PA-824 alone or in combination

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Initiation of treatment

3.94

Death of untreated controls

6.025.835.53

Selection of drug-resistant mutants

Proportion of colonies resistant to:

Regimen

INH (0.2 µg/ml)

PA-824(2 µg/ml)

No treatment1 1.3 x 10-6 9 x 10-7

INH alone 2.5 x 10-4 --------

PA-824 alone -------- 3.8 x 10-3

INH + PA-824 < 5 x 10-6 5 x 10-6

1Stover et al, Nature (2000);405:962

012

34567

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No treatment

3.94

Initial phase regimen:

RHZ

Continuation phase regimen:

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0.600

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CFU counts in the lungs of mice treated with PA-824 in the continuation phase

1) the impact of rifampin

2) the impact of pyrazinamide

Assessment of activity in combination therapy: Two past examples

Comparative bactericidal activity of INH + SM vs. INH + RIF in Comparative bactericidal activity of INH + SM vs. INH + RIF in mice … as in humansmice … as in humans

((Tubercle 1967;48:11-26; Tubercle 1962;43: 201-67; Tubercle 1969; 50 (march suppl):12-21)Tubercle 1967;48:11-26; Tubercle 1962;43: 201-67; Tubercle 1969; 50 (march suppl):12-21)

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INH + RIF

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Failure and relapse rates after INH+SM and INH+RIF*

Regimen Duration (mo.)

Proportion of Mice and (Humans) with Positive Cultures:

On completion of treatment

3-6 mo. after treatment

INH+SM 6 100% (0) 100% (29)

INH+SM 18 35% (0) 75% (~10)

INH +RIF 6 0% (0) 20% (6-7)

INH+RIF 9 0% (0) 0% (1-3)

Conclusion: because the mouse model is a pessimistic model, results achieved in the mouse are likely achievable in humans

*From Mitchison; and Grosset & Ji; in Gangadharam & Jenkins, Chapman & Hall, 1998

Comparative bactericidal activityComparative bactericidal activity of INH + SM, INH + RIF, and of INH + SM, INH + RIF, and INH + RIF + PZA in mice… as in humansINH + RIF + PZA in mice… as in humans(Grosset, Tubercle 1978: 59:287; EA/BMRC, Tubercle 1986;67:5)(Grosset, Tubercle 1978: 59:287; EA/BMRC, Tubercle 1986;67:5)

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Failure and relapse rates after INH+RIF (HR) and INH+RIF+PZA (HRZ)

Drug regimen

Proportion of Mice and (Humans) with Positive Cultures:

On completion of treatment

3-6 mo. after treatment

6HR 0-10% (0) 40-60% (6-7)

2HRZ/4HR 0% (0) 10-30% (1-2)

From Mitchison; and Grosset & Ji; in Gangadharam & Jenkins, Chapman & Hall, 1998

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

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

1. Assessment after incorporation in the first-line regimen (2RHZ/4RH*)

• Activity of moxifloxacin

* R, rifampin; H, isoniazid; Z, pyrazinamide

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

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

2. Assessment after incorporation in new regimens

• Activity of PA-824 in the RMZ* regimen

* R, rifampin; M, moxifloxacin, Z, pyrazinamide

CFU counts after 2 months of treatment

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Pre-Rx RMZ RMZPa PaMZ RPaZ RMPaRegimens

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Proportion of mice relapsing after 3 months of therapy

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2PaMZ + 1PaM 2RPaZ + 1RPa 3RMPa

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*p<0.05 vs. RMZ

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46% 79% 78%

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.

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)

AcknowledgementsAll 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).