issues in testing regimens containing multiple novel agents i. preclinical testing jacques grosset...
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Issues in testing regimens containing multiple novel agents
I. Preclinical Testing
Jacques Grosset
Johns Hopkins University School of Medicine, Baltimore, MD
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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 .
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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
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Log10 kill of M. tuberculosis by INH in 7H9 broth
0
1
2
3
4
5
6
7
0 0.01 0.03 0.06 0.12 0.25 0.5 1 2 4 8concentrations
log1
0 k
ill
Exp Expt 2, inoculum 5.41 log10cfu; Expt 3, inoculum 6.27 log10
EmaxC
MBC
MIC
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Log10 kill of M. tuberculosis by RIF in 7H9 broth
0
1
2
3
4
5
6
7
0 0.12 0.25 0.5 1 2 4 8 16 32
concentration (mcg/ml)
Log
10 c
fu k
ill
MIC
MBC
EmaxC
Inoculum 6.27 log10 cfu; CFU counts after 2 weeks of culture in 7H9+OADC
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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 )
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Example of a dose fractionation studyJayaram et al, AAC (2003); 47:2118-2124
RIF pharmacodynamic parameter
CF
U c
ou
nts
in
mo
use
lu
ng
CFU counts after 6 days of treatment
After 10mg/kg
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An example
What should not be done….
(to jump to mouse experiments without solid data)
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Vehicle Control 4wks after infectionCMC
many visible lesions
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INH 25mg/kg (after 4wks treatment)
no visible lesions
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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
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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”
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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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0
1
2
3
4
5
6
7
8
log
CFU
in lu
ngs
Dose-ranging activity of PA-824
MEDMBD
2 logs
MED
0
1
2
3
4
5
6
7
8
log
CFU
in lu
ngs
MED
MBD
After 4 weeks of daily (5/7) treatment in mice aerosol infected with 5x103 CFU
EmaxD≥200
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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.
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CFU counts in the lungs of mice treated with PA-824 alone or in combination
0
2
4
6
8
10
12
-20 0 56Days
Lo
g 1
0 C
FU
co
un
t
PA-824
H + PA
H
RHZ
Initiation of treatment
3.94
Death of untreated controls
6.025.835.53
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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
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012
34567
89
10
-3 0 8 16 24 Weeks
Log
10 C
FU
cou
nt MHPA-824RH
No treatment
3.94
Initial phase regimen:
RHZ
Continuation phase regimen:
1.92
0.600
2.48
CFU counts in the lungs of mice treated with PA-824 in the continuation phase
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1) the impact of rifampin
2) the impact of pyrazinamide
Assessment of activity in combination therapy: Two past examples
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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)
0
1
2
3
4
5
6
7
8
0 1 2 3 6 9 12 18
INH + SM
INH + RIF
months
Lo
g1
0 cfu
in
lu
ng
s
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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
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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)
0
1
2
3
4
5
6
7
8
0 1 2 3 6 9 12 18
INH + SM
INH + RIF
INH + RIF + PZA
months
Lo
g1
0 cfu
in
lu
ng
s
22
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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
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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
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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
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1. Assessment after incorporation in the first-line regimen (2RHZ/4RH*)
• Activity of moxifloxacin
* R, rifampin; H, isoniazid; Z, pyrazinamide
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Results of log10 CFU counts from lung homogenates in mice treated with MXF and standard regimen 2RHZ/4RH.
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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
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2. Assessment after incorporation in new regimens
• Activity of PA-824 in the RMZ* regimen
* R, rifampin; M, moxifloxacin, Z, pyrazinamide
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CFU counts after 2 months of treatment
0
1
2
3
4
5
6
7
8
Pre-Rx RMZ RMZPa PaMZ RPaZ RMPaRegimens
Lo
g(1
0)
CF
U c
ou
nt
Lung
Spleen
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Proportion of mice relapsing after 3 months of therapy
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
2RMZ + 1RM 2RMZPa +1RMPa
2PaMZ + 1PaM 2RPaZ + 1RPa 3RMPa
% r
elap
se
* *
3
2
*p<0.05 vs. RMZ
1
46% 79% 78%
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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.
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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)
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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).