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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
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
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
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
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
CF
U c
ou
nts
in
mo
use
lu
ng
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.
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
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
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
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
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
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)
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
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)
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
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
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
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%
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).