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Animal Efficacy Studies for Antitubercular Agents
V. Balasubramanian
ASTRAZENECA R&D BANGALORE, INDIA
Some questions…...
 Do we need animal model(s) for determining
antitubercular efficacy?
 Is the determination of blood levels sufficient
to predict the outcome against infection?
Some generalizations…..
Drug Class
In vitro mode
of killing
in vivo efficacy
parameter
• b-lactams
Time dependent
T > MIC
• Aminoglycosides
Concentration dependent
AUC/AUIC ratio
• Fluoroquinolones
Concentration dependent
AUC/AUIC ratio
• Macrolides
Time dependent
AUC [half-lives and
PAE high]
T > MIC [half lives
and PAE low]
Pharmocokinetic Properties of Anti-mycobacterial Drugs
Drug [mg/kg]
MIC Cmax Fold
[ug/ml] [ug/ml]
T1/2 T > MIC
[h]
[h]
Isoniazid [25]
0.02
7
350
2
18
Rifampicin [25]
0.1
10
100
3.5
28
Rifabutin [25]
0.05
10
200
45
10 d
Rifapentine [25]
0.02
15
750
13.2
5d
Pyrazinamide [100] 6
30
5
10
36
Ethambutol [100]
4
2
3
6
2
Pharmocokinetic Properties of Anti-mycobacterial Drugs
Drug [mg/kg]
MIC Cmax Fold
[ug/ml] [ug/ml]
Ethambutol [100]
2
Ethionamide [100] 1
Streptomycin [200] 0.5
Amikacin [200]
1
Kanamycin [200]
6
Sparfloxacin [50] 0.12
Ofloxacin [200]
1.0
Linezolid [50]
PNU-100480 [50]
4
2
3
30
30
2.8
10
17.7
7
2
2
6
30
5
5.6
20
T1/2 T > MIC
[h]
[h]
3
2
2
2
2
5
7
1.4
0.7
6
4
8
12
6
18
35
Effect of Chemotherapy on Survival - Kradolfer et al
Design
• Intravenous infection with M. bovis Ravenal strain
• Oral administration of drugs - day 11 & 12 post infection (Sm - s.c.)
• Minimal effect: Prolonging the survival of 50% mice in the treated groups - ED50
7
H
R
E
A S
Probit
6
5
4
3
2
1
10
100
1000
ED50 [mg/kg]
Isoniazid (H)
2.7 - 3.4
Rifampicin (R)
4.5 - 6.5
Ethambutol (E)
35 - 55
Ethionamide (A)
75 - 107
Streptomycin (S)
75 - 108
Dose [mg/kg]
Data from: Kradolfer, F. 1970. Antibiotica et Chemotherapia. 16:352-360
Bactericidal Effect of Chemotherapy - Kradolfer et al
Design
• Intravenous infection with M. bovis Ravenal strain
• Bactericidal effect: Culture negativity after prolonged oral treatment
8
Ref: Kradolfer, F & Schnell, R. 1971. Chemotherapy. 16:173-182
Log 10 cfu / lungs
7
6
5
Inh 25
4
Rif 40
Rif 40 + Emb 150
Rif 40 + Sm 250 s.c
3
2
1
0
Rif 40 + Inh 25
0
150
200
Days
250
Bactericidal Effect: An Initial Measure for Comparison
Iv infection (~107 cfu); treatment by gavage started 1 wk pi., for 4 wks.
Isoniazid, Rifamycins
Oxazolidinones
9
9
Log 10 cfu / lungs
8
7
8
6
7
5
6
4
5
3
4
2
3
1
0
Ctrl Rif Rbt Rlz Inh Pza Emb Lev
20 20 20 25 150 125 200
2
Ctrl
Inh
25
100480
100
• Cynamon, M. H., Klemens, S. P., Sharpe, C. A., Chase, S. 1999. A. A. C. T. 43:1189-1191
• Klemens, S. P., Grossi, M. A., Cynamon, M. H. 1994. A. A. C. T. 38: 2245-2248
Lin
100
Epre
100
Bactericidal Effect: An Initial Measure for Comparison
Iv infection (~107 cfu); treatment by gavage started 1 day pi., for 4 wks.
7
Aminoglycosides & Quinolones
Log 10 cfu / spleen
6
5
• Baohong Ji, Lounis, N., Truffot-Pernot C., Grosset,
J. 1995. A. A. C. T. 39: 1341 - 1344
4
• Lounis, N., Baohong Ji, Truffot-Pernot C., Grosset,
J. 1997. A. A. C. T. 41: 607 - 610
3
2
1
0
Ctrl Inh Sm Kan Ami Ise Spf Ofl Lev
25 200 200 200 200 50 200 200
Bactericidal Effect: Infection Dose As a Variable
Treatment by gavage started 1 day pi., for 4 wks.
iv infection (~105 cfu)
iv infection (~107 cfu)
6 Lungs
Spleen
7
Log 10 cfu / organ
Log 10 cfu / organ
5
8 Lungs
4
3
2
Spleen
6
5
4
3
2
1
1
0
0
Ctrl
Inh
25
Mox
100
Inh25
Mox 100
• Miyazaki, E., R. E., Bishai, W. R. et al. 1999. A. A. C. T. 43: 85-89
Ctrl
Mox
100
Inh25
Mox 100
PK Parameters
Significant bactericidal activity in mice
H >>> Z > Emb > Eth [Cmax, T > MIC]
H > Z > Emb > Eth
Rfp = Rlz = Rbu > Rif [Cmax, T > MIC]
Rlz = Rfp > Rbu > Rif
Ami > Sm = Kan [Cmax]
Ami > Sm = Kan
Spar > Lev = Ofla [Cmax]
Spar > Lev = Ofla
H > Linezolid > PNU100480 [?]
H > PNU100480 > Linezolid
No single parameter can independently
Rlz = Rfp > Rbu > Rif = Spar > H
predict across drug classes
H > PNU100480> Lin > Z
Z = Ofla > Emb > Eth
Clinical Efficacy in Tuberculosis
Markers
Factors
• Early Bactericidal Activity • Combination Regimen
• Sputum Conversion
• Duration of Rx
• Emergence of resistance
• Frequency of Dosing
• Relapse Rates
Why combination is needed and why is it at least six months long?
Inh, Rif, Emb
108
Cfu/ml in sputum
107
[Early Bactericidal Activity]
106
105
104
Pza
[Sterilizing Activity]
103
Rif
102
2 Time (months) 4
6
Early Bactericidal
Activity [EBA]
Sterilizing Activity
Measured by
Cfu from sputum for
1st 2 days after onset
of treatment
Relapse rates, 30
mo. after onset of
treatment
Importance
Community
Individual
Drug
Isoniazid
Rifampicin
Pyrazinamide
Ethambutol
Streptomycin
+++
++
+
+
+
+
+++
+++
-
Model for the Initial and Continuation Phases of Rx
iv infection (107 cfu/animal)
Initial Phase
[once daily for 2 months]
Rx: Oral gavage started 14 days pi.
Log10 cfu /spleen Spontaneous Relapse
(6 mo. post)
Untreated
0
6.65 ± 0.19
Inh+Rif
25 + 10
3.00  0.54
Rif+PZA
10 + 150
0.86  0.44
*Inh+Rif+PZA
25 + 10+ 150
2.79  0.73
Continuation Phase
[once daily for 4 months]
*+*
25+10+150
0
% mice with
+ve spleen cultures
35%
*+Inh+Rif
25+10
0
38%
*+Rif+PZA
10+150
0
9%
Ref: J. Grosset, Truffot-Pernot, C., Lacroix, C., Ji. B. 1992. A.A.C.T. 36: 548-551
Shortened Course - Daily Treatment with Rifapentine
Spleen
Log10 cfu / organ
9
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
0
0
0
4
8
12
16
Lungs
9
20
24
28
Inh 25 + Rif 20
Inh + Rif +Pza 150
Inh + Pza +Rfp 20
0
4
8
12
16
20
24
Inh+Rif
7/7
7/7
7/7
7/7
Inh+Rif+Pza
6/7
7/7
6/7
7/7
Inh+Pza+Rfp
0/8
3/8
0/8
3/8
Ref: Cynamon, M. H. et al. 1999. A. A. C. T. 43: 2356-2360
28
Intermittent treatment with Rifapentine
iv. infection ~107 cfu / animal
8
Log10 cfu / organ
7
Lungs
Rx 8 weeks
Spleen
8 Lungs
Spleen
6
Rx 12 weeks
4
6
Rifr mice
47% 61%
5
3
4
2
3
2
1
1
0
0
Ctrl
Rif6
Rfp1 Rif6 Rfp1 Rfp1
HZ6 HZ6 HZ1
ctrl
Rfp2
H2
Rfp1
H1
Rfp2
HZ2
Grosset J. et al. 1998. Am J Respir
Cynamon, M. H. et al. 1999.
Crit Care Med. 157:1436-1440
A. A. C. T. 43: 2356-2360
Rfp1
HZ1
Prophylaxis
0
28
44
100
128
8 wk
BCG
Infection
Log10 cfu / spleen
7
6
12 wk
Dosing
Ref: Jabes, D., Bruna, C. D., Rossi, R., Olliaro, P.
unvaccinated
5
1994. A. A. C. T. 38:2346-2350
untreated
4
HRb (1/wk)
3
*
2
1
0
0
5
10
15
Weeks post vaccination
H (6/wk)
HRb (2/wk)
Rb (6/wk)
20
Cornell Model for Effect on Reactivation Disease
Wks.
2
9
Inh+Pza
7 wks
15
Test Rx
6 wks
23
26
Cort
3 wks
Infect iv
8.8x105
•
•
•
•
Sac
32
35
Cort
3 wks
Sac
Question: After the initial phase, what confers sterilization?
Test regimens were R [15mg/kg]; RH [H25]; RZ [Z150]; RHZ
% positive organs : R 81; RH 63; RZ 65; RHZ 71 [p = 0.3]
However, trend chi-square suggested than addition of H or Z
improved the sterilization effect of R
• Answer: At least in the Cornell model, none of the existing
regimens confer complete sterilization
Ref: Dhillon, J., Dickinson, J. M., Sole, K., Mitchison, D. A. 1996.
A. A. C. T. 40: 552-555
Cornell Model for Effect on Reactivation Disease
0
12
Inh+Pza
12 wks
Infect iv
3.95x105
Treatment
16
No
Rx
34
Test Rx
18 wks
41
No
Rx
Culture -ve
organs
Dosing
Placebo
44
Cort
48
No
Rx
Sac
% Positive
organs
Log10cfu
spleen
Log10cfu
lungs
100
3.242.2
5.770.11
Inh 25
daily
50
1.932.11
0.711.34
Rif 10
twice weekly
100
4.110.40
5.390.49
Rpt 10
once weekly
100
3.631.18
4.672.35
Inh+Rpt
once weekly
37.5
1.462.04
1.782.49
Ref: Miyazaki, E., Chaisson, R. E., Bishai, W.R. 1999.
A. A. C. T. 43:2126-2130
Rifapentine
Animal Data
• Daily dosing with Rfp offers the possibility of reduced Rx.
• However, due to long half life, possibility of accumulation
• Based on animal studies, twice weekly better than once weekly
• Based on daily dosing in animals, HZRfp > HZRif (relapse rates)
• However, relapse rates not determined in the case of intermittent Rx!)
• Intermittent Rfp approved by FDA in 1988 (1st in >15 yrs for TB)
Clinical Trial data (surprises!)
• 2HRZ/4H2R2 better than 2HRpZ/H1Rp1, based on relapse data
• 4/5 HIV+ patients in 2HRpZ/H1Rp1 developed Rifres
Ref: Vernon, A. et al. 1998. Am. J. Resp. Crit. Care. Med. 157: A467
ROUTE OF INFECTION AS A VARIABLE
Infection Route
Intravenous
Respiratory
Infection Dose
106 cfu/animal
10 cfu/animal
Quantitation
cfu / spleen
cfu / lungs
Infrastructure
Simple
Specialized
14.6%
Peritoneal 3.7%
Meningeal 4.2%
Bone/Joint 8.5%
Other 9.3%
Tuberculosis
Pulmonary
84.5%
•
•
•
•
Miliary 9.8%
Genitourinary 16.0%
Pleural 21.5%
Lymphatic 27.0%
Comparison of M. tuberculosis iv. vs. Respiratory Infection in Mice
Lungs
Spleen
6
resp.
i.v.
8
Log 10cfu/lungs
i.v.
5
6
4
resp.
4
3
2
0
2
0
50
100
150
Time (days)
200
250
1
0
25
50
75
100
125
Time (days)
Ref:
1. Robert J. North. 1995. Mycobacterium tuberculosis is strikingly more virulent for mice when given via the
respiratory route than via the intravenous route. J. Inf. Dis. 172:1550-1553.
2. Orme, I. M. & F. M. Collins. 1994. Mouse Model of Tuberculosis. In:Bloom, B. R. (ed) Tuberculosis:
Pathogenesis, Protection and Control. ASM, Washington D.C., pp113-134.
Model for Evaluating Primary Efficacy
Infect with
M. tuberculosis
Intravenous Challenge
(106 cfu / animal)
10
Respiratory Challenge
(10 cfu / animal)
7
untreated
*
Log10 cfu / lungs
8
untreated
6
5
6
4
Rif 25 mg/kg
4
3
Rif 20 mg/kg
2
2
0
0
56
28
84
1
1
84
15 18
21
24
27
14
Time (days)
30
33
Combination Rx in the Respiratory Infection Model
8
8
CSU 93
6
6
Lungs
Untreated
Inh+Rif
Erdman
Inh+Rbt
4
4
2
2
Inh+Rpt
Log10cfu / organ
Inh+Rlz
0
0
20
40
60
0
0
20
40
60
35
Spleen
8
8
CSU 93
6
6
4
4
2
2
0
0
20
40
60
0
0
Days
• Resp infection (100 cfu / animal)
• Once a week oral Rx
Erdman
Brooks, J & Orme, I. 1998.
A. A. C. T. 42: 3047-3048
Similar results
1/wk; 5 wks (resp) vs
20
40
60
6/wk;12 wks (iv)
Model for Evaluating Sterilization
[20 wk. assay]
Infect with
M. tuberculosis
10
Untreated
Log10cfu /lungs
8
6
[Untreated+Cortisone]
Rif+PZA
4
2
[Test Compound+Cortisone]
0
-2
0
56
112
126
133
140
Days
RZ:8wk
Test 3wk.
1wk
Bactericidal Effect Determined in Guinea Pigs
Log10cfu/primary lesion
6
5
Untreated
4
3
2
1
0
0
Inh+Rif
28
56 84
Days
112 140
Ref: Smith, D. W., Balasubramanian, V., Wiegeshaus, E. H.
1991. Tubercle. 72:223-2310
Towards clinical use...
Compounds exhibiting statistically significant reduction in cfu
PK predictive of efficacy within
but not across
• Rlz = Rfp > Rbu > Rif = Spar > H
• H > PNU100480 > Lin > Z = Ofla = Lev
• Z > Emb > Eth
• All significantly better than untreated control
Culture negativity
•Limitation: poor thera. Index
•Not predictable from PK/PD
•No drug given singly
•HRZ > HR > RZ > HZ
•RE, RS, HE, not effective
Relapse rates
2HRZ/4RZ > 6HRZ > 2HRZ/4HR
[8%]
[34%]
[38%]
•In humans 2HRZ/4HR = 6HRZ [~8%].
•2HRZ/4RZ not given due to Z toxicity
Clinical Trial?
Clues from Animal Efficacy for Clinical Efficacy
Humans
Expt. animals
• EBA
• ??
• Sputum conversion
• Rate of culture negativity
• Drug resistance
• Resistance of survivors
• Relapse rates @ 30 mo.
• Relapse rates @ 6 mo.
• Duration of Rx
• Time to culture negativity +
relapse rates @ 6 mo.
• Frequency of Rx
• PK profiles such as T1/2
Efficacy Models During the Course of Development
• Effect (singly or combination) during early phase of infection
* Survival
* Bacterial counts
• Spontaneous Relapse after initial and continuation phase
• Reactivation following immunosuppression - Cornell
• Prophylaxis
• Effect on immunocompromised hosts
• All of the above in animals infected via the airway will be more
efficient and relevant
Summary of important variables for the different efficacy models
Variables
Bactericidal effect
Spontaneous relapse*
Vaccination
-
-
Vaccination-infection interval
-
-
Route of infection
i.v. or resp
i.v. or (resp)
Infection inoculum
~107cfu or 100 cfu
~107cfu or (100 cfu)
Infection-Drug interval
2-4 weeks
2-4 weeks
Duration of initial treatment
8 wks (iv); 2 wks (resp)
8 wks; (4 wks)
Duration of continuation Rx
-
16 wks; (8 wks)
Duration of immunosuppression
-
-
Post treatment interval
1 day
1 day, 3 & 6 months
Measure of efficacy
Cfu / spleen or lungs
Cfu / spleen or lungs
% mice with +ve organ cultures
*conditions shown in parentheses not yet established
Summary of important variables for the different efficacy models
Variables
Sterilization*
Prophylaxis
Vaccination
-
BCG
Vaccination-infection interval
-
4 weeks
Route of infection
i.v. or (resp)
i.v. or resp
Infection inoculum
~107cfu or (100 cfu)
~107cfu or 100 cfu
Infection-Drug interval
2-4 weeks
2-4 weeks
Duration of initial treatment
12 weeks
8-12 weeks
Duration of continuation Rx
12 weeks
-
Duration of immunosuppression
3 weeks
-
Post treatment interval
1 day, 2-4 weeks
1 day
Measure of efficacy
Cfu / spleen or lungs
Cfu / spleen or lungs
% mice with +ve organ cultures
*conditions shown in parentheses not yet established