Animal Efficacy Studies for Antitubercular Agents

V. Balasubramanian

ASTRAZENECA R&D BANGALORE, INDIA

Do we need animal model(s) for determining

antitubercular efficacy?

Is the determination of blood levels sufficient

to predict the outcome against infection?

Some questions…...

Drug Class In vitro mode in vivo efficacyof killing parameter

• -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]

Some generalizations…..

Drug [mg/kg] MIC Cmax Fold T1/2 T > MIC[ug/ml] [ug/ml] [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 5 d

Pyrazinamide [100] 6 30 5 10 36

Ethambutol [100] 2 4 2 3 6

Pharmocokinetic Properties of Anti-mycobacterial Drugs

Drug [mg/kg] MIC Cmax Fold T1/2 T > MIC[ug/ml] [ug/ml] [h] [h]

Ethambutol [100] 2 4 2 3 6

Ethionamide [100] 1 2 2 2 4

Streptomycin [200] 0.5 3 6 2 8

Amikacin [200] 1 30 30 2 12

Kanamycin [200] 6 30 5 2 6

Sparfloxacin [50] 0.12 2.8 5.6 5 18

Ofloxacin [200] 1.0 10 20 7 35

Linezolid [50] 17.7 1.4

PNU-100480 [50] 7 0.7

Pharmocokinetic Properties of Anti-mycobacterial Drugs

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

1 10 100 10002

3

4

5

6

7

Dose [mg/kg]

Prob

it

H R E A S

Data from: Kradolfer, F. 1970. Antibiotica et Chemotherapia. 16:352-360

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

Bactericidal Effect of Chemotherapy - Kradolfer et al

Design• Intravenous infection with M. bovis Ravenal strain• Bactericidal effect: Culture negativity after prolonged oral treatment

Ref: Kradolfer, F & Schnell, R. 1971. Chemotherapy. 16:173-182

0 1500

1

2

3

4

5

6

7

8

Rif 40

Inh 25

Rif 40 + Inh 25

Rif 40 + Emb 150Rif 40 + Sm 250 s.c

200 250

Days

Log

10

cfu

/ lun

gs

Bactericidal Effect: An Initial Measure for Comparison

Iv infection (~107 cfu); treatment by gavage started 1 wk pi., for 4 wks.

• 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

0

1

2

3

4

5

6

7

8

9

Ctrl Rif20

Rbt20

Rlz20

Inh25

Pza150

Emb125

Lev200

Log

10

cfu

/ lu

ngs

Isoniazid, Rifamycins

2

3

4

5

6

7

8

9

Ctrl Inh25

100480100

Lin100

Epre100

Oxazolidinones

• Baohong Ji, Lounis, N., Truffot-Pernot C., Grosset,

J. 1995. A. A. C. T. 39: 1341 - 1344

• Lounis, N., Baohong Ji, Truffot-Pernot C., Grosset,

J. 1997. A. A. C. T. 41: 607 - 610

Aminoglycosides & Quinolones

Iv infection (~107 cfu); treatment by gavage started 1 day pi., for 4 wks.

0

1

2

3

4

5

6

7

Ctrl Inh25

Sm200

Kan200

Ami200

Ise200

Spf50

Ofl200

Lev200

Log

10

cfu

/ spl

een

Bactericidal Effect: An Initial Measure for Comparison

Bactericidal Effect: Infection Dose As a VariableTreatment by gavage started 1 day pi., for 4 wks.

0

1

2

3

4

5

6 Lungs

Spleen

Ctrl Inh25

Mox100

Inh25Mox 100

Log

10

cfu

/ org

an

iv infection (~105 cfu) iv infection (~107 cfu)

0

1

2

3

4

5

6

7

8 Lungs

Spleen

Ctrl Mox100

Inh25Mox 100

Log

10

cfu

/ org

an

• Miyazaki, E., R. E., Bishai, W. R. et al. 1999. A. A. C. T. 43: 85-89

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

• Early Bactericidal Activity

• Sputum Conversion

• Emergence of resistance

• Relapse Rates

Markers Factors

• Combination Regimen

• Duration of Rx

• Frequency of Dosing

Inh, Rif, Emb

Pza

Rif

[Early Bactericidal Activity]

[Sterilizing Activity]

108

107

106

105

104

103

102

2 4 6

Cfu

/ml i

n s

pu

tum

Time (months)

Why combination is needed and why is it at least six months long?

Drug

Isoniazid +++ +

Rifampicin ++ +++

Pyrazinamide + +++

Ethambutol + -

Streptomycin + -

Early Bactericidal Activity [EBA]

Sterilizing Activity

Measured byCfu from sputum for 1st 2 days after onset

of treatment

Relapse rates, 30 mo. after onset of

treatment

Importance Community Individual

Model for the Initial and Continuation Phases of Rx

Initial Phase Log10 cfu /spleen Spontaneous Relapse

[once daily for 2 months] (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 % mice with [once daily for 4 months] +ve spleen cultures

*+* 25+10+150 0 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

iv infection (107 cfu/animal) Rx: Oral gavage started 14 days pi.

Shortened Course - Daily Treatment with Rifapentine

0 4 8 12 16 20 24 280

1

2

3

4

5

6

7

8

9

Inh + Pza +Rfp 20

Inh 25 + Rif 20

Inh + Rif +Pza 150

0 4 8 12 16 20 24 280

1

2

3

4

5

6

7

8

9L

og10

cfu

/ or

gan

Spleen Lungs

Inh+Rif

Inh+Rif+Pza

Inh+Pza+Rfp

7/7

6/7

0/8

7/7

7/7

3/8

7/7

6/7

0/8

7/7

7/7

3/8

Ref: Cynamon, M. H. et al. 1999. A. A. C. T. 43: 2356-2360

Intermittent treatment with Rifapentine

0

1

2

3

SpleenLungs

ctrl Rfp2H2

Rfp1H1

Rfp2HZ2

4

6

8

Rfp1HZ1

Rx 12 weeks

iv. infection ~107 cfu / animal

Cynamon, M. H. et al. 1999.

A. A. C. T. 43: 2356-2360

Rx 8 weeks

0

1

2

3

4

5

6

7

8

Ctrl Rif6 Rfp1 Rif6HZ6

Rfp1HZ6

Rfp1HZ1

Spleen

Lungs

Log

10 c

fu /

orga

n

47%

Rifr mice

61%

Grosset J. et al. 1998. Am J Respir

Crit Care Med. 157:1436-1440

0 5 10 15 200

1

2

3

4

5

6

7

untreated

Rb (6/wk)

H (6/wk)HRb (2/wk)

HRb (1/wk)

unvaccinated

Weeks post vaccination

Log

10 c

fu /

sple

en0

BCG Infection

28

Dosing

44 100 128

8 wk12 wk

Prophylaxis

Ref: Jabes, D., Bruna, C. D., Rossi, R., Olliaro, P.

1994. A. A. C. T. 38:2346-2350

*

Cornell Model for Effect on Reactivation Disease

Inh+Pza7 wks

Test Rx6 wks

Infect iv8.8x105

Cort3 wks

Sac

Cort3 wks

Sac

2 9 15 23 26 32 35Wks.

• 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

Inh+Pza12 wks

NoRx

Test Rx18 wks

NoRx

NoRx

Cort

Infect iv3.95x105

Sac

0 12 16 34

Culture -veorgans

41 44 48

Treatment Dosing % Positive Log10cfu Log10cfuorgans spleen lungs

Placebo 100 3.242.2 5.770.11

Inh 25 daily 50 1.932.11 0.711.34

Rif 10 twice weekly 100 4.110.40 5.390.49

Rpt 10 once weekly 100 3.631.18 4.672.35

Inh+Rpt once weekly 37.5 1.462.04 1.782.49

Ref: Miyazaki, E., Chaisson, R. E., Bishai, W.R. 1999. A. A. C. T. 43:2126-2130

RifapentineAnimal 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

Tuberculosis Genitourinary 16.0%

Peritoneal 3.7%Meningeal 4.2%Bone/Joint 8.5%

Other 9.3%

Miliary 9.8%

Pleural 21.5%

Lymphatic 27.0%

Pu

lmon

ary

84.5

%

14.6%

0 50 100 150 200 2500

2

4

6

8

i.v.

resp.

Time (days)

Log

10cf

u/l

un

gs

Comparison of M. tuberculosis iv. vs. Respiratory Infection in Mice

Ref:1. Robert J. North. 1995. Mycobacterium tuberculosis is strikingly more virulent for mice when given via therespiratory 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.

0 25 50 75 100 1251

2

3

4

5

6

resp.

i.v.

Time (days)

Lungs Spleen

1 151

2

3

4

5

6

7

untreated

Rif 25 mg/kg

18 21 24 27 30 330 28 56 840

2

4

6

8

10

untreated

Rif 20 mg/kg

*

Model for Evaluating Primary Efficacy

Time (days)

84

Respiratory Challenge(10 cfu / animal)

Infect with M. tuberculosis

Intravenous Challenge (106 cfu / animal)

Log

10 c

fu /

lun

gs

14

350 20 40 60

0

2

4

6

8 CSU 93L

un

gs

0 20 40 600

2

4

6

8 Erdman

0 20 40 600

2

4

6

8 CSU 93

Sp

leen

0 20 40 600

2

4

6

8 Erdman

Untreated

Inh+Rif

Inh+Rbt

Inh+Rpt

Inh+Rlz

Log

10cf

u /

orga

n

Days

• Resp infection (100 cfu / animal)• Once a week oral Rx

Brooks, J & Orme, I. 1998. A. A. C. T. 42: 3047-3048

1/wk; 5 wks (resp) vs

6/wk;12 wks (iv)

Similar results

Combination Rx in the Respiratory Infection Model

Infect with M. tuberculosis

0 56 112-2

0

2

4

6

8

10

Untreated

Rif+PZA

126 133 140

[Untreated+Cortisone]

[Test Compound+Cortisone]

Days

Log

10cf

u /

lun

gs

RZ:8wk Test 3wk. 1wk

Model for Evaluating Sterilization [20 wk. assay]

Bactericidal Effect Determined in Guinea Pigs

0 28 56 84 112 1400

1

2

3

4

5

6

Untreated

Inh+Rif

Days

Log

10cf

u/pr

imar

y le

sion

Ref: Smith, D. W., Balasubramanian, V., Wiegeshaus, E. H. 1991. Tubercle. 72:223-2310

Culture negativity

•Limitation: poor thera. Index•Not predictable from PK/PD

Relapse rates

2HRZ/4RZ > 6HRZ > 2HRZ/4HR [8%] [34%] [38%]

Towards clinical use...

•No drug given singly •HRZ > HR > RZ > HZ•RE, RS, HE, not effective

•In humans 2HRZ/4HR = 6HRZ [~8%].•2HRZ/4RZ not given due to Z toxicity

• Rlz = Rfp > Rbu > Rif = Spar > H

• H > PNU100480 > Lin > Z = Ofla = Lev

• Z > Emb > Eth

• All significantly better than untreated control

Compounds exhibiting statistically significant reduction in cfu

PK predictive of efficacy within

but not across

Clinical Trial?

Clues from Animal Efficacy for Clinical Efficacy

• EBA

• Sputum conversion

• Drug resistance

• Relapse rates @ 30 mo.

• Duration of Rx

• Frequency of Rx

Humans• ??

• Rate of culture negativity

• Resistance of survivors

• Relapse rates @ 6 mo.

• Time to culture negativity +

relapse rates @ 6 mo.

• PK profiles such as T1/2

Expt. animals

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

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

Summary of important variables for the different efficacy models