improved efficacy of a cofactor-independent inha inhibitor...

Improved efficacy of a cofactor-independent InhA

inhibitor revealed by the C3HeB/FeJ mouse model

Gregory T. Robertson, Ph.D.

Assistant Professor, Mycobacteria Research Labs

Michael S. Sherman, Matthew B. McNeil, David S. Carter, Yi Xia, Yasheen

Zhou, Wai Choi, Jason Halladay, Pam Berry, Weimin Mao, Vincent

Hernandez, Victoria A. Ektnitphong, Anthony J. Smith, Lisa Woolhiser, Devon

D. Dennison, Tanya Parish, M.R.K. Alley, Anne J. Lenaerts

Outline

• Behavior of INH and AN12855 in conventional mice

• The C3HeB/FeJ model as a tool to assess the role of advanced lung pathology

1. Impact on efficacy

2. Contributions to drug resistance

3. Altered regional PK

• Conclusions and questions

C3HeB/FeJ (Kramnik) mouse model

• Heterogeneity of pulmonary lesions similar to that seen in human disease*

• Develops highly organized necrotic lesions (unlike conventional mice, ie BALB/c)

– Higher ratio of extracellular/intracellular

– Altered drug penetration

– Heterogeneity in bacterial phenotypes

– Higher incidence of emergence of drug resistance in vivo**

f

c

n

a

*Irwin et al., Dis. Model Mech. 2015, 8:591; **Driver et al., AAC. 2012, 56(6):3181.

H/E SYBR-Gold

Mtb in green

In vitro improvements of AN12855 over Isoniazid

Lys 165

Tyr 158

Glu 219

Arg 195

Ile 194

AN12855

Activity of diazaborines against M. tuberculosis drug resistant clinical isolates

Strain ResistanceMIC (µg/ml)

AN12855 INH MOXI RIF

H37Rv None (WT) 0.13 ≤ 0.06 0.13 ≤ 0.06

M70 FQ, STR, INH, RIF, PZA 0.25 > 16 1 > 16

M28 FQ, INH, RIF, EMB, PZA 0.25 > 16 2 > 16

TN5904 STR, INH, RIF, PZA 0.13 1 0.13 > 16

AN12855

• Direct inhibitor of InhA

• Co-factor independent (IC50 0.03 µM)

• Retains activity vs MDR strains

• ~ 100x lower frequency of resistance

Activity of AN12855 and isoniazid in conventional mice

GKO Acute Physical Chemical Properties: AN12855

Mol Wt 441

LogD 7.5 -0.07

In vitro ADME

Metabolic Stability (Mo/Hu S9) t½ >350 min

PPB 95% @ 20 µM

Aqueous Solubility >200 µM

72 hr Cytotox (HepG2/Jurkat) 194 µM, 55.2 µM

In vivo Safety

MTD > 200 mg/kg/d

PK Properties

CL (mL/h/kg) 348 (IV @5)

Vss (mL/kg) 1270 (IV @5)

Bioavailability (%) 53% (oral @10)

Plasma AUC (h*ug/mL) 15.4 (oral @10)

Lung AUC (h*ug/mL) 10.4 (oral @10)

BALB/c Chronic

0 5 10 15 20 250

2

4

6

8

10

Days

log

10 C

FU

/Lung

Untreated

1 mg/kg

25 mg/kg50 mg/kg

INH (25 mg/kg)

AN12855

0 2 0 4 0 6 0 8 0

0

2

4

6

8

1 0

D a y s

log

10

CF

U/L

un

gU n t r e a t e d

5 m g / k g

2 5 m g / k g

1 0 0 m g / k g

I N H ( 2 5 m g / k g )

A N 1 2 8 5 5

A.

B.

• AN12855 exhibits dose dependent efficacy that is comparable to INH in conventional mouse models

Possible explanations:

1. Microenvironment does not support conversion of INH to an active form?

2. Emergence of INH-R and lower resistance frequency of AN12855?

3. Altered lesion-specific PK owing to advance lung pathology?

2

3

4

5

6

7

8

Lo

g1

0 C

FU

/Lu

ng

4

5

6

7

8

9

1 0

Lo

g1

0 C

FU

/Lu

ng

AN12855 outperforms isoniazid in C3HeB/FeJ mice

56 days Rx 37 days Rx

BALB/c Mice C3HeB/FeJ Mice

1.5 log1.7 log2.5 log

0.5 log

Treatment start

Isoniazid (25 mpk)

AN12855 (100 mpk)

Experimental set up

Aerosol infection (100 CFU/mouse)

Treatment (2, 4, 8 weeks, 5/7)

Day 15 mice

Day 68 Start of Rx

Day 82 Day 96 Day 124

9 weeks (for pathology)

Sacrifice 8 mice/grp

Plate for CFU

Co-plate for drug-R (5x MIC)

C3HeB/FeJ chronic model

BALB/c chronic modelAerosol infection (100 CFU/mouse)

Treatment (2, 4, 8 weeks, 5/7)

Day 15 mice

Day 28 Start of Rx

Day 42 Day 56

4 weeks

Sacrifice 6 mice/grp

Plate for CFU

Co-plate for drug-R (5x MIC)

Day 84

Comparative efficacy in BALB/c and C3HeB/FeJ mice

0 1 4 2 8 4 2 5 6 7 0 8 4

1

2

3

4

5

6

7

8

D a y s p . i .

Lo

g1

0 C

FU

/Lu

ng

0 2 8

2

3

4

5

6

7

8

9

5 6 7 0 8 4 9 8 1 1 2

D a y s p . i .L

og

10

CF

U/L

un

g

U n t C n t r

I N H ( 2 5 )

A N 1 2 8 5 5 ( 1 0 0 )


• AN12855 retains activity in C3HeB/FeJ mice with advanced lung pathology

Emergence of drug resistance in C3HeB/FeJ mice

0 1 4 2 8 4 2 5 6 7 0 8 4

1

2

3

4

5

6

7

8

D a y s p . i .

Lo

g1

0 C

FU

/Lu

ng

0 2 8

2

3

4

5

6

7

8

9

5 6 7 0 8 4 9 8 1 1 2

D a y s p . i .L

og

10

CF

U/L

un

g

U n t C n t r

I N H ( 2 5 )

A N 1 2 8 5 5 ( 1 0 0 )

I N H - R

A N 1 2 8 5 5 - R



• Appreciable INH-R, and minimal AN12855 Drug-R in C3HeB/FeJ mice

• No appreciable emergence of INH or AN12855 Drug-R in BALB/c mice

Emergence of drug resistance in C3HeB/FeJ mice

0 1 4 2 8 4 2 5 6 7 0 8 4

1

2

3

4

5

6

7

8

D a y s p . i .

Lo

g1

0 C

FU

/Lu

ng

0 2 8

2

3

4

5

6

7

8

9

5 6 7 0 8 4 9 8 1 1 2

D a y s p . i .L

og

10

CF

U/L

un

g

U n t C n t r

I N H ( 2 5 )

A N 1 2 8 5 5 ( 1 0 0 )

I N H - R

A N 1 2 8 5 5 - R



• Appreciable INH-R, and minimal AN12855 Drug-R in C3HeB/FeJ mice

• No appreciable emergence of INH or AN12855 Drug-R in BALB/c mice

• AN12855 is more effective than isoniazid and exhibits lower emergence of

drug resistance in C3HeB/FeJ mice with advanced lung pathology

Plate Description Strain FabGI-PinhA KatG SNP InhA SNP

Pre Rx-on INH ED-DPR2-RM2 none W328R none

Pre Rx-on INH ED-DPR3-RM1 none H108P none

Pre Rx-on INH ED-DPR3-RM2 none D189V none

INH 8 week ED-DPR7-RM2 none A172T (sib) none

INH 8 week ED-DPR7-RM3 none A172T (sib) none

INH 8 week ED-DPR8-RM1 none R571H none

INH 8 week ED-DPR8-RM2 none W477Stop none

INH In vitro ED-DPR16-RM1 none R104Q none

INH In vitro ED-DPR16-RM2 none Q525Fs none

INH In vitro ED-DPR17-RM1 none G440Fs none

INH In vitro ED-DPR17-RM2 none N637Fs none

Fs: Frameshift from residue position.

in vitro and in vivo Isoniazid resistance alleles

Matthew B. McNeil, Devon D. Dennison, Tanya Parish (IDRI)

• Isoniazid resistance in vitro or in vivo in C3HeB/FeJ mice is correlated with

loss of function mutations in katG

Plate Description Strain FabGI-PinhA KatG SNP InhA SNP

12855 8 Week ED-DPR9-RM1 none none G96V (sib)





12855 in vitro ED-DPR18-RM1 none none G96A

12855 in vitro ED-DPR18-RM2 C-15T none none

12855 in vitro ED-DPR18-RM3 none none G96A

12855 in vitro ED-DPR18-RM4 none none G96V

in vitro and in vivo AN12855 resistance alleles

Strain Relevant genotype

Liquid MIC (µM)

INH AN12855

Erdman-WT WT 0.8 0.16

ED-DPR18-RM1 InhA (G96A) 0.9 > 1.0

ED-DPR18-RM4 InhA (G96V) 1.1 > 1.0

ED-DPR18-RM2 fabG1-PinhA (C-15T) 5.4 > 1.0

G96

AN12855

Matthew B. McNeil, Devon D. Dennison, Tanya Parish (IDRI)

Possible explanations:

1. Microenvironment does not support conversion of INH to an active form?

2. Emergence of INH-R and lower resistance frequency of AN12855?

3. Altered lesion-specific PK owing to advance lung pathology?

2

3

4

5

6

7

8

Lo

g1

0 C

FU

/Lu

ng

4

5

6

7

8

9

1 0

Lo

g1

0 C

FU

/Lu

ng

AN12855 outperforms isoniazid in C3HeB/FeJ mice

56 days Rx 37 days Rx


1.5 log1.7 log2.5 log

0.5 log

Treatment start

Isoniazid (25 mpk)

AN12855 (100 mpk)

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Lesion pharmacokinetic analysis: (7 days of Rx)

Necrotic

Lesion

AN12855 (100)Isoniazid (25)

C

Cmax (0.5h or 2h)

AN12855 (100)Isoniazid (25)

Trough (24 h)

Aerosol infection (100 CFU/mouse)

Treatment (7 consecutive days)

Day 15 mice

Day 70 Day 77

10 weeks

LOQ LOQ

Conclusions

▪ The C3HeB/FeJ mouse model allows for the more dynamic assessment of TB antimicrobial agents

➢Assessment of in vivo activity

➢Assessment of the development and expansion of Drug-R

➢Assessment of drug exposures in necrotic lesions

Acknowledgements

AnacorM. R. K. AlleyYi XiaYasheen ZhouDavid S. Carter Wai ChoiJason HalladayPam BerryWeimin Mao Vincent Hernandez

Colorado State UniversityAnne J. LenaertsMichael S. SchermanAnthony J. Smith Victoria A. EktnitphongLisa K. WoolhiserVeronica Gruppo

Infectious Disease Research InstituteTanya ParishMatthew B. McNeilDevon Dennison

Funding: Bill and Melinda Gates Foundation

improved efficacy of a cofactor-independent inha inhibitor...

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