improved efficacy of a cofactor-independent inha inhibitor...
TRANSCRIPT
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 )
BALB/c Mice C3HeB/FeJ Mice
• 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
BALB/c Mice C3HeB/FeJ Mice
• AN12855 retains activity in C3HeB/FeJ mice with advanced lung pathology
• 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
BALB/c Mice C3HeB/FeJ Mice
• AN12855 retains activity in C3HeB/FeJ mice with advanced lung pathology
• 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 8 Week ED-DPR9-RM2 none none G96V (sib)
12855 8 Week ED-DPR9-RM3 none none G96V (sib)
12855 8 Week ED-DPR9-RM5 none none G96V (sib)
12855 8 Week ED-DPR9-RM6 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
BALB/c Mice C3HeB/FeJ Mice
1.5 log1.7 log2.5 log
0.5 log
Treatment start
Isoniazid (25 mpk)
AN12855 (100 mpk)
P
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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