Predicting, Identifying and Managing Aldehyde

Oxidase Metabolism in Drug Discovery

Aaron C. Burns

251st ACS National Meeting

March 13, 2016

Structural and Mechanistic Aspects of Aldehyde Oxidase Metabolism

HN

N NH

HN

O

S

MoS

OPO3Na2H2N

OH

H

O S

OH

molybdenum cofactor (MoCo)

Nucleophilic molybdate reacts with electrophilic substrates: heteroarenes and P450 metabolites (e.g., aldehydes or iminium species)1

Water is the source of oxygen atom, in contrast to O2 with P450 (H2

18O)2

AO and xanthine oxidase (XO) homology (1°, ~50%)

1Pryde, D. C.; Dalvie, D.; Hu, Q.; Jones, P.; Obach, S.; Tran, T.-D. J. Med. Chem. 2010, 53, 8441. 2Hutzler, J. M.; Yang, T.-S.; Albaugh, D.; Fullenwider, C. L.; Schmenk, J.; Fisher, M. B. Drug Metabol. Disp. 2012, 40, 267

Comparison of Aldehyde and Xanthine Oxidases

1Sodhi, J. K.; Wong, S.; Kirkpatrick, D. S.; Liu, L.; Khojasteh, S. C.; Hop, E. C. A.; Barr, J. T.; Jones, J. P.; Halladay, J. S. Drug Metab. Disp. 2015, 43, 908.

Poor Human PK in Clinical Trials Due to AO Metabolism

Essentially all of these clinical failures (after carbazeran) could have been avoided if more comprehensive DMPK studies were conducted prior to human dosing (Hutzler et al. Expert Opin. Drug Metab. Toxicol. 2013, 9, 153)

The industry has been learning the same lessons about AO over and over again (Di, L. Expert Opin. Drug Metab. Toxicol. 2014, 10, 379)

Man’s Best Friend Gets His Revenge (Clinical Trial Failures)

Rat (variable): depends on specific rat strain Dog (none) Human (high)

With Respect to IV Metabolism:

Garattini, E.; Terao, M. Expert Opin. Drug Metab. Toxicol. 2012, 8, 487

★

Dramatic Variation Among Species, Gender and Substrates

To make matters worse, the rank order of reactivity with respect to species can vary with the substrate1,2

Makes allometric scaling to predict human dose difficult

1Dalvie, D.; Xiang, C.; Kang, P.; Zhou, S. Xenobiotica 2013, 43, 399. 2Choughule, K. V.; Barr, J. T.; Jones, J. P. Drug Metab. Disp. 2013, 41, 1852.

Issues Associated with In Vitro Determination of Intrinsic Clearance

Differences between manufactures and lots (isolation/stability)1

Donor to donor differences have been observed with hepatocytes2

Single nucleotide polymorphs (SNPs) have been identified, but the clinical relevance is still being debated1

1Zientek, M. A.; Youdim, K. Drug Metab. Dispos. 2013, 43, 163. 2Hutzler, J. M.; Yang, Y.-S.; Brown, C.; Heyward, S.; Moeller, T. Drug Metab. Dispos. 2014, 42, 1090.

In Vitro Human Clearance Prediction

Use pooled cytosol/S91 or cryopreserved hepatocytes2

All methods tend to underpredict in vivo clearance (extrahepatic expression: kidney & lung)

By comparing the Cint to compounds with human PK data available, one can classify into low, moderate and high groups1

1Zientek, M.; Jiang, Y.; Youdim, K.; Obach, R. S. Drug Metab. Disp. 2010, 38, 1322. 2Hutzler, J. M.; Yang, Y.-S.; Albaugh, D.; Fullenwider, C. L.; Schmenk, J.; Fisher, M. B. Drug Metab. Disp. 2012, 40, 267.

Pfizer AO Empirical Calibration Curve to Predict Human Clearance

Cheng, H.; Li, C.; Bailey, S.; Baxi, S. M.; Goulet, L.; Guo, L.; Hoffman, J.; Jiang, Y.; Johnson, T. O.; Johnson, T. W.;Knighton, D. R.; Li, J.; Liu, K., K.-C.; Liu, Z.; Marx, M. A.; Walls, M.; Wells, P. A.; Yin, M.-J.; Zhu, J.; Zientek, M. ACS Med. Chem. Lett. 2013, 4, 91

Some Additional Issues Associated with AO

AO inhibitors (DDIs) Only one clinical DDI has been linked to AO

(cimetidine/zaleplon), Ki = 155 μM in cytosol)1

Solubility of metabolites Representative Photomicrograph of Primate Renal Histology

Photograph Provided by Christopher Smith

1Hutzler, J. M.; Obach, R. S.; Dalvie, D.; Zientek, M. A. Expert Opin. Drug Metab. Toxicol. 2013, 9, 153.

Identifying AO Metabolism In Vitro (Easy Part)

In vivo clearance is higher than expected based on LM Cl int (cytosolic metabolism)Beware (human AO subs can be stable to rat AO and vice versa)Beware (LMs can be contaminated with cytosolic enzymes, +/- NADPH)

Substrate possesses a possible AO reactive site (i.e., 2-unsubstituted quinoline) Incubate substrate in cytosol or S9 (w/o NADPH) Confirm and determine fm(AO) by using an appropriate AO inhibitor (raloxifene/S9 or

hydralazine/HEPs) (Tier II Assays)

Identify early to influence medicinal chemistry designs

Prioritizing Medicinal Chemistry Designs with In Silico Models

Electrostatic Potential Charge (ESP) -Predicts site relatively well, but not reactivity1

Lowest Unoccupied Molecular Orbital (ELUMO) -Similar to ESP1

Tetrahedral Intermediate Energy (ΔG) -Predicts relative reactivity with respect to two bins (high and low, ~90%)2

Homology Model Docking -Seems to be used retrospectively, but can influence future designs1

-human AOX1 now available3

1Dalvie, D.; Sun, H.; Xiang, C.; Hu, Q.; Jiang, Y.; Kang, P. Drug Metab. Dispos. 2012, 40, 1575. 2Torres, R. A.; Korzekwa, K. R.; McMasters, D. R.; Fandozzi, C. M.; Jones, J. P. J. Med. Chem. 2007, 50, 4642. 3Coelho, C.; Foti, A.; Hartmann, T.; Santos-Silva, T.; Leimkühler, S.; Romão, M. J. Nat. Chem. Biol. 2015, 11, 779.

O’Hara, F.; Burns, A. C.; Collins, M. R.; Dalvie, D.; Ornelas, M. A.; Vaz, A. D. N.; Fujiwara, Y.; Baran, P. S. 2014, 57, 1616.

Only requires a few mg material and very little time About 90% accuracy in predicting AO metabolism (no

false negatives) Obtain a new compound with AO site blocked Probes underlying reactivity (i.e. non-enzymatic) Caution in interpreting results where DFM radical adds to

a non-AO site (contributes to false positives)

Chemical Test to Probe Potential AO Metabolism (Litmus Test)

t = 30 min

“False Positives” in The AO Litmus Test

M+H M+50

SM

Graeme Freestone (Dart)

In Vitro Aldehyde Oxidase Assay

methotrexate zaleplon carbazeran DNS-3935

T1/2 (min) stable 319 7.3 stable

Clint,cytosol (mL/min/kg) stable 1.7 7.10 0.23 (Low)

Medicinal Chemistry Strategies to Remove or Attenuate AO Metabolism

Strategies to Remove or Attenuate AO Metabolism: Remote Interactions and Isosteres

Homology Model Docking

Electrostatic Potential Charge (ESP) -Predicts relative site well -Not effective at predicting AO reactivity Lowest Unoccupied Molecular Orbital (ELUMO) -Similar to ESP Tetrahedral Intermediate Energy (ΔG) -Predicts relative reactivity wrt two bins (higher or lower, 87%)

Dalvie, D.; Sun, H.; Xiang, C.; Hu, Q.; Jiang, Y.; Kang, P. Drug Metab. Dispos. 2012, 40, 1575.

Blocking or Removing the Site of AO Metabolism

1Linton, A.; Kang, P.; Ornelas, M.; Kephart, S.; Hu, Q.; Pairish, M.; Jiang, Y.; Guo, C. J. Med. Chem. 2011, 54, 7705. 2Jia, H.; Dai, G.; Weng, J.; Zhang, J.; Zhang, Z.; Wang, Q.; Zhou, F.; Jiao, L.; Cui, Y.; Ren, Y.; Fan, S.; Zhou, J.; Qing, W.; Gu, Y.; Wang, J.; Sai, Y.; Su, W. J. Med. Chem. 2014, 57, 7577.

Addressing AO Metabolism in TLR7 Program (Pfizer)

Incorporation of pyridine led to AO (rat) Three strategies worked:

Removal of AO site Isosteric replacement Remote steric effect

Pryde, D. C.; Tran, T.-D.; Jones, P.; Duckworth, J.; Howard, M.; Gardner, I.; Hyland, R.; Webster, R.; Wenham, T.; Bagal, S.; Omoto, K.; Schneider, R. P. Bioorg. Med. Chem. Lett. 2012, 22, 2856

Predicting, Identifying and Managing Aldehyde Oxidase Metabolism in Drug Discovery

Issues Associated with AO in Drug Discovery Cytosolic enzyme (i.e., does not show up in LMs) Species variation in structure and activity

In Vitro Identification of AO Metabolism Turnover in cytosol or S9 (w/o NADPH) Determine fm(AO) using specific inhibitors

Predicting human clearance remains a challenge Tools for Prioritizing Medicinal Chemistry Designs

Litmus test and computational methods (hAOX1 structure) Medicinal Chemistry Strategies to Remove or Attenuate AO Metabolism

Blocking/removing site, isosteric ring replacement and remote effects

Acknowledgements

Deepak Dalvie (Pfizer DMPK) Michael Zientek (Pfizer DMPK) Phil Baran and Michael Collins (Litmus Test) Graeme Freestone (Dart) Nicholas Meanwell

Back-Up Slides

Aldehyde Oxidase and Xanthine Oxidase/Dehydrogenase

With HetAr almost always at the two position For AO, almost always 6-membered rings

(Vmax/Km)

Species Differences in the Metabolism of Carbazeran

Kaye et al. Xenobiotica 1985, 237

Was discovered using dog as a pharmacology model PK in dog prior to human studies

Man’s Best Friend Gets His Revenge (Clinical Trial Failures)

Rat (variable) Dog (none) Human (high)

With Respect to IV Metabolism:

Garattini, E.; Terao, M. Expert Opin. Drug Metab. Toxicol. 2012, 8, 487

★

Various Hepatic Fractions

Source: Thermo Fisher Scientific website

Frequency of AO Manuscripts by Year

In Vitro Human Clearance Prediction and Future Outlook

Use pooled cytosol/S91 or cryopreserved hepatocytes2

All methods tend to underpredict in vivo clearance (extrahepatic expression: kidney & lung)

By comparing the Cint to compounds with human PK data available, one can classify into low, medium and high groups1

Recombinant expression of AOXs that lead to reproducible and robust enzymes could help3

Better tissue analysis for PK modeling PXB Mice with humanized livers4

1Zientek, M.; Jiang, Y.; Youdim, K.; Obach, R. S. Drug Metab. Disp. 2010, 38, 1322. 2Hutzler, J. M.; Yang, Y.-S.; Albaugh, D.; Fullenwider, C. L.; Schmenk, J.; Fisher, M. B. Drug Metab. Disp. 2012, 40, 267. 3Foti, A.; Hartman, T.; Coelho, C.; Santos-Silva, T.; Romão, J.; Leimkühler, S. Drug Metab. Disp. Fast Forward, DOI: 10.1124/dmd.115.068395. 4Sanoh, S.; Nozaki, K.; Murai, H.; Terashita, S.; Teramura, T.; Ohta, S. Drug Metab. Disp. 2012, 40, 76.

AO Tissue Distribution in Various Species

Human AOX1 Tissue Expression

Identifying AO/XO Metabolism In Vitro

In vivo clearance is higher than expected based on LM Cl int (cytosolic metabolism) Substrate possesses a possible AO/XO reactive site (i.e., 2-unsubstituted quinoline) Compare LM Clint with cytosol, S9 or HEPs Clint (tier II assays)

Beware (human AO subs can be stable to rat AO and vice versa)Beware (LMs can be contaminated with cytosolic enzymes, +/- NADPH)

Determine fm(AO) by using an appropriate AO/XO inhibitor (raloxifene/S9 or hydralazine/HEPs) (clearance, TI, 1st in class, etc.)

Rule out XO (human XO very narrow in scope, but there are examples of rat XO substrates that are not metabolized by human XO)1

Identify early to influence medicinal chemistry designs, while you still have the chance

1Harrel, A. W.; Wheeler, S. M.; East, P.; Clarke, S. E.; Chenery, R. J. Drug Metab. Dispos. 1994, 22, 189

Chemical Test to Probe Potential AO Metabolism (Litmus Test)

O’Hara, F.; Burns, A. C.; Collins, M. R.; Dalvie, D.; Ornelas, M. A.; Vaz, A. D. N.; Fujiwara, Y.; Baran, P. S. 2014, 57, 1616.

Het 12 mg DFMS

10 Lt-BuOOH, 2 L TFA150 L DMSO, rt, 2 h

5 mg

Het Het HetF2HC

Development of the AO Litmus Test

Regiochemistry in the Sulfinate-Minisci Reaction

N

NHN

DFMS, TFA

DCM, H2O50%

N

NHN

CHF2

CF3SO2Na

DCM, H2O50%

N

NHN

CF3

varenicline"elecrophilic radical" "nucleophilic radical"

N

(PhCO2)2

N N NPh

PhPh

t-Bu

solventNN

benzene:water:

7123

2977

w/o AcOH:AcOH:

6280

3820

TL 1965, 14, 897JOC 1987, 52, 730

Solvent and pH:

Nature of the radical:

+ + +

N

CO2Et

N

CO2Et

CF2HN

CO2Et

CF3

C2:C3 (4:1)

, H+

t-Bu

t-Bu

CF3SO2Na

DCM, H2O53%

DFMS, TFA

DCM, H2O66%

*Navin Fluorine

Deuterium Incorporation to Attenuate AO Metabolism

Has not found its place yet, but may be a reasonable strategy for low to moderate AO subtrates

Sharma, R.; Strelevitz, T. J.; Gao, H.; Clark, A. J.; Schildknegt, K.; Obach, S. R.; Ripp, S. L.; Spracklin, D. K.; Tremaine, L. M.; Vaz, A. D. N. Drug Metab. Disp. 2012, 40, 625