BioreclamationIVTBaltimore,MD

• AldehydeoxidaseinhumansisrepresentedbyasinglegeneAOX1andtwopseudogenes allonchromosome2q.AOX1covers85kbwith35exons.

• Thereisvariabilityinotherspeicies, mouseandratcontain4genes.Withhighexpressionvariationacrossstrain.Caninescontainnofunctionalcopy.

• Largenumberofpolymorphisms 264,NCBIlisted acrossthegenewithminimalcharacterization.• FuC.et.al.showedProteinexpressionvariationinhumans3.5foldandnotwellcorrelatedwithactivity.PossiblyrelatedtoSNPsorotherproteinfunctionvariation.

• Smith,M.,et.al. 2009 .Novelpharmacogenetic markersfortreatmentoutcomeinazathioprine‐treatedinflammatoryboweldisease.AlimentaryPharmacology&Therapeutics,30 4 ,375–384.

‐ rs55754655N1135Sreducedresponseratetotreatment.

• Hartmann,T.,et.al. 2012 .TheImpactofSingleNucleotidePolymorphismsonHumanAldehydeOxidase.DrugMetabolismandDisposition,40 5 ,856–864.

‐ Sequencedthe35exonsofhAOX1in180patientsamplestoidentifypolymorphicsites.‐ Generatedrecombinantenzymesforselectvariantsandassayedwithmultiplesubstrates.‐ Sawcatalyticincreaseswithrs3731722andrs55754655.‐ Hypothesizedthatthesesurfaceaminoacidchangesmayaffectstability

• Ramírez J,et.al.. 2014 Apharmacogenetic studyofaldehydeoxidaseIinpatientstreatedwithXK469.Pharmacogenet Genomics. Feb;24 2 :129‐32

‐ Analyzed41SNPsand7eQTL in92solidtumorandleukemiapatients.‐ Foundacorrelationwithrs10931910insolidpatientswithreductioninclearanceofXK469in

mutantcarriers,howevertrendreversedinleukemiapatients. smallersamplesize

• Hutzler JM,et.al.. 2014 Aldehyde oxidaseactivityindonor‐matchedfreshandcryopreservedhumanhepatocytesandassessmentofvariabilityin75donors..DrugMetab Dispos. Jun;42 6 :1090‐7

Reference SNP Exon Nucleotide Change Amino Acid Change

rs199984835 5 378C>G Y126Stop

rs41309768 22 2404C>T R802C

rs56199635 25 2762G>A R921H

rs10931910 Intron 27 A>G N/A

rs55754655 30 3404A>G N1135S

rs141786030 34 3812C>T S1271L

rs3731722 34 3890A>G H1297R

• Polymorphisms selected for this study, by NCBI reference SNP number• The majority are protein changes with one generating truncated protein

and a single intronic SNP who’s impact is poorly understood.

• Humanlivertissuesamplesandcryopreservedhumanhepatocytesfrom221donorswerecollectedfromtheBioreclamationIVTinventoryandgenomicDNAwaspreppedfromeach.

• 7SNPswereselectedwhichcontainedliteraturereferencesofpossiblesignificance.Datageneratedbytaqman realtimePCRassays.

• Incubationswithcarbazeran andO‐6‐benzylguaninewereperformedtogenerateinvitrointrinsicclearancevaluesfromthehepatocytesinasuspensionassay.

• Polymorphismsandclearancevalueswerelinkedandanalyzedforcorrelations.StatisticalanalysiswasperformedwithPrismv5.02 .AllsamplesweretestedforHardy‐WeinbergEquilibrium.

Reference SNP ExonNucleotide Change

Amino Acid Change

Cohort Minor AllelicFrequency

dbSNP Minor AllelicFrequency

rs10931910Intron 27

A>G N/A .466 1 .475 3

rs55754655 30 3404A>G N1135S .153 1 .189 3

rs3731722 34 3890A>G H1297R .075 1 .056 3

rs199984835 5 378C>G Y126Stop 0 1 .026 4

rs41309768 22 2404C>T R802C 0 2 .006 4

rs56199635 25 2762G>A R921H 0 2 .003 4

rs141786030 34 3812C>T S1271L 0 2 0 31 n=380,  2 n=254, 3 ESP_Cohort_Population n=4552, 4 COLL2006 n=360, n=chromosomes samples analyzed

Frequencies were similar to those reported in other studies in NCBI. However rs199984835,rs41309768, rs56199635, rs141786030 were monomorphic.

0

0.2

0.4

0.6

AA C H

Allelic Freq

uency

G A

0

0.2

0.4

0.6

0.8

1

AA C H

G A

0

0.2

0.4

0.6

0.8

1

AA C H

G A

rs55754655:N1135S rs3731722:H1297Rrs10931910

AA =African American n=16C= Caucasian n=142H=Hispanic n=24

No significant differences by race of any of the SNPs

G/G A/G A/A

0

200

400

600

Cl in

trin

sic

(mL/

min

/kg)

Carbazeran

228.9 241.3 235.9ml/min/kgMean

GG

A/G A/A

0

20

40

60

80

100

Cl in

trin

sic

(mL/

min

/kg)

36.1ml/min/kg40.0 39.4

O‐6‐BenzylguanineP 0.518

P 0.707

P 0.8449

P 0.913

n 4 n 30 n 55 n 4 n 23 n 44

A/G A/A

0

200

400

600

Cl in

trin

sic

(mL/

min

/kg)

224.29ml/min/kg302.18Mean:n 15 n 74

A/G A/A

0

20

40

60

80

100

Cl in

trin

sic

(mL/

min

/kg)

35.42ml/min/kg46.28n 13 n 58

P 0.0233 P 0.0770

O‐6‐BenzylguanineCarbazeran

Haplotypes rs55754655 rs3731722 G rs10931910Donors with haplotype

1 G/G A/A G/G 42 A/G A/A G/G 73 A/A A/G G/G 14 A/A A/A G/G 95 A/G A/G A/G 56 A/G A/A A/G 187 A/A A/G A/G 58 A/A A/A A/G 199 A/A A/G A/A 5

10 A/A A/A A/A 16

• Analysis of haplotypes may reveal conflicting mechanisms• Stratifying by haplotype may require more samples• Possible linkage between rs55754655 and rs10931910

G/G A/G A/A

0

200

400

600

Cl in

trin

sic

(mL/

min

/kg)

G/G A/G A/A

0

200

400

600

Cl in

trins

ic (m

L/m

in/k

g)

rs10931910A for rs3731722

202.3 212.3 279.2ml/min/kg

P 0.061

P 0.0379

n 20 n 37 n 16

rs10931910

204.9 234.5 278.9ml/min/kgMean

P 0.0504

P 0.0978

n 21 n 47 n 20

G/G A/G A/A

0

200

400

600

Cl in

trins

ic (m

L/m

in/k

g)

rs10931910A for rs3731722

G/G A/G A/A

0

200

400

600

Cl in

trin

sic

(mL/

min

/kg)

rs10931910A for rs3731722 and rs55754655

202.3 212.3 279.2ml/min/kgMean

P 0.061

P 0.0379

n 20 n 37 n 16

181.6 192.7 279.2ml/min/kg

n 9 n 19 n 16

P 0.0466

P 0.047

G/G A/G A/A

0

20

40

60

80

100

Cl in

trin

sic

(mL/

min

/kg)

G/G A/G A/A

0

20

40

60

80

100

Cl in

trin

sic

(mL/

min

/kg)

Rs10931910A for rs3731722

rs10931910A for rs3731722 and rs55754655

36.0 34.1 38.2ml/min/kgMean

P 0.766

P 0.534

n 16 n 38 n 17

26.2 31.3 38.2ml/min/kg

n 6 n 15 n 12

P 0.385

P 0.133

A/G A/A

0

200

400

600

Cl in

trin

sic

(mL/

min

/kg)% of Control Activity

6.0%

IC50 1.4±.6 uM

Plasma concentations in fatal overdose of 25-65uM

Quetiapine

Obach RS, et. al.,(2004) Human Liver Aldehyde Oxidase Inhibition by 239 Drugs. J Clin Pharmacol. Jan;44(1):7-19..

Carbazeran Clearancers3731722:H1297R

Seroquel IR (quetiapine fumarate)

CryopreservedHumanHepatocytes

N

N N

N

H2N

O

O6-Benzylguanine

Pooled Cryopreserved Hepatocytes34.2 ± 1.4 mL/min/kg (n=19 donors, mixed gender)

Individual Donor Cryopreserved Hepatocytes39.9 ± 19 mL/min/kg (mean ± SD, n=70 donors)

Hutzler et al. (2014) Drug Metab Dispos. 42(6), 1090-97.

• Significantdonorvariabilityexistsinourhepatocytedonors.• SNPsrs199984835,rs41309768,rs56199635,rs141786030werenotfoundinour

population.• rs5576455showednoeffectonclearancewitheithercarbazeran orO‐6‐

benzylguanine• rs3731722presentatlowfrequencyhadanincreaseinclearancewithboth

substratestested.Thoughresultsonlyreachedstatisticalsignificancewithcarbazeran.

• rs10931910showedadecreaseinexpressionwhichwassignificantwithcarbazeran.Thiswasseenonlyinhaplotypeswildtype forrs3731722

• Geneticpolymorphismsplayaroleinaldehydeoxidaseclearance.Witheffectswhichcanincreaseordecreaseclearancerates.Thoughthosedifferencesdonotexplainthetotalvariabilityseen.

• Donortodonorvariabilityinaldehydeoxidaseactivityinisolatedhepatocytescancomefrommanyfactors.

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