Endogenous Bile Acid Metabolites as Biomarkers for … · Endogenous Bile Acid Metabolites as Biomarkers ... Endogenous Bile Acid Metabolites as Biomarkers for CYP3A Tommy B. Andersson

Endogenous Bile Acid Metabolites as Biomarkers for CYP3ATOMMY B. ANDERSSON, ASTRAZENECA,

CARDIOVASCULAR AND METABOLIC DISEASES

Endogenous Bile Acid Metabolites as Biomarkers for CYP3A

Tommy B. Andersson, AstraZeneca, Cardiovascular and Metabolic Diseases

14th European ISSX Meeting, 26 - 29 June 2017, Cologne,

3iMED CVMD | DMPK

Biomarker 4 b OH

cholesterol/c

holesterol

(1)

6b OH

cortisol/cor

tisol

(2)

1b-

hydroxyde

oxycholic

acid

6b OH cort

Isol

(3)

N-methyl

nicotinamide

(4)

Thiamine

(5)

Tryptophan

(6)

Serum

bilirubin

(unconjuga

ted)

(7,8)

Conjugated

or direct

Bilirubin

(9)

Creatinine

(10, 11)

Uric

Acid

(12)

Target CYP3A CYP3A CYP3A OAT3

MATE1

MATE-2k

OCT2

MATE1

MATE-2K

OCT2

MATE

OCT2 OATP1B1 MRP2,

MRP3

UGT1A1

OCT2 BCRP

(URAT

1URA

T1v)

In vitro

validation

and

specificty

Specificty

needs ti be

valdiated

Specificty

needs to be

validated

Specificty

needs to be

validated

Specificity

needs to be

validated

Validated in

vivo

Indu/Inhib?

induction No

(probenencid

inhibition)

(probenecid

inhibition)

Pyrimetham

ine

inhibition

No Yes

Rif induction

UGT and

transporter

Physiological

impact

Cholesterol

lowering

drugs

Circadian

rythms

Further

validation

needed

Further

valdiation

needed

Further

valdiation

neded

Further

valdiation

neded

Further

valdiation

neded

Yes, Gilbert

syndrome

Yes Gilbert

Syndrome

Liver tox

GFR

Correlation

with other

metrics

(several

including

midazolam)

(several

including

midazolam)

No No No No No Not No No

PGx impact (CYP3A5) ? ? No No No Yes Yes Yes No

Sample Plasma Urine Urine Plasma Plasma and

urine

Plasma and

urine

Urine Plasma Plasma Plasma

Comments Stable levels

over time.

Low

sensitivity for

inhib

Confounded

by renal

clearance

and further

metabolism

Sensitive

and rapid

response.

Potential for

CYP3A

inhibition

Only one

publication

Only one

publication

Only one

publiaction

Only one

publication

Difficult to

separate

between

enzyme/trp

interaction/liv

er tox

On top of

GFR

Examples of ADME biomarkers

Requirements for ADME protein biomarkers

4 iMED CVMD | DMPK

•Endogenous metabolite or substrate for one specific transporter or drug metabolizing

enzymes

•Well understood in vitro specificity of the biomarker

•Validated analytical method

•Changes in metric in vivo when subjects are treated with inducers/inhibitors of the

enzyme/transporter

•Metric reflects known genetic polymorphisms of the specific transporter/DME

•Metrics does not depend on other factors not related to enzyme/transporter activity (e.g.

urine pH, urinary flow, renal function, etc)

•Reproducibility (low coefficent of variation of repeated sampling)

•Correlation of biomarker with other validated metrics

How can a biomarker be found

14th European ISSX Meeting

• Systematic analysis using -omic technologies or by

serendipity?

The case of 4ß-Hydroxycholesterol as an endogenous blood

biomarker for CYP3A

How 4ß-Hydroxycholesterol was found

Generating the Hypothesis

The story of how the original hypothesis was generated

•Karolinska University Hospital has a long standing interest in sterol metabolism (within clinical chemistry)

•Studies were being conducted to investigate sterol metabolism in human volunteers

•In one study an outlier was observed in 4ß-hydroxycholesterol concentrations (out of 4 subjects in total)

•The outlier was identified as someone working closely with the principal investigator (PI)

•The PI recognised that this person was on anti-epileptic therapy

•The idea that the outlier concentrations was generated by CYP3A induction was a logical explanation as the anti-epileptic therapy was a well known inducer

6 iMED CVMD | DMPK

4b hydroxy cholesterol is a CYP3A specific

metabolite that shows a remarkable stable plasma

profile over time

DayWeek

Month

Diczfalusy et al (2008) British J Clin Pharmacol (67) 38-43

730 March 2012

4b hydroxycholesterol is a sensitve biomarker for

CYP3A induction

A dose response study

Kanebratt et al. CPT 84, 589 (2008)8 30 March 2012

0

50

100

150

200

Before RIF After RIF

34

81

p<0.001

(p<0.01)

0

50

100

150

200


3146

0

50

100

150

200


37

141

p<0.001

(p<0.01)

p<0.01

(p<0.01)

0

50

100

150

200


34

81

p<0.001

(p<0.01)

0

50

100

150

200


3434

8181

p<0.001

(p<0.01)

0

50

100

150

200


31314646

0

50

100

150

200


37

141

p<0.001

(p<0.01)

0

50

100

150

200


3737

141141

p<0.001

(p<0.01)

p<0.01

(p<0.01)

0.1

1.0

10.0

100.0


7.46

1.93

0.1

1.0

10.0

100.0


10.7

3.87

0.1

1.0

10.0

100.0


8.945.81

p<0.001

(p<0.05)

p<0.001

(p<0.01)

p<0.01

(p<0.05)

0.1

1.0

10.0

100.0


7.467.46

1.931.93

0.1

1.0

10.0

100.0


10.710.7

3.873.87

0.1

1.0

10.0

100.0


8.948.945.815.81

p<0.001

(p<0.05)

p<0.001

(p<0.01)

p<0.01

(p<0.05)

500 mg

RIF

Quinine/

3’-hydroxy-

quinine

Effect of

induction:

100 mg

RIF

20 mg

RIF

CYP3A4 activity

4β-hydroxy-

cholesterol

Effect of

induction:

CYP3A4 activity

0

50

100

150

200


34

81

p<0.001

(p<0.01)

0

50

100

150

200


3146

0

50

100

150

200


37

141

p<0.001

(p<0.01)

p<0.01

(p<0.01)

0

50

100

150

200


34

81

p<0.001

(p<0.01)

0

50

100

150

200


3434

8181

p<0.001

(p<0.01)

0

50

100

150

200


31314646

0

50

100

150

200


37

141

p<0.001

(p<0.01)

0

50

100

150

200


3737

141141

p<0.001

(p<0.01)

p<0.01

(p<0.01)

0.1

1.0

10.0

100.0


7.46

1.93

0.1

1.0

10.0

100.0


10.7

3.87

0.1

1.0

10.0

100.0


8.945.81

p<0.001

(p<0.05)

p<0.001

(p<0.01)

p<0.01

(p<0.05)

0.1

1.0

10.0

100.0


7.467.46

1.931.93

0.1

1.0

10.0

100.0


10.710.7

3.873.87

0.1

1.0

10.0

100.0


8.948.945.815.81

p<0.001

(p<0.05)

p<0.001

(p<0.01)

p<0.01

(p<0.05)

500 mg

RIF

Quinine/

3’-hydroxy-

quinine

Effect of

induction:

100 mg

RIF

20 mg

RIF

20 mg

RIF

CYP3A4 activity

4β-hydroxy-

cholesterol

Effect of

induction:

CYP3A4 activity

Rifampicin dose response induction study in vivo

4b hydroxycholesterol comparison with quinine in the same subjects

0

2

4

6

8

0 2 4 6 8

Quinine MRbefore

Quinine MRafter

4β-h

ydro

xychole

ste

rol a

fte

r

4β-h

ydro

xychole

ste

rol b

efo

re y = x

0

2

4

6

8

0 2 4 6 8

Quinine MRbefore

Quinine MRafter

4β-h

ydro

xychole

ste

rol a

fte

r

4β-h

ydro

xychole

ste

rol b

efo

re

0

2

4

6

8

0 2 4 6 8

Quinine MRbefore

Quinine MRafter

4β-h

ydro

xychole

ste

rol a

fte

r

4β-h

ydro

xychole

ste

rol b

efo

re

Quinine MRbefore

Quinine MRafter

Quinine MRbefore

Quinine MRafter

4β-h

ydro

xychole

ste

rol a

fte

r

4β-h

ydro

xychole

ste

rol b

efo

re y = x

Spearman rank rs=0.71

95% C.I.=0.52-0.90

p<0.001; n=22.

Kanebratt et al. CPT 84, 589 (2008)

Fold inductions are almost identical

Rif dose Quinine 4b OHC

20 mg 1.5 1.5

100 mg 2.7 2.4

500 mg 3.8 4.0

A good

correlation

between the

two probes

Midazolam AUC

Plasma

4b hydroxycholesterol/

cholesterol ratio

Urine, 14 h collection

6b OH Cortisol/Cortisol

ratio

Comparison of induction response by rifampcine

Björkhem-Bergman et al. DMD 2013

Comparison of Induction and inhibition of 4β OH cholesterol

levels after rifampicin and ketoconazole treatment

11

• 4βHC levels significantly increased by Day 4 after rifampin induction

• Modest decrease in 4βHC levels after ketoconazole inhibition that plateau by Day 3

• No effect of placebo on 4βHC levels

Kasichayanula BJCP, 2014

= Beginning or End of probe drug/placebo administration

Inhibition signal

using 4βHC is

narrow

compared with

induction

4b hydroxycholesterol as a CYP3A biomarker

• Specifically formed by CYP3A

• Stable biomarker over time (long half life)

• Takes long time to reach steady state

• Blood sampling is needed

• Useful for CYP 3A induction but less useful for CYP inhibition

Slow response due to long half life – need long treatment periods

The dynamic range for detecting inhibition is limited

iMED CVMD | DMPK

An old finding caught our interest when we discussed a good new

endogenous in vivo biomarker for CYP3A inhibition

iMED CVMD | DMPK

Bodin et al, 2005

The hydroxylation was found to be catalyzed by CYP3A4

Elevated levels of 1b-Hydroxydeoxycholic acid in urine samples from

carbamacepine, a CYP3A inducer, treated subjects

Studies needed to investigate the option to use 1b-hydroxydeoxycholic

acid as an endogenous biomaker

1. Reference substances for quantitative analysis

2. Specific analytical method to separate and quantify compounds

3. Determine the CYP specific biotransformation of deoxycholic acid by CYP

enzymes

4. Hydrolyse conjugated analytes in urine

5. Determine the variability of urine excretion of 1b-Hydroxydeoxycholic acid

6. Determine the dynamic range of urine excretion of 1b-Hydroxydeoxycholic

acid in subjects exposed to CYP3A inducers and inhibitors

7. Compare the biomarker with accepted probes (midazolam kinetics)

Enzymatic synthesis of 1b-OH deoxycholic acid synthesis by a

bacterial CYP mutant

OOH

CH3

CH3

OH

OH

CH3

H

H

H

H H

OOH

CH3

CH3

OH

OH

CH3

H

H

H

H H

OH

Codexis MCYP0029

GDH, NADP+

Codexis MCYP0029

GDH, NADP+

OOH

CH3

CH3

OH

CH3

H

H

H

H H

D D

D

D

OH

OOH

CH3

CH3

OH

CH3

H

H

H

H H

D D

D

D

OH

OH

AZ13837350-001 unlabeled

10.5mg

AZ13837350-002 7.23mg D4 labeled

AZ13837350-003 13.46mg99 atom % D, CDN isotopes

1b-OH Deoxycholic acids fully characterised by 1H, 13C, COSY, ROESY NMR

Unlabeled

Deuterium

labels

Reference

substances were

purified by mass-

directed preparative

HPLC

Hayes et al. Drug Metab Dispos, 44:1480–1489, 2016

Analysis of reference metabolites


1b-Hydroxylation of deoxycholic acid

Is it specifically catalyzed by CYP3A?

18iMED CVMD | DMPK

OH

O

CH3

OH

OH

CH3

CH3

OH

OH

O

CH3

OH

OH

CH3

CH3

DCA 1β-OH-DCA

CYP3A?

0

100

200

300

400

500

600

700

CYP

1A

1

CYP

1A

2

CYP

1B

1

CYP

2A

6

CYP

2A

13

CYP

2B

6

CYP

2C

8

CYP

2C

9

CYP

2C

18

CYP

2C

19

CYP

2D

6

CYP

2E1

CYP

2J2

CYP

3A

4

CYP

3A

5

CYP

3A

7

CYP

4A

11

CYP

4F2

CYP

4F3

CYP

17

A1

CYP

46

A1

HLM

% 1β

-OH

-DC

A F

orm

atio

n(N

orm

aliz

ed

to

HLM

)

19

Identify CYP enzymes catalyzing 1β-hydroxylation of deoxycholic acid

DCA=2 µM

rCYP=100 pmol/mL protein

HLM=0,5mg/mL protein

1 mM NADPH

60 min

CYP3A4, 3A5, 3A7 were identified as the enzymes that catalyzing DCA 1b-hydroxylation.

CYP3A7 is mainlyhuman fetal liver.

CYP46A1 also catalyzes the formation of 1β-OH-DCA, and it mostly expresses in brain.


20

Inhibition of 1β-hydroxylation of deoxycholic acid by ketoconazole

0

20

40

60

80

100

Control Ketoconazole 0.1 µM

Ketoconazole 1 µM

Ketoconazole 10 µM

No NADPH

% 1β

-OH

-DC

A F

orm

atio

nvs

Co

ntr

ol

HLM

HLM=0,5mg/mL protein, 60 min. DCA=2 µM.


21

Deoxycholic acid and 1β OH deoxycholic acid is partially excreted in urine

as conjugates

SulfationAmidation Glucuronidation

Phase II metabolites

Glycine conjugate Taurine conjugate

DCA 1β-OH-DCA Iso OH-DCA

(including CA)

Dehydro-DCA

OH

O

CH3

OH

OH

CH3

CH3

OH

OH

O

CH3

OH

OH

CH3

CH3

Phase I metabolites

OH

O

CH3

OH

O

CH3

CH3

OH

O

CH3

OH

OH

CH3

CH3

OH

O

CH3CH3

NH

O

OH

O

CH3CH3

NH

S

O

O

OH

OS

O

OOH

O

O

OH

OH

OH

OH O

O

CH3CH3

O

O

OH OH

OH

OH

O


22

Urine sample preparation to hydrolyse the conjugates

5 µL of β-glucuronidase/arylsulfatase

5 µLof choloylglycine hydrolase (2 U/µL)

Urine 200 µL

37°C

Over night

LC/HRMS

0.1 M NaHPO4 (pH 5)

Quench by AcN

(containing IS)

LC/HRMS

Oasis MAX 96-well µElution Plate

Solvolysis

37°C

1 hr

NaOH (4 M)

i-propanol

SPE

N2

SPE

N2

Solvolysis

HCL (1 M)

Acetone

70°C

Over night

N2

FA to neutralize

Enzymetic deconjugation

(Bodin K, et al, 2005)


23

Urine analysis from healthy subjects and a carbamazepine treated patient

Sample Prep.: Urine samples undergo enzymetic deconjugation and chemical solvolysis.

2.00 3.00 4.00 5.000

2.00e5Standard

XIC: -m/z 407.280

x4

1b-OH-DCA

2.00 3.00 4.00 5.000

2.00e5Control Urine

1b-OH-DCA

Time (min)

2.00 3.00 4.00 5.00

Patient urine

0

2.00e5

Inte

nsity

1b-OH-DCA

2.00 3.00 4.00 5.00

%

0

6.00e4

XIC: -m/z 391.285

Standard

x4

DCA

2.00 3.00 4.00 5.000

6.00e4Control Urine

DCA

Time (min)2.00 3.00 4.00 5.00

%

0

6.00e4Patient urine

DCA

A B


Comparison of enzymatic hydrolysis and hydrolysis followed by

solvolysis of urine samples


25

Comparisons between 24 h urine collections and spot samples

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1 2 3 4 5 6 7 8 9 10 11 13

1b

-OH

-DC

A /

DC

A

Subject no.

24 h

spot

Day 1 Day 2 Day 30

1b-OH-DCA / DCA ratios for spot samples and 24 h

samples

20 juni 2017Namn Efternamn26

y = 1.0112x + 0.0136R² = 0.8183

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Rati

o f

or

sp

ot

sam

ple

Ratio for 24 h sample

Urine levels of 1b-OH deoxycholic acid biomarker affected by moderate and strong CYP3A inhibitors (preliminary data)


Itraconazole (strong inhibitor) inhibits formation of a 1b-OH deoxycholic acid

in all 13 subjects analysed. Verapamil (moderate inhibitor) gave a weaker

inhibition in 10 of 13 subjects.

Possible interaction with kidney transporters


Deoxycholic acid

Glycin

Taurine

Sulfate

Glucuronide

1β OH deoxycholic acid

Glycin

Taurine

Sulfate

Glucuronide

Will Interactions with

kidney transporters

impact the urine

secretion?

Clinical pharmacology & Therapeutics | VOLUME 92 NUMBER 5 | NOVEMBER 2012 553

Zamek-Gliszczynski et al.

Further validation of 1b deoxycholic acid as a CYP3A

biomarker

Correlation with other accepted metrics, e.g. midazolam kinetics. Direct

comparison of CYP3A activity in the same subjects

Effect of other factors not related to enzyme activity (e.g. urine pH, urinary flow,

renal function)

Effect by transporter activity

Acknowledgements

Karolinska Institutet

• Ulf Diczfalusy

• Leif Bertilsson

AstraZeneca

• Martin Hayes

• Xueqing Li

• Eric Mason

• Ulf Eriksson

• Phil Gardner

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Documents

Endogenous Bile Acid Metabolites as Biomarkers for … · Endogenous Bile Acid Metabolites as Biomarkers ... Endogenous Bile Acid Metabolites as Biomarkers for CYP3A Tommy B. Andersson