Hiroyuki Kusuhara, Ph.D. Professor, Laboratory of Molecular Pharmacokinetics,

Graduate School of Pharmaceutical Sciences, the University of Tokyo

Endogenous biomarkers for Renal Transporters

International Transporter Consortium Workshop3 (ITCW3) March 13&14, 2017 Omni Shoreham Hotel, Washington DC, USA

Organic anion and cation transport systems in the kidney

Wright SH, et al. Physiol Rev. 2004

proximal tubules

tubular secretion

fB・GFR (size selective)

CLsec

reabsorption

Oct1 OC+

MATE1 OC+(

(creatinine, NMN) OCT2

P-gp OC+

OC+

OC+

H+ MATE2K

OCTN2

OCTN1

H+

OC+

Rodent only

OC+ H+

H+

tubular secretion

epithelial cells

OAT1 OA-

blood side （basolateral）

luminal side （brush border）

OA- OAT3

MRP2 OA-

OAT4 ?

NPT4 ?

MRP4 OA-

BCRP OA-

OA-

OA-

OA-

Organic Anion

Organic Cation

Bulky organic anions Endogenous substrates:uremic toxins, steroid conjugates (DHEAS, E217βG and E1S) Drugs: β-lactam antibiotics (PCG), metotrexate , HMG-CoA reductase inhibitors (pravastatin) and H2 receptor antagonists (famotidine), Ro64-0802, fexofenadine Inhibitor:probenecid

Hydrophilic organic anions with low MW Endogenous substrates: cyclic nucleotides, prostaglandins, uric acid and dicarboxylates Drugs: PAH, β-lactam antibiotics, antiviral drugs (adefovir, tenofovir), NSAIDs, and diuretics (furosemide) Inhibitor:probenecid

OC+

(pyriamine) ？

Hydrophilic organic anions with low MW Endogenous substrates: creatinine, NMN Drugs: metfromin, nadolol, atenolol sumatriptan, frovatriptan Inhibitor:DX-619

various organic cations with low MW Endogenous substrates: creatinine, NMN, thiamine, carnitine Drugs: metfromin, cephalexin, MIBG, ASP, Rhodamine123 Inhibitor:cimetidine, pyrimethamine

OA- OAT2

Selected human transport proteins for drugs and endogenous substances. Giacomini KM. et al Nature Reviews Drug Discovery 9, 215-236 (March 2010)

DDI (CysA, rifampicin)

DDI ? (rifampcin, probenecid)

DDI with GFJ constituents

SNP&DDI(rifampicin, ritonavir)

DDI (probenecid)

DDI (pyrimethamine,cimetidine, trimethoprim)

DDI(quinidine, tariquidar, elacridar)

DDI (GF120918, curcumin, eltrombopag)

DDI (quinidine)

Plas

ma

Conc

.

Time

Plas

ma

Conc

.

Time

Kidn

ey C

onc.

Time

Kidn

ey fu

nctio

n Time

Increases

small impact

Inhibition of uptake process (OCT2)

Inhibition of efflux process (MATE)

nephrotoxicity

Importance of evaluation of DDI in the efflux process

Transporters mediate tissue uptake and subsequent efflux.

pharmacological effect↑ risk of adverse reaction↑

NH2

N H

N

H

N H

N

11CH3

Only PET can detect such interaction!

Increases

Calm on the surface, but big fire in the core（内輪は火の車）

pharmacological effect↑ risk of adverse reaction↑

Pyrimethamine

Opravil M, et al, Antimicrob Agents Chemother. 37:1056-60, 1993

・Anti-malarial and anti-toxoplasmic drug ・Inhibit the dihydrofolate reductase ・protein binding : 87% (human) 92% (mouse) ・ Unbound plasma concentration: 200 nM (at the clinical dose 50 mg, po) ・t1/2 : 96hr (human) 4hr (mouse) ・Decrease renal secretion of creatinine

efflux (brush border membrane) uptake(basolateral) MATE1 MATE2-K OCTN1 OCTN2 OCT2

Ki Mouse 145±36 nM ― > 10µM > 30µM 6.0±1.5 µM human 77±13 nM 46±6 nM ? ? 10±1 µM

Inhibition constant (in vitro)

Ito S et al J Pharmacol Exp Ther 333:341-350, 2010.

MD MD+PYR ThD ThD+PYR AUC/dose (h/mL*10-6) 21±2 23±2 (110%) 22±4 30±3 (139%) fe0-24 (%) 78±6 65±5 (84%) 55±2 50±4 (91%) CLr (mL/min) 623±97 451±55 (77%) 395±31 255±27 (66%)

*,# * # # * # *,#

*p < 0.05; compared with control. #p < 0.05; compared with MD.

Effect of metformin on the plasma concentration time profile and urinary excretion of metformin in healthy subjects

T im e (m in )

%D

os

e/t

iss

ue

0 1 0 2 0 3 00

1 0

2 0

3 0

T im e (m in )

%D

os

e/g

tis

su

e

0 1 0 2 0 3 00

1 0

2 0

3 0

T im e (m in )%

Do

se

/g t

iss

ue

0 1 0 2 0 3 00

5 0

1 0 0

1 5 0

T im e (m in )%

Do

se

/g t

iss

ue

0 1 0 2 0 3 00

2

4

6

8

1 0

(B) Liver

(D) Corticomedulla region (E) Renal pelvis (C) Whole kidney

Coronal plane

RC LC

RP LP RWK LWK

Li

RC LC

RP LP RWK LWK

Li

Frame #6 [30sec]

Frame #20 [500sec]

Time profiles of the radioactivities of 11C-metformin in the liver and kidney in mice

T im e (m in )

%D

os

e/m

L i

n B

loo

d

0 1 0 2 0 3 00 .1

1

1 0

1 0 0

(A) Blood (Heart)

Nephrotoxic effect of cisplatin was enhanced by pyrimethamine treatment in mouse

Nakamura T, et al Biochem Pharmacol. 2010 Dec 1;80(11):1762-7.

Drug interactions involving the efflux transporter may enhance the drug response of victim drugs with greater magnitude than expected from the plasma concentrations

Transporter Function and Metabolome

*Clayton TA et al., Nature 440, 1073 (2006)

Drug-drug interaction

Alteration in the transporter function

PK profiles of drug (exogenous substance profiles in

body fluids)

Inter-subject variation in drug response

Influence

Diagnosis of variation of transporter function for personalized medicine

Genetic mutation Disease state

Influence

Predictable? (Pharmaco-metabonomic Hypothesis* )

Metabonome (endogenous substance profiles

in body fluids)

Diagnosis?

advantage: Multiple transporters’ activity can be evaluated without administration of exogenous probes

(C)

Plasma kin CLtot

1. Kinetics of endogenous metabolites in the body

C)IK

I(1Ck

dtdC

pi

ptotin ⋅

+−−= LVd

The impact of DDI caused by single- or multiple dose of inhibitors on the plasma concentrations of endogenous metabolites depends on not only the magnitude of reduction in the clearance, but also on the synthesis rate and plasm concentrations before inhibitor administration (amount produced during the study versus amount present in the body before inhibitor administration)

Inhibition Ki Inhibition constant Ip Plasma concentrations of inhibitors

Synthesis Clearance

10

2. Other factors needs to be considered

diurnal variation relatively large inter-individual difference (substrate-dependent inhibition constant) lack of quantitative information on the clearance mechanisms and mass balance Continuous synthesis in the body

Plural blood and urine sampling points (AUC evaluation) Clinical study in a crossover fashion (same subjects)

In vitro inhibition study

Clinical studies to show the specificity/sensitivity of the endogenous compounds as probe of specific transporter/enzyme by PGx or DDI studies

OUR APPROACH 1. Conduct clinical DDI studies using probe drugs in healthy

subjects with established inhibitors (the base line was defined in the same subjects)

2. Measure known endogenous or food-derived substrates in the plasma and urine, or comprehensive analysis to identify compounds the concentration of which is affected by drug administration using LC-MS/MS (so-called metabolome analysis)

3. Compare the impact of DDI between the probe drugs and endogenous compounds in the same subjects.

12

Role of MATEs in the tubular secretion of endogenous metabolites ☆Creatinine: a metabolite of creatine, and biomarker for kidney function

☆N-methylnicotinamide (NMN): a metabolite of nicotinamide which undergo extensive tubular secretion in humans. It has been used as biomarker for renal organic cation transport system.

NMN is a substrate of hOCT2 and hMATE2-K. NMN may be an in vivo probe for renal organic cation transporters.

Serum/plasma creatinine can be a biomarker of OCT2, MATE1 and MATE2-K.

0 4 8 120.0

0.2

0.4

0.6

0.8

1.0

Time (h)

Plas

ma

conc

entr

atio

n (m

g/dl

)

0

100

200

300

400

500

0-6 6-12

Control+PYR

Collection Interval (hr)X u

rine

(mg)

control +PYR0

50

100

150

200

Ren

al c

lear

ance

(mL/

min

)

* *

N

NH2

O

CH3

control

+PYR **

Weber W et al. J Pharmacokinet Biopharm. 19:553-74. 1991

Gorboulev V et al. DNA Cell Biol. 16:871-81. 1997 Masuda S et al. J Am Soc Nephrol. 17:2127-35. 2006

Kusuhara H et al. Clin Pharmacol Ther. 89:837-44. 2011

NMN

Mean±S.E. (N=8) *p < 0.05, **p < 0.01

13 Effects of PYR on the plasma concentrations and urinary excretion of NMN in healthy subjects

0 4 8 120

5

10

15

20

25

Time (h)Plas

ma

conc

entra

tion

(ng/

mL)

0

500

1000

1500

2000

2500

0-6 6-12 12-24

Control+PYR

Collection Interval (hr)

X urin

e (µ

g)

Control +PYR0

200

400

600

800

GFR

Ren

al c

lear

ance

(mL/

min

)

** ** * **

**

control

+PYR

Mean±S.E. (N=8) *p < 0.05, **p < 0.01

☆N-methylnicotinamide (NMN): a metabolite of nicotinamide which undergo extensive tubular secretion in humans. It has been used as biomarker for renal organic cation transport system.

NMN is a substrate of hOCT2 and hMATE2-K. NMN may be an in vivo probe for renal organic cation transporters.

N

NH2

O

CH3

Weber W et al. J Pharmacokinet Biopharm. 19:553-74. 1991

Gorboulev V et al. DNA Cell Biol. 16:871-81. 1997 Masuda S et al. J Am Soc Nephrol. 17:2127-35. 2006

NMN

Ito S et al Clin Pharmacol Ther, 92:635-41, 2012

14

0

1

2

3

pHout(6.0)=pHin(6.0)pHout(8.0)>pHin(6.0)

PYR (nM) 0 100 1000

v/[N

MN

] (µ

L/30

s/m

g pr

otei

n)

0 500 1000 1500 2000 25000

1

2

3

[metformin] = 0[metformin] = 500 µM

v (pmol/30s/mg protein)

v/[N

MN

] (µ

L/30

s/m

g pr

otei

n)

Metformin and NMN share the H+ coupled transport in human brush border membrane vesicles

Control +metformin Km (μM) 360 ± 55 758 ± 128

Vmax (pmol/30s/mg protein) 1002 ± 113 959 ± 140

Mean±S.E. (N=4)

**

**

**

** p<0.01 (One-way ANOVA followed by Dunnet’s Multiple comparison test)

In vitro data to support NMN as MATE probe H+

NMN

BBMV

Metformin competitively inhibits NMN uptake in human kidney BBMV

15/22

Workflow of Metabolomic Approach

Peak extraction and alignment

Sample preparation LC/MS* analysis

Selection of potential markers

Structural identification of potential markers

Group A Wild type Drug administration Healthy subject

Group B Genetic deficient type Non-administration Patient

Plasma Urine Tissue

0000000 0000000

0000000 0000000

0000000 0000000

0000000 0000000

0000000 0000000

0000000 0000000

0000000 0000000

0000000 0000000

000 ・ ・ ・ 000 000 Met2

・ ・ ・

・ ・

・ ・ ・

・ ・ ・

・ ・ ・

000

000

000 ・ ・ ・ 000 Met Y

000 ・ ・ ・ 000 Met1

・ ・ ・

000 ・ ・ ・ 000 000 Met2

・ ・ ・

・ ・

・ ・ ・

・ ・ ・

・ ・ ・

000

000

000 ・ ・ ・ 000 Met Y

000 ・ ・ ・ 000 Met1

B-n ・ ・ ・ A-2 A-1

000.00

000.00

000.00

000.00 000.00

000.00

000.00

000.00

000.00 000.00

STD STD

Database

Comprehensive analysis of low molecular weight components in a biological sample Group A Group B

Peak intensity Components

* Liquid chromatography/ mass spectrometry

Discovery the potential biomarkers for diagnosis of genetic polymorphism, drug side-effect, and disease by metabolomics

R2

R1

OH

SIMCA-P+

OPLS-DA S-plot

・ODS column ・HILIC for polar compounds

Ref: Wiklund S, Anal Chem 80,115(2008)

Workflow of Untargeted Metabolomic Approach

Peak extraction and alignment

Urine Samples

LC/MS analysis

Selection of compounds of interest

Structural identification

• A total of ca. 2,000 peaks in human urine

• Criteria: 1) [peak response x urine volume] was decreased by

more than half of the control in both humans and mice by PYR treatment.

2) The decrease was seen in at least 7 of the 8 volunteers

3) The peak intensity was strong enough for the product ion spectrum to be obtained.

• Column, Waters XBridge HILIC (4.6 x 50 mm) • Mobile phase, 10 mM ammonium acetate (pH 5.0)

and acetonitrile • Positive ion-heated electrospray ionization • Scanning range, m/z 100 – 800 • Resolving power, 30,000

16 /10

MeCN/MeOH

LTQ Orbitrap XL

MZmine software

Online DB search Authentic standards

w/o PYR with PYR 0.5 mg/kg, i.v. w/o PYR

vs. vs. with PYR 50 mg, p.o.

Summary of probe compounds for MATEs-mediated drug interaction studies

Compound Urinary

excretion mechanism

%Decrease of renal CL Advantage Disadvantage

Metformin

secretion type

(CLR>fpGFR)

34%

•Straightforward approach •No variation of

intake

• Needs to be administered •Low impact of MATE

inhibition on CLr

Creatinine 20% •High

concentration

•Small change in renal CL

NMN 70% •Need to determine the

renal CL due to large diurnal variation of Cplasma

Thiamine Reabsorption type

(CLR<fpGFR)

80% •Plasma concentrations show small diurnal variation

• Low concentration in the plasma

Carnitine 90% • Lack of in vitro support to be substrates of MATEs Acetylcarnitine 90%

6β-hydroxycoritsol

secretion type

(CLR>fpGFR) Not-affected -

More than 80% inhibition of MATE could occur by pyrimehtamine.

Selected human transport proteins for drugs and endogenous substances. Giacomini KM. et al Nature Reviews Drug Discovery 9, 215-236 (March 2010)

DDI (CysA, rifampicin)

DDI ? (rifampcin, probenecid)

DDI with GFJ constituents

SNP&DDI(rifampicin, ritonavir)

DDI (probenecid)

DDI (pyrimethamine,cimetidine, trimethoprim)

DDI(quinidine, tariquidar, elacridar)

DDI (GF120918, curcumin, eltrombopag)

DDI (quinidine)

Outline of clinical DDI study in healthy subjects

benzylpenicillin (OAT3 probe)

adefovir (OAT1 probe)

Test substrates

PAH (OAT1 inhibitor)

probenecid (OAT1 & OAT3 inhibitor)

Inhibitors

probenecid (oral) PAH (i.v. infusion)

- 2 - 0.5 1 0 0.5 2 3 4 6 8 12 24

adefovir or PCG benzathine (oral)

Blood sampling

Urine sampling

( hr)

PAH (i.v. infusion)

- 2 - 0.5 1 0 0.5 2 3 4 6 8 12 24 ( hr)

* The protocol of this clinical study was approved by the ethics committees in the Faculty of Pharmaceutical Sciences, the University of Tokyo and the Kitasato University East Hospital.

(non, low, middle, high dose)

6β-hydroxycortisol is found to be a OAT3 substrate

day1 day15 day1 day150

50

100

150

CL r

enal

,6β

-OH

F(m

l/min

)

Placebo DX-619

** fu*GFR = 20 ml/min

Renal clearance of 6β-OHF involves tubular secretion (>fpGFR) 6β-OHF is a OAT3 and OCT2 substrate.

Renal clearance of 6β-OHF

Imamura Y et al, Pharm Res., 30, 447-457 (2013)

O

O

O

O

H H

H

O

O

O

O

O

H H

H

O

O

CYP3A4

Cortisol (F) 6β-hydroxycortisol (6 β-OHF)

Biomarker for estimating CYP3A4 actitvity

Conventional method

Current method

R6β-OHF/F = U6β-OHF / UF

CL6β-OHF = X6β-OHF / AUCF

(X6β-OHF and AUCF are amount of 6β-OHF in urine and cortisol in plasma for designated time interval)

(U6β-OHF and UF are amount of 6β-OHF and cortisol in urine for designated time interval)

hOAT1 hOAT3 hOCT2

* *

*

mock

TP expressed

TP expressed + inhibitor

Endogenous Compounds of Interest on Metabolome Analysis of plasma and urine specimens from subjects who received probenecid

Peak

number

Identity Retention

time (min)

Protonated

molecule

(m/z)

Fold change

(probenecid-treated/control)

Plasma Urine Urine/plasma

1 Taurine 7.32 124.0073 0.9±0.3 0.2±0.1 0.3±0.1

2 U305a 7.33 206.0100 1.0±0.1 0.3±0.2 0.2±0.2

3 U118a 7.29 297.9724 0.9±0.3 0.2±0.2 0.2±0.2

4 GCDCA-S 4.28 528.2634 1.9±1.8 0.2±0.3 0.2±0.4

5 Inosineab 5.03 267.0731 5.6±2.0 0.8±0.5 0.13±0.08

6 2-Acetylthiazolea,b 7.28 126.0031 1.0±0.3 0.13±0.06 0.13±0.08

7 U67a 7.30 271.0036 1.0±0.3 0.12±0.07 0.12±0.06

8 4,4'-Sulfonyldiphenola,b 7.30 249.0216 1.0±0.4 0.06±0.04 0.06±0.04

9 U682a 7.28 286.9776 0.9±0.3 0.04±0.05 0.04±0.05

Correlations between the renal clearance of the endogenous substrates and probe drugs with or without probenecid treatment

0 .0 0 0 .0 2 0 .0 4 0 .0 6 0 .0 80 .0

0 .1

0 .2

0 .3

0 .4

0 .5

G C D C A -S C L R ,p la s m a [m L /m in /k g ]

tau

rin

e C

LR

,pla

sm

a [

mL

/min

/kg

]

r= 0 .9 9 5 7

0 2 4 60 .0

0 .1

0 .2

0 .3

0 .4

0 .5

b e n z y lp e n ic illin C L R ,p la s m a [m L /m in /k g ]

tau

rin

e C

LR

,pla

sm

a [

mL

/min

/kg

]r= 0 .9 6 7 2

0 2 4 60 .0

0 .1

0 .2

0 .3

0 .4

0 .5

6β -O H F C L R ,p la s m a [m L /m in /k g ]

tau

rin

e C

LR

,pla

sm

a [

mL

/min

/kg

]

r= 0 .9 7 2 5

0 .0 0 0 .0 2 0 .0 4 0 .0 6 0 .0 80 .0

1 .5

3 .0

4 .5

6 .0

G C D C A -S C L R ,p la s m a [m L /m in /k g ]

6β

-OH

FC

LR

,pla

sm

a [

mL

/min

/kg

]

r = 0 .9 7 5 1

0 2 4 60 .0

1 .5

3 .0

4 .5

6 .0

6β -O H F C L R , p la s m a [m L /m in /k g ]

be

nzy

lpe

nic

illi

n C

LR

,pla

sm

a

[mL

/min

/kg

]

r= 0 .9 9 4 5

0 .0 0 0 .0 2 0 .0 4 0 .0 6 0 .0 80 .0

1 .5

3 .0

4 .5

6 .0

G C D C A -S C L R , p la s m a [m L /m in /k g ]

be

nzy

lpe

nic

illi

n C

LR

,pla

sm

a

[mL

/min

/kg

]

r= 0 .9 6 0 7

Evaluation of DDI risk involving drug metabolizing enzymes and transporters during drug development

single dose

multiple dose

DDI risk using probe substrates

Phase I Phase II

Pharmacokinetic DDI risks caused by the investigational new drug can be evaluated using endogenous biomarkers even during Phase I helping decision of

conducting clinical DDI study

Plasma and urine specimens are subjected to the pharmacokinetic analysis of metabolites

setting exclusion criteria of drugs to avoid DDI

EMA and MHLW also published draft DDI guidelines in 2013 and 2014, respectively.

strong Moderate Weak or none

drug-endogenous compound interaction study

In vivo DDI study with a sensitive

substrate

In vivo DDI study is recommended

not-needed

A proposed amendation of decision tree

Cost and labor for conducing DDI study can be saved.

We need further studies

• Dose-dependence in the effect of inhibitors • Ethnic difference in the impact on transporter

endogenous biomarker • Effect of other drug transporter inhibitors from

weak to strong ones

Summary We could identify the endogenous biomarkers for

kidney drug transporters OCT2：creatinine, N-methylnicotinamide MATEs: creatinine, N-methylnicotinamide, thiamine OAT1:taurine OAT3: 6β-hydroxycortisol, glycochenodeoxycholate sulfate OCT1:thiamine (needs investigation in humans） Red colored compounds: Both plasma concentrations as well as renal clearance can be biomarkers for drug transporters.

Using these endogenous biomarkers, DDI risks involving multiple renal transporters can be evaluated in the same subjects even during phase I studies without administration of probe substrates.

Endogenous biomarkers for hepatic drug transporters remains to be determined (bilirubin, bile acids and their conjugated metabolites?)

Acknowledgement: Dr. Yuichi Sugiyama (Professor emeritus, University of Tokyo, & Sugiyama laboratory,

RIKEN) •OCT2 and MATE study Sumito Ito, Yushun Kuroiwa, Chunyong Wu, Tomoko Kitoh (The University of Tokyo) Yuichiro Imamura, Takashi Izumi (Daiichi Sankyo, Tokyo, Japan) Yoshinori Moriyama, Ph.D. (PYR Okayama Univ) Katsuhisa Inoue, Ph.D., Hiroaki Yuasa, Ph.D. (MATE1-HEK, Nagoya City Univ.) Yuji Kumagai (Kitasato University East Hospital Tomoyoshi Shiroshita (Sekisui Medical Co.,Ltd.) •Probenecid study Yuri Tsuruya, Kazuya Maeda (The University of Tokyo) Yuji Kumagai (Kitasato University East Hospital •Rifampicin study Hanano Terashima, Takeshi Nakayama, Daiki Mori, Tadahaya Mizuno (University of Tokyo) Issei Takehara, Nobuaki Watanabe (Daiichi Sankyo) •Metabolome analysis Koji Kato (Taisho Pharmaceuticals)