Implication of the carrier-mediated transport of nicotine ... · Implication of the...

Implication of the carrier-mediated transport of nicotine in lung and other tissues

Ken-ichi Hosoya, Ph.D.Workshop on Drug Transporters in the LungsSeptember 22, 2016

Graduate School of Medicine and Pharmaceutical SciencesUniversity of Toyama

NicotinePrimary alkaloid contained in tobaccoAgonist of nicotinic acetylcholine receptor (nAChR)Addictive drug causing smoking-dependence

(MW: 162.23)

Nicotine is absorbed from thealveoli of the lung into thecirculating blood.

Pharmacodynamic actions

Absorption Metabolism

Nicotine is mainly metabolized inthe liver by cytochrome P450(CYP) localized in endoplasmicreticulum.

Nicotine binds to nAChR of theneurons in the central nervoussystem (brain and retina), andleading various effects.

• Rewarding effect• Cognitive enhancement• Analgesic effect. etc.

Brain

• Neuroprotecton. etc.Retina

Pulmonary absorption of nicotineis rapidly because of …• Large alveolar surface area• Extensive capillary system in

lung

Nicotine Cotinine70-80%1)

t1/2 (nicotine): 100-150 min2)

1) Clin Pharmacol Ther. 56:483-493, 1994.2) Pharmacol Rev. 57:79-115, 2005.

CNS (brain , retina)

Lung Liver

Nicotine

(MW: 162.23)

Alveolarepithelial cell

Capillary Endothelial cell

Astrocyte (Müller cell)

Pericyte

Capillary endothelial cell

Sinusoidal endothelial cell

Hepatocyte

Nicotine

Primary alkaloid contained in tobaccoAgonist of nicotinic acetylcholine receptor (nAChR)Addictive drug causing smoking-dependence

Log DC = 0.95

Intracellular

+ pKa1 = 3.021) pKa2 = 8.021)

19%81%

pH 7.4

1) Yang and Smeta. Chromatographia. 40: 375-378,1995.

+

+

Ionized molcules are impermeable across the cell membrane lipid bilayer.

Extracellular

Extracellular

Intracellular

+ pKa1 = 3.021) pKa2 = 8.021)

19%81%

pH 7.4

1) Yang and Smeta. Chromatographia. 40: 375-378,1995.

？

+

+

Specific transport system(s) is needed to nicotine transport across the cell membrane lipid bilayer.

1

2

3

Each point represents the mean ± S.E.M. (n = 3)

Kp, app(t) = CLinf × AUC(t)/Cp(t) + Vi

Kp, app : apparent brain-to-plasma concentration ratioCLinf : apparent blood-to-tissue clearanceAUC : area under the plasma concentration time curveCp : plasma concentrationVi : rapidly equilibrated distribution volume

[3H]Nicotine

Kp,

app

(mL/

g br

ain)

00 1 2 3 4

1

2

3

4

5 6AUC(t)/Cp(t) (min)

Integration plot method

Femoral vein

Kp,

app

(mL/

g re

tina)

00 1 2 3 4 5 6

AUC(t)/Cp(t) (min)

CLinf, brain= 272 ± 69 µL/(min・g brain)

CLinf, retina= 131 ± 25 µL/(min・g retina)

Brain

Retina

Blood-to-tissue transportIn vivo

Each point represents the mean ± S.E.M. (n = 3).

Slic

e/m

ediu

m ra

tio

(mL/

g br

ain)

0 10 20 30 40 50 600

0.5

1.0

1.5

2.0

2.5

Vd, brain = 2.30 mL/g brain

Time (min)

Tracer solution

Brain sliceBlood

BBBBrain

0 5 10 15 20The

perc

enta

ge o

f [3 H

]nic

otin

e re

mai

ning

in

the

ipsi

late

ralc

ereb

rum

20

30

40

50

60708090

100

t1/2 = 11.6 minkel = 5.95×10-2 min-1

Time (min)

: [3H]Nicotine : [14C]D-Mannitol(Impermeable marker)

Par2 region

Brain efflux index (BEI) methodBrain efflux index (BEI) method Brian slice uptakeBrian slice uptake

Brain-to-blood transport

CLeff,brain= 137 ± 24 µL/(min･g brain)

In vivo

Each point represents the mean ± S.E.M. (n = 3).

Slic

e/m

ediu

m ra

tio

(mL/

g br

ain)

0 10 20 30 40 50 600

0.5

1.0

1.5

2.0

2.5

Vd, brain = 2.30 mL/g brain

Time (min)

Tracer solution

Brain sliceBlood

BBBBrain

0 5 10 15 20The

perc

enta

ge o

f [3 H

]nic

otin

e re

mai

ning

in

the

ipsi

late

ralc

ereb

rum

20

30

40

50

60708090

100

t1/2 = 11.6 minkel = 5.95×10-2 min-1

Time (min)

: [3H]Nicotine : [14C]D-Mannitol(Impermeable marker)

Par2 region

Brain efflux index (BEI) methodBrain efflux index (BEI) method Brian slice uptakeBrian slice uptake

Brain-to-blood transport

CLeff,brain= 137 ± 24 µL/(min･g brain)

In vivo

Brain

Blood

Blood-brainbarrier

Possible existence of nicotine influx transport system(s) at the blood-brain barrier

Efflux Influx

＞Two-fold greater

(137 µL/(min･g brain)) (272 µL/(min・g brain))

Brain and retinal uptake index(BUI and RUI) method

Common carotid artery

BUI, RUI (%) = [3H]/[14C] (in tissue)

[3H]/[14C] (in injectate)× 100

Blood-to-tissue transportIn vivo

[3H]Nicotine[14C]n-Butanol

Inhibitors


Inhibitors

3

200

Log DC

BU

I (%

)

210-1-2-4

100

50

10

5

1

VerapamilL-Arg

Vincristine

Digoxin

T4

L-Phe

L-DopaL-Leu

D-Glucose

Gly

BiotinAdenosine

Acetyl-L-carnitine

-3

Nicotine

3

500

Log DC

RU

I (%

)

210-1-2-4

400300200

100

50

10

5

VerapamilL-Arg

VincristineDigoxin

T4

L-Phe

L-DopaL-Leu

D-Glucose

Gly

BiotinAdenosine

Acetyl-L-carnitine

-3

Nicotine

Brain

Retina

: Substrate for SLC transporter : Substrate for P-glycoproteinHosoya et al., Pharm Res. 27:2715-2724, 2010.

Each bar represents the mean ± S.E.M. (n = 3-6). *p < 0.05, **p < 0.01, significantly different from control.

Pyrilamine(40 mM)

*

**

0 4020 60 80 100

Control

Nicotine(100 mM)

Verapamil(3 mM)

Percentage of control

**

120

Pyrilamine(40 mM)

**

0 4020 60 80 100

Control

Nicotine(30 mM)

Verapamil(3 mM)


**

**

**

BUI RUI

Blood-to-brain and retina transportIn vivo

Each bar represents the mean ± S. E. M. (n = 3-5). *p < 0.05, **p < 0.01, significantly different from control.(TEA, tetraethylammonium.)

L-Carnitine(50 mM)

Control

Nicotine(50 mM)

Pyrilamine(50 mM)

0 4020 60 80 100Percentage of control

**

TEA(50 mM)

120

*

LUILUI

Blood-to-liver transportIn vivo

Liver uptake index(LUI) method

Liver uptake index(LUI) method


Inhibitors


Inhibitors

LUI (%) = [3H]/[14C] (in liver)

[3H]/[14C] (in injectate)× 100

LiverPortal vein

TR-BBB13 cells(Conditionally immortalized rat brain capillary endothelial cell)

TR-iBRB2 cells(Conditionally immortalized rat retinal capillary endothelial cell)

BBB

Inner BRB

Isolated rat hepatocytes

Liver

Radioactivity and protein content were measured.

Radioactivity and protein content were measured.

Spin down

Hepatocyte

Tracer solution

：[3H]Nicotine

Oil layer

KOH layer

：[3H]Nicotine

Cells

A549 cells(Human alveolar adenocarcinoma cell line)

Lung

Cell uptake across the plasma membrane In vitro

Tracer solution


Cel

l/med

ium

ratio

(µ

L/m

g pr

otei

n)60

40

20

00 10 20 30

Time (sec)

TR-BBB13 cellsTR-BBB13 cells

0 10 20 30

Cel

l/med

ium

ratio

(µ

L/m

g pr

otei

n)

10

6

2

0

Time (sec)

8

4

Cel

l/med

ium

ratio

n(µ

L/m

g pr

otei

n) 60

40

20

00 1 2 3

Time (min)

80HepatocytesHepatocytes

Cel

l/med

ium

ratio

(µL/

mg

prot

ein)

40

30

20

00 20 40 60

Time (sec)

50

10

A549 cellsA549 cells

TR-iBRB2 cellsTR-iBRB2 cells

Time-courseIn vitro

Concentration-dependence


In vitro

5

10

15

00 100 200 300 400 500

[Nicotine] (µM)

Nic

otin

e up

take

rate

(nm

ol/(m

in・m

g pr

otei

n))

Km = 92.4 μM

TR-BBB13 cellsTR-BBB13 cells TR-iBRB2 cellsTR-iBRB2 cells

0 500 1000 1500 2000

10

20

30

0

[Nicotine] (µM)

Nic

otin

e up

take

rate

(nm

ol/(m

in・m

g pr

otei

n))

Km = 492 μM

40

1000 1500 2000500

5

10

15

00

[Nicotine] (µM)

Nic

otin

e up

take

rate

(nm

ol/(m

in・m

g pr

otei

n))

Km = 50.4 μM

20 A549 cellsA549 cells

Saturable

Non-saturable

HepatocytesHepatocytes

Nic

otin

e up

take

rate

(nm

ol/(m

in・m

g pr

otei

n))

00

100 200 300 400 500[Nicotine] (µM)

1

2

3

4 Km = 141 μM

5

Saturable

Non-saturable

Substrates for

OCTsOCTNsPMAT MATE OATs

Each inhibitor was applied at 1 mM. Each bar represents the mean ± S.E.M. (n = 3-6). **p < 0.01, significantly different from control.(MPP+, 1-methyl-4-phenylpyridinium; TEA, tetraethylammonium; PAH, p-aminohippurate.)

In vitro

PAH

MPP+

0 20 40 60 80 100 120 140

Control

Nicotine

Pyrilamine

Verapamil

Quinidine

Amantadine

Propranolol

Clonidine

Choline

TEA

L-Carnitine


**************

TR-BBB13 cellsTR-BBB13 cells

0 50 100 150 200

Control

Nicotine

Pyrilamine

Verapamil

Desipramine

Choline

L-Carnitine


**

**

PAH

MPP+

Timolol

Propranolol

Clonidine

TEA

******

**

**

TR-iBRB2 cellsTR-iBRB2 cells

**

*

Inhibitory effects

Hydrophobicorganic cation

drugs

Each inhibitor was applied at 1 mM. Each bar represents the mean ± S.E.M. (n = 3-9). *p < 0.05, **p < 0.01, significantly differentfrom control. (MPP+, 1-methyl-4-phenylpyridinium; TEA, tetraethylammonium; PAH, p-aminohippurate.)

In vitroHepatocytesHepatocytes

Control

Pyrilamine

Verapamil

Quinidine

Amantadine

Propranolol

0 50 150

Nicotine

Choline

L-Carnitine


****

****

******

PAH

MPP+

Clonidine

TEA

100

****

**

**

Inhibitory effects

A549 cellsA549 cells

0 50 100 150 200

Control

Nicotine

Verapamil

Pyrilamine

Propranolol

TEA

MPP+

L-Carnitine

Salbutamol


********

Choline **

*

Procaterol **

250

Substrates for



drugs

PAH

*

Each inhibitor was applied at 1 mM. Each bar represents the mean ± S.E.M. (n = 3-9). *p < 0.05, **p < 0.01, significantly differentfrom control. (MPP+, 1-methyl-4-phenylpyridinium; TEA, tetraethylammonium; PAH, p-aminohippurate.)

In vitroHepatocyteHepatocyte

Control

Pyrilamine

Verapamil

Quinidine

Amantadine

Propranolol

0 50 150

Nicotine

Choline

L-Carnitine


****

****

******

PAH

MPP+

Clonidine

TEA

100

****

**

**

Inhibitory effects

A549 cellA549 cell

0 50 100 150 200

Control

Nicotine

Verapamil

Pyrilamine

Propranolol

TEA

MPP+

L-Carnitine

Salbutamol


********

Choline **

*

Procaterol **

250

Substrates for



drugs

PAH

*

Pyrilamine

TR-BBB13 cellsOkura et al., Drug Metab Dispos. 36:2005-2013, 2008.

H+

Each column represents the mean ± S.E.M. (n = 3-6). *p < 0.05, **p < 0.01, significantly different from control.

**

0

20

40

60

80

100

120

140

Perc

enta

ge o

f con

trol

pH 6.4 pH 7.4(Control)

pH 8.4

*

Extracellular pH-dependenceExtracellular pH-dependence Intracellular pH-dependenceIntracellular pH-dependence

In vitro pH-dependence (TR-BBB13 cells)pH-dependence (TR-BBB13 cells)

**

100

50

0Pe

rcen

tage

of c

ontr

ol

150

**

pHi

pH 8.4

AcuteUntreated(Control)

Pretreated

Each column represents the mean ± S.E.M. (n = 3-6). *p < 0.05, **p < 0.01, significantly different from control.

Intracellular pH-dependenceIntracellular pH-dependenceAcute-treatment with NH4Cl

Pre-treatment with NH4Cl

Alkalization

Cell

H+

NH3 NH4+NH3

Cell

H+

NH3 NH4+NH3

Acidification

In vitro pH-dependence (TR-BBB13 cells)pH-dependence (TR-BBB13 cells)

**

100

50

0Pe

rcen

tage

of c

ontr

ol

150

**

pHi

pH 8.4


Pretreated

Each column represents the mean ± S.E.M. (n = 3). *p < 0.05, **p < 0.01, significantly different from control.

0

40

80

120

160

Perc

enta

ge o

f con

trol


pH 8.4

***

0

300

100

200

Perc

enta

ge o

f con

trol


Pretreated**

pHi

pH 7.4


In vitro pH-dependence (TR-iBRB2 cells)pH-dependence (TR-iBRB2 cells)

Each column represents the mean ± S.E.M. (n = 3). *p < 0.05, **p < 0.01, significantly different from control.

0

**

20

40

60

80

100

120

140

Perc

enta

ge o

f con

trol


pH 8.4

* **

0

400

300

100

200

Perc

enta

ge o

f con

trol


Pretreated

*

pHi

pH 7.4


In vitro pH-dependence (Hepatocytes)pH-dependence (Hepatocytes)

*

0

20

40

60

80

100

120

Perc

enta

ge o

f con

trol


pH 8.4

Each column represents the mean ± S.E.M.(n = 3). *p<0.05, **p<0.01, significantlydifferent from control.

Type I-like (TIL) cells and type II (TII) cells wasincubated in MES or HEPES buffer at different pHcontaining 50 µM [3H]nicotine for 15 sec at 37°C.Each point represents the mean ± SEM (n = 3-6).

5 6 7 8

1.0

2.0

3.0

0

pH

Nic

otin

e up

take

rate

(nm

ol/m

g pr

otei

n)

Rat primary alveolar epithelial cells1)

Rat primary alveolar epithelial cells1)

1) Takano et al., J Pharm. Sci. 105:982-988, 2016.

In vitro pH-dependence (A549 cells)pH-dependence (A549 cells)

Extracellular pH-dependenceExtracellular pH-dependence

:Type I-like cells :Type II cells

TR-BBB13 cells

Blood

NicotinePyrilamine

Competitive

Each point represents the mean ± S.E.M. (n = 3). **p < 0.01, significantly different from control.1) Okura et al., Drug Metab Dispos. 36:2005-2013, 2008. ([3H]Pyrilamine uptake by TR-BBB13 cells)

Ki = 15 μM

200

150

100

50

00 5 10 15

V (nmol/(min・mg protein))

V/S

(µL/

(min・m

g pr

otei

n))

≒ Km = 28 μM 1)

: Control: +Pyrilamine (50 µM)

Control (mean ± SD) + Pyrilamine (mean ± SD)

Vmax (nmol/(min・mg protein)) 16.7 ± 1.5 19.5 ± 3.4

Km (µM) 92.4 ± 13.1 371 ± 80**

In vitro Eadie-Scatchard plot (TR-BBB13 cells)Eadie-Scatchard plot (TR-BBB13 cells)

30 40V (nmol/(min・mg protein))

20

80

0 100

20

V/S

(µL/

(min・m

g pr

otei

n))

60

40


Vmax (nmol/(min・mg protein)) 35.3 ± 2.0 38.7 ± 5.1

Km (µM) 492 ± 40 848 ± 257**

TR-iBRB2 cells

Blood

NicotinePyrilamine

Ki = 67.7 μM


Each point represents the mean ± S.E.M. (n = 3). **p < 0.01, significantly different from control.

Competitive

In vitro Eadie-Scatchard plot (TR-iBRB2 cells)Eadie-Scatchard plot (TR-iBRB2 cells)


Vmax (nmol/(min・mg protein)) 1.78 ± 0.71 0.468 ± 0.087**Km (µM) 141 ± 63 180 ± 50

Kd (mL/(min・mg protein)) 5.69 × 10-3 ± 0.67 × 10-3 6.06 × 10-3 ± 0.03 × 10-3

Each point represents the mean ± S.E.M. (n = 3-6). **p < 0.01, significantly different from control.

30V (nmol/(min・mg protein))

10

20

0 100

20

V/S

(µL/

(min・m

g pr

otei

n))


0 0.5 1.5 210

0.01

V-KdS (nmol/(min・mg protein))

(V-K

dS)/S

(mL/

(min・m

g pr

otei

n))

Blood

NicotinePyrilamine

Non-competitive

Hepatocytes

In vitro Eadie-Scatchard plot (Hepatocytes)Eadie-Scatchard plot (Hepatocytes)

NicotineH+

Novel H+/organic cation antiporter is involved in nicotine transport in BBB, inner BRB, liver and lung.

Novel H+/organic cation antiporter is involved in nicotine transport in BBB, inner BRB, liver and lung.

SummaryBrain

Retina

Lung

Liver

H+

Summary

Cationic drugs

The use of novel H+/organic cation antiporter offers thepossibility of delivering cationic drugs to the target organvia lung.

Cationic drugs Nicotine

Acknowledgements

Dr. Yoshiyuki Kubo , Dr. Shin-ichi Akanuma, Yuma Tega, Chihiro Yuzurihara

Implication of the carrier-mediated transport of nicotine ... · Implication of the...

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