efflux transporters in the lungs - siminhale cost · 2019. 9. 29. · p-gp can increase the...

Efflux transporters in the lungs

Dr. rer. nat. Carsten Ehrhardt, M.A., F.T.C.D.Professor in Pharmaceutics and BiopharmaceuticsSchool of Pharmacy and Pharmaceutical Sciences

SimInhale International Conference on Current Challenges and Future Opportunities for Inhalation TherapiesAthens, GreeceDate 02/10/2019

Trinity College Dublin, The University of Dublin

Outline

1. Membrane transporters

2. Case study – P-glycoprotein (MDR1/P-gp)

3. Case study – Multidrug resistance-associated protein 1 (MRP1)

Membrane transporters

o More than 450 different drug transporters have been identified in humans (ABC and SLC family).

o Approx. 5% (> 2000 genes) of the human genome is associated with drug transporters.

o 48.5% (50 / 103) of FDA-approved new molecular entities (2013 ‐ 2016) contain patient information leaflet warnings about transporter-related drug-drug interactions.

o Since 2013, transporter-drug interactions have to be tested in a panel of 7 (EMEA) or 9 (FDA/Japan) different transporters.

Conformational changes of P-gp

Adapted Li et al. (2018) J Biol Chem


Transporters in lung epithelium may influence

o Airway residence/intra-luminal kinetics [1]

o Access to intracellular targets of inhaled drug to EPI [2] or systemic drug to ENDO [5]

o Airway to interstitium [3] or blood to interstitium [6]

o Interstitial residence/kinetics [4]

o Interstitium to blood [7] and interstitium to airway [8]

o Composites of above.

Adapted from Mark Gumbleton with permission

MRP1

P-gp


Outline





Transporter expression analysis (RT-PCR)

Endter et al. (2009) J Pharm Pharmacol


The first report of P-gp expression in human bronchus dates back to 1997 (Lechapt-Zalcman et al. Eur Respir J)

Expression and function in human alveolus and cultured alveolar epithelial cells was demonstrated in 2007 (Endter et al. Cell Tissue Res)

P-gp localisation in human lung

tissues and cells


P-gp 170 kDa Bi-directional transport of rhodamine 123

Salomon et al. (2014) Mol Pharm

P-gp localisation and activity in NCI-H441 cells

BA/AB ratio: 3.43 in NCI-H441 cells3.09 in ATI-like cells


Tissue Mdr1a+/+ Mdr1a -/- Ratio (-/-) : (+/+)

Brain 5 ± 7 112 ± 18 22.4

Muscle 22 ± 9 148 ± 14 6.7

Heart 64 ± 15 219 ± 39 3.4

Kidney 399 ± 120 932 ± 75 2.3

Liver 115 ± 61 281 ± 75 2.4

Gall Bladder 342 ± 143 466 ± 69 1.4

Lung 517 ± 47 1058 ± 191 2.1

Stomach 320 ± 96 563 ± 81 1.8

Sm. Intestine 197 ± 25 577 ± 102 2.9

Colon 214 ± 43 444 ± 51 2.1

4 hours following 1 mg/ kg I.V. dose

Schinkel et al. (1994) Cell

P-gp effect on vinblastine organ distribution


Absorption of P-gp substrates from

the isolated-perfused mouse lung

Al-Jayyoussi et al. (2013) J Pharm Sci

Trinity College Dublin, The University of Dublin Al-Jayyoussi et al. (2013) J Pharm Sci

Absorption of P-gp substrates from the

isolated-perfused mdr1a/1b-/- mouse lung


32 subjects were randomised to one of two cohorts, each subjecttaking part in two 13-day treatment periods, as follows:

Cohort 1, Period 1: GSK573719 (500 μg) once daily for 8 days,immediately followed by Period 2: 5 days of GSK573719 (500 μg) andverapamil SR 240 mg, 1 hour prior to inhalation.

Cohort 2, Period 1: GSK573719 (500 μg) and GW642444 (25 μg) for 8days, immediately followed by Period 2: 5 days of GSK573719 (500 μg),GW642444 (25 μg) and verapamil SR 240 mg, 1 hour prior to inhalation.

Approved dose: 62.5 µg

tmax = 5.2 h

Trinity College Dublin, The University of Dublin Mehta et al. (2013) Int J COPD

123%

126%

127%

104%

119%

138%


Outline





MRP

TransportersGSH

Glutathion S-transferases (GSTs)

GS X

Nucleophilic substitutionNucleophilic additionPeroxide reduction

TransacetylationDouble bond isomerisation

MRP1’s role in cellular detoxification

Trinity College Dublin, The University of Dublin van der Deen et al. (2006) Virchows Arch

MRP1 abundance is linked to

Chronic Obstructive Pulmonary Disease


Transporter expression analysis (RT-PCR)

Endter et al. (2009) J Pharm Pharmacol


MRP1 expression in human alveolar

epithelial cells

AT1-likeAT2

MRP1 expression inn AT2 and AT1 -like and H441 cells

AT2

AT1

-like

NCI-H

441

0

200

400

600**

Rela

tive e

xp

ressio

n level

Data represent means ± SD, n = 3 - 6, **P <0.01


AIC LCC

MRP1 expression in the NCI-H441 cell line

Data represent means ± SD, n = 6


MRP1 is localised to the basolateral cell membrane of AT1-like and NCI-

H441 cells and mediates net absorption of carboxyfluorescein (CF)

NCI-H441

MRP1

AT1-like

Surface protein biotinylation. Ap and Bs, indicate apically and basolaterally sulfo-NHS-biotin treated cells, respectively, and C indicates whole cell lysate cfda transport AT1 cells 13 04 16 100um

a to

b

b to a

a to

b

b to a

0

1.010-7

2.010-7

3.010-7

**

*

**

+MK-571 (20µM)

Pa

pp (

cm

/s)

pooled cfda transport h441 100uM

a to

b

b to a

a to

b

b to a

0

1.010-7

2.010-7

3.010-7 **

+MK-571 (20µM)

**

Pa

pp (

cm

/s)

Data represent means ± SD, *P <0.05, **P <0.01, n = 9


MRP1-mediated CF efflux from AT1-like and NCI-H441

cells occurs mainly across the basolateral membrane

Bi-directional CF efflux, data represent means ± SD, n = 6, **p ≤ 0.01, ***p ≤ 0.001

0 15 30 45 60 75 90

0

200

400

600C

F (

pg

/µ

g p

rote

in)

Time (min)

***

***

*

NCI-H441

0 30 60 90

0

100

200

300

CF

(p

g/µ

g p

rote

in)

Time (min)

*****

AT1-like


Budesonide reduces MRP1 activity

without changing its expression level

0 20 40 60 800

20

40

60

Control

Budesonide 10 µM

Budesonide 5 µM

time (min)

CF

eff

lux

(as %

of

intr

acellu

lar

co

nc.

at

T0)

0 15 30 45 60 75 90

0

20

40

60

Control

5 µM budesonide

10 µM budesonide

**

***

time (min)

CF

eff

lux

(as %

of

intr

acellu

lar

co

nc.

at

T0)

0 15 30 45 60 75 90

0

20

40

60

Control

5 µM budesonide

10 µM budesonide

**

***

time (min)

CF

eff

lux

(as %

of

intr

acellu

lar

co

nc.

at

T0)

NCI-H441 cells AT1-like cells

Data represent means ± SD, n = 3 - 9, **P <0.01, ***P <0.001


Salbutamol salts have no effect on MRP1

activity and expression levels in NCI-H441 cells

0 20 40 60 800

20

40

60

Control

Salbutamol 100 µM

Salbutamol sulphate 100 µM

R-Salbutamol HCl 100 µM

S-Salbutamol HCl 100 µM

time (min)

CF

(%

in

tracellu

lar

co

nc.

at

T0)

Data represent means ± SD, n = 3 - 6


Data represent means ± SD, n = 3, **P <0.01

Tobacco smoke extract reduces MRP1 activity but up-

regulates its expression in vitro

NCI-H441 cells AT1-like cells

0 15 30 45 60 75 90

0

200

400

600

800

CF

(p

g /

µg

pro

tein

)

Time (min)

Control

10% fresh CSE

10% aged CSE

0 15 30 45 60 75 90

0

100

200

300

400

500

CF

(p

g /

µg

pro

tein

)

Time (min)

Control

5% CSE

Legend

0 15 30 45 60 75 90

0

100

200

300

400

500

CF

(p

g /

µg

pro

tein

)

Time (min)

Control

10 µM Sulforaphan

5% CSE

and Nrf2 activation with sulforaphane can reverse the effect

0 -

5 -

10 -

0

50

100

150

200

250***

Treatment [CSE %]

Rela

tive e

xp

ressio

n level


Summary and Conclusions

P-gp and MRP1 are functionally expressed in human lung (epithelial) cells

P-gp can increase the retention time of inhaled drugs in the lungs, whilst MRP1 is involved in drug clearance

Inhaled drugs can interact with ABC efflux transporters in vitro, ex vivo and in vivo

Tobacco smoke extract up-regulates MRP1 expression but reduces its activity

Nrf2 activation can rescue MRP1 function after tobacco smoke exposure

Efflux transporters might be novel drug targets in COPD (and other lung diseases)


Downstream Consequences

Pulmonary drug recycling/ sequestration / accumulation with toxicity/ altered systemic PD

Inter-individual variation (SNPs), drug-drug interactions (also with orally taken drugs)

NCEs can alter physiologic function of transporters

Ehrhardt LabJohannes SakeMohammed Ali Selo

Former Lab-MembersLeonie BaginskiStephen BuckleyCaoimhe ClerkinSibylle EndterManuela GasparStephany MicallefSabrina NickelJohanna SalomonElena SchwagerusJanani SwaminathanSabrina Nickel

Brigham Young UniversityPaul Reynolds

University of KanazawaYukio Kato

St. James’s HospitalJoseph Keane

UMC GroningenIrene HeijinkAlen Faiz

University of ToyamaKen-ichi Hosoya

Cardiff UniversityMark Gumbleton

HIPS SaarbrückenClaus-Michael LehrNicole Daum

AITOliver Langer


Open questions

What are the exact (sub)cellular localisations of drug transporters in the lung?

How are drug transporters in the lung regulated in health and disease?

What is the effect of smoking on drug transporters?

What are the physiological substrates of drug transporters in the lung?

Are species differences important?

Are the currently used in vitro models appropriate?

efflux transporters in the lungs - siminhale cost · 2019. 9. 29. · p-gp can increase the...

Documents