Transporters and the lung

Mark Gumbleton

June 2010

APS “Biopharmaceutics of inhaled drug delivery”

Iceland

Transporter families

• ABC superfamily– 48 transporters in Human

– Seven families • e.g. ABCC2 (C family & 2 individual isoforms)

• SLC/SLCO superfamily– approx 300 human

– 47 families • e.g. SLCO1A2 (1 family ; A subfamily ; 2 individual isoform)

• Amino-acid sequence identity, e.g. SLCO– Family members, e.g. OATP1 and Oatp1, with >40% amino acid sequence

identity

– Subfamily members, e.g. OATP1A and Oatp1a when > 60% amino acid sequence identity

– Individual isoforms within a subfamily, e.g. OATP1A2 and Oatp1a1

Gene Protein

ABCB1 MDR1/P-glycoprotein

ABCB4 MDR3/P-glycoprotein

ABCC1 MRP1

ABCC2 MRP2

ABCC3 MRP3

ABCC4 MRP4

ABCC5 MRP5

ABCC6 MRP6

ABCC10 MRP7

ABCC11 MRP8

ABCC12 MRP9

ABCG2 BCRP

SLC15A1 PEPT1

SLC15A2 PEPT2

SLC22A1 OCT1

SLC22A2 OCT2

SLC22A3 OCT3

SLC22A4 OCTN1

SLC22A5 OCTN2

SLC22A6 OAT1

SLC22A7 OAT2

SLC22A8 OAT3

SLCO1A2 OATP1A2

SLCO1B1 OATP1B1

SLCO1B3 OATP1B3

SLCO2B1 OATP2B1

Context for small molecule drug transporters

in the lung Following inhalation the rate and extent of drug lung trans-epithelial transport/membrane permeation involves an interplay between:

• dose and deposition site of drug within the lung• drug availability/exposure to the epithelial surface• physiological variables operational at the epithelial-luminal interface.

The latter would include:

• Passive permeability of the barrier(s) to drug• Competitive and sequential clearance mechanisms (e.g.

mucocilliary clearance, metabolic, active transport) • Regioselectivity

• Consider impact of pulmonary transporters upon uptake of drugs from systemic circulation

Active transporters in lung epithelium may influence:

• drug airway residence times/intra-luminal kinetics and airway to

systemic absorption profiles.

• access of inhaled or systemically administered drugs to intracellular

targets

• drug extraction from the systemic circulation into the lung with

consequent pulmonary accumulation of drug, pulmonary toxicity /

altered systemic PD

• inter-individual variation, drug-drug interactions

Active transporters in lung epithelium may be subject to modulation by:

• disease

• NCEs leading to alterations in the physiologic function

Potential Impacts

Physiological roles

• P-gycoprotein (MDR1)– Xenobiotic defense

– Phospholipid translocation

• OCTN2– Co-transport of Na+ and carnitine into cell

– Carnitine transports (cytosol to mitochondrial matrix) long chain acyl groups (from fatty acids) leading to ATP production

– Increased foetal lung carnitine appears to enhance phospholipid synthesis and DPPC content in developing alveolar epithelial cells

What kind of information do we have?

What kind of information do we need ?

(q)RT-PCR mRNA datain whole tissues

Whole rat lung

Campbell et al 2003, Francombe et al

Brady et al 2002

Beneficial:

Confirms an absence of a particular mRNA

• ‘Cognisant of false negatives’

Potential to gauge relative levels of same transcript across tissues

•Basis for further protein and functional assessments •Corroboration of functional data

mdr1a

mdr1b

Total lung mdr1 levels low• vs ileum x60-fold lower [1a]• vs x2-fold lower [1b]

Lung mdr1b > mdr1a

Microarray mRNA screening whole tissue

Beneficial but:

• Need multi-laboratory screen on normal (diseased) human lungs

• Need protein data

• Need spatial perspective

• Total lung epithelial cell population that contributes less than 20% of total lung parenchymal cell number

• Comparative data for lungs of various genera, e.g. rats, mice

Bleasby et al 2006

• Human data as absolute Intensity expressed as percentiles against reference set of 19,000 genes in 100 tissues

• Probes against exons and exon-exon junctions

• Whole tissues – cadaver tissue pooled from greater 12 individuals

Extracted Bleasby data: inter-genera lung

Relative expression levels in lung (Log10 ratio Lung:Pool)

The reference pool was a species-specific combination of at least 20 different, disease-free, adult tissues.

[Note: NOT ABSOLUTE expression between genera]

Dog=Rat=Mouse ≠ Human

Dog=Rat=Mouse ≠ Human

Generally Higher levels in lung vs pooled

Mouse Higher levels in lung vs pooled

Rat ≠ Human?

Human Higher levels in lung vs pooled

Human Higher levels in lung vs pooled

SLC29A1 (ENT1)

SLCO2A1 (PGT)

SLCO2B1 (OATP)

MDR1/mdr1a

OAT4

OCTN1

SLCO4C1 (OATP)

Spatial perspectives on transporter protein expression

AS

AS

ASAS

CP

BE

AS

MDR1

Campbell et al 2003

Reviews• van der Deen et al 2005• Bosquillon 2009

Key Issues • Driven initially by positive genes in RNA

screening

• AIM: Consensus mapping of the spatial pattern in drug transporter protein expression across genera

• Involve panel (at least 2) of antibodies* against single antigen

• Incorporate ‘quantitative’ scoring with comparison across tissues

• Immunoelectron microscopy for issues on resolution

* In-situ hybridization may augmentNewman et al 1999

Human expression at spatial level in intact lung

P-gp: Campbell et al 2003; Cordon-Cardo et al 1990; Endter et al 2007;

Lechapt-Zalcman et al 1997; van der Valk et al 1990; Scheffer et al 2002

MRP: Brechot et al 1998; Flens et al 1996; Scheffer et al 2002

BCRP: Fetsch et al 2006; Scheffer et al 2002

OCT: Lips et al 2005

OCTN: Horvath et al 2007

PEPT: Groneberg et al 2001

Conducting airway epithelium

Capillaries

Serous cells

Mucus cells

Alveolar epithelium

Alveolar macrophageAIRWAY

VASCULAR LUMEN

2

1,21,2,3

van der Deen 2005 ABC expression

AE type I-like

Campbell et al 2003

In-vitro lung epithelial cells (ideally primary) can corroborate issues of expression and functionality at the cellular level

For example: At immuno-histochemical level in intact lung the expression of P-gp in alveolar epithelium remains controversial

200 kDa

130 kDa

AE cells MDCK-MDR1

Horvath et al 2007

In-vitro lung epithelial cells (ideally primary) can corroborate issues of expression and functionality at the cellular level

Figure A: Organic cation transporter RNA expression in scraped airway epithelium and primary cultures at an ALI

Figure B: Uptake of 10mM organic cation ASP [4-(4-(dimethylamino)styryl)-N-methylpyridinium] into human airway epithelial cells for 15min @37oC

Other independent investigators & studies ………..

Figure C: Uptake of 10mM ASP into human airway epithelial cells when incubated for 15 min with b2-adrenoreceptor agonists

In-vitro epithelial cells (not necessarily lung cells) can identify transporters thatmay affect absorption and distribution in the intact lung tissue

HEK OCTN 1 Transfectants

OCTs OCTN1 OCTN2

Ergothioneine mM - 21 -

TEA mM 76-1300 195-1300 300

L-carnitine mM - 24 5

HEK OCTN 2 Transfectants

Nakamura et al 2010

Judging impact of transporters on absorption and pulmonary disposition

• Empiric in-vitro models may find a role in prediction

• Intact lung architecture required:

• In-vitro/in-vivo predictions

• Sequential and parallel clearance mechanisms

• Variation in functional epithelial phenotypes down the respiratory tract coupled with varying deposition patterns

Complicating Issues:

1.Inter-species correlation (and issues that go with this)

2.Redundancy in functional interactions between transporters and substrates

3.Chemical inhibitors that are selective but not specific*

Intact lung architecture

2003 - Tronde et al 2003 a,b In-vivo rat & IPRL - Losartan (Caco-2 ER 3-4)

• Pulmonary F% high, e.g. IPRL 94% in 2 hrs with t1/2 absorption 26 min.

2008 - Manford et al 2008 In-vivo mice (CF-1 spontaneous mdr1a –deficient)

• Intra-tracheal instillation of ≈ 80-90 ng Digoxin (Caco-2 ER 10-20)

• No difference in rate or extent of clearance of Digoxin from lung.

2009 - Madlova et al 2009 IPRL rat

• Instillation of Digoxin ± GF120918

• No difference in rate or extent of transfer of digoxin

Explicitquestion

Byron & Niven

Intact lung architecture

2008 - Francombe et al 2008 IPRL rat

Instillation of 5 nmoles RH123 ± GF120918

Hydrochloride salt of Rhodamine123

MW 381, pKa 7.2, Log P 1.06. Delocalised cationic compound

Rat endogenous lung fluid volume (based on surface area): Alveolar volume ~ 80 mL

/ conducting ~ 200 mLWith assumptions, a dose

of 5 nmoles approxairway conc of 5 – 15 mM

Disodium salt of Fluorescein MW 376 (F-Na)

Tetramethyl Rosamine< 1 % absorbed

Rhodamine 123

Intra-luminal kinetics

Intact lung architecture

P-gp abundance membrane dwell time P-gp afinity

Uptake into Lung from systemic circulation:

mdr1 KO mice studiesAdministering drugs i.v. or orally indicate that the KO has no affect upon the accumulation of respective substrates in lung tissue [based upon plasma : lung ratios]

GF used @ 2-70 mM Pulmonary airspace

Capillary lumen

Summary• Challenge - gain a consensus of the circumstances under which transporters may

impact upon pulmonary drug absorption and/or disposition

Some issues for pulmonary drug transporter research:

• Presence, and spatial pattern of protein expression, of drug transporters within normal human lung, and the lungs of various genera, e.g. rats, mice etc.

• Make appropriate use of models of varying complexity. Ultimately studies in intact lung tissue needed even if simple solution/suspension instillation.

• For a given transporter exploit a range of substrates of differing physico-chemical properties together with different inhibitors that possess dissimilar inhibitory profiles.

• Consider intra-luminal kinetics and lining fluid concentrations – Note for drugs retained in the lung to high extents

• Transgenic knockout animals should significantly aid progress. – Studies in small animals will require consideration of the performance of small animal pulmonary delivery

devices.

• Consider drug accumulation by the lung from systemic circulation

• ‘Pulmonary’ context of disease impact upon transporter function, the potential for drug-drug interactions, the potential of transporters contributing to inter-individual variation

Acknowledgements

• Ghaith Al-Jayyoussi

• Adam Crandon-Lewis

• Danielle Francombe

• Chris Morris

• Masahiro Sakagami

• Mathew Smith

• Glyn Taylor

• Collaborators at GSK

– Graham Somers

– Chris Edwards

– John Keogh

– Peter Eddershaw