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