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2010_TSRC38_Robinson.pdf

INFLUENCE OF ON THE PERMEABILITY OF ARTIFICIAL CELL MEMBRANES TO ACROLEIN, BENZO(A) (BAP), AND 4- (METHYLNITROSAMINO)- 1-(3-PYRIDYL)-1- BUTANONE (NNK) E.A. Robinson and F.R. Perini Lorillard Company Greensboro, NC

1 th 64 TSRC, Hilton Head, SC TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed Overview 2010_TSRC38_Robinson.pdf

• Background • PAMPA Method • PAMPA Procedure • Data

• Conclusions 2 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed Background 2010_TSRC38_Robinson.pdf

• Azzi et al. (2006) – at 0.08% (5 mM) menthol, did not affect the permeation of BaP and NNK test compounds but did increase the membrane reservoir formation for NNK

– Porcine esophagus

– Morphologically different from lung tissue

– Significantly higher menthol concentration than

delivered in (1 µM) 3 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed Background 2010_TSRC38_Robinson.pdf

• Squier et al. (2010) – at 0.08% (5 mM) menthol, permeability to both 3H-N-Nitrosonornicotine (NNN) and 3H- was significantly increased (p < 0.05)

– Porcine buccal and floor of mouth mucosa

– Morphologically different from lung tissue

– Significantly higher menthol concentration than

delivered in cigarette smoke (1 µM) 4 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed Cellular Permeation 2010_TSRC38_Robinson.pdf

Transcellular

Paracellular

Active Passive transport transport Endocytosis 5 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed PAMPA Method 2010_TSRC38_Robinson.pdf

• Parallel Artificial Membrane Permeability Assay – Developed in 1998 by Kansy et al. as a screening alternative to cell-based assays

– Allows prediction of passive transport properties

– Quantitation by plate reader 6 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed PAMPA Method 2010_TSRC38_Robinson.pdf

• No cell culture • Membrane created at time of experiment – About 300 bilayers thick

• Good prediction for passively

transported compounds 7 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed PAMPA Plate Design 2010_TSRC38_Robinson.pdf

Membrane

Acceptor (pH 7.4 buffer)

Donor (analyte in pH 7.4 buffer)

• 125 µm-thick microfilter disc, coated with a 2% wt/vol

dodecane solution of dioleoylphosphatidylcholine (DOPC) 8 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed PAMPA Procedure

• Analyte to menthol concentrations 2010_TSRC38_Robinson.pdf represent amounts of analyte class relative to menthol in 3R4F reference cigarette smoke

• Analytes dissolved in a pH 7.4 buffer from pION

• DMSO added at 1.56% for BaP and 12.48% for NNK and acrolein. Measurement using Varioskan • Dioleoylphosphatidyl-choline in •Acrolein - absorbance 300 nm dodecane pipetted onto the filter to •BaP- fluorescence 504 nm produce an artificial membrane •NNK - absorbance 328 nm

• Incubation at 37 oC 9 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed Effective Permeability Calculation 2010_TSRC38_Robinson.pdf

-6 Pe = -218.3 log 1 - 2 CA(t) 10 cm/s

t CD(0)

t = time in hours

CA(t) = acceptor concentration at time t CD(0) = donor concentration at time 0

Equation from pION, Inc PAMPA Protocol 10 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed 2010_TSRC38_Robinson.pdf 10 mM Acrolein PAMPA Membrane Retention at 3 hours

0.002

0.0015

0.001

0.0005

0

[acrolein], M *

-0.0005 Acrolein Acrolein + 0.26 mM Menthol -0.001

-0.0015

Acrolein + Acrolein + Acrolein + Acrolein

Acrolein Acrolein + 0.13 mM Menthol Acrolein + 0.260.26 mM MentholmM menthol Acrolein0.521 + 0.521 mM mM menthol Menthol 0.13 mM menthol TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed 2010_TSRC38_Robinson.pdf Effective Permeability for 10 mM Acrolein, 3 hours

7.00E-06

6.00E-06

5.00E-06

4.00E-06

3.00E-06 *

2.00E-06 Effective Perm eability, cm /s 1.00E-06

0.00E+00

Acrolein Acrolein + 0.13 AcroleinmM + 0.26 mM Acrolein + 0.521 mM

Menthol Menthol Menthol TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed 2010_TSRC38_Robinson.pdf 1.75 µM BaP PAMPA Membrane Retention at 4 hours

1.80E-06 1.60E-06 1.40E-06 1.20E-06 1.00E-06 8.00E-07

[BaP], M [BaP], 6.00E-07 4.00E-07 2.00E-07 0.00E+00

1.75 micro M 1.75 micro M 1.75 micro M 1.75 micro M

BaPBaP BaP/BaP 0.065 + mM BaP/BaP 0.13 + mM BaP/ 0.26BaP + mM 0.065 mM menthol 0.13 mM menthol 0.26 mM menthol Menthol Menthol Menthol TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed 2010_TSRC38_Robinson.pdf Effective Permeability for 1.75µµµM BaP at 4 hours

1.40E-06

1.20E-06

1.00E-06

8.00E-07

6.00E-07

4.00E-07

2.00E-07

0.00E+00

Effective Permeability,-2.00E-07 cm/s BaP BaP + 0.13 mM Menthol -4.00E-07

-6.00E-07

BaP + BaP + BaP + BaP

Effective Permeability0.13 mM menthol 0.26 mM menthol 0.065 mM menthol TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed 2010_TSRC38_Robinson.pdf 1.284 mM NNK PAMPA Membrane Retention at 6 hours

0.0084

0.0083

0.0082

0.0081

0.008 [NNK], M

0.0079

0.0078

0.0077

NNK NNK + 0.13 mM NNK + 0.26 mM NNK + 0.521 mM

Menthol Menthol Menthol TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed 2010_TSRC38_Robinson.pdf Effective Permeability for 1.284 mM NNK at 6 hours, cm/s

1.20E-06

1.00E-06 * 8.00E-07

6.00E-07

4.00E-07

Effective Permeability, cm/s 2.00E-07

0.00E+00

NNK NNK + 0.13 mM NNK +0.26 mM NNK + 0.521 mM

Menthol Menthol Menthol TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed 2010_TSRC38_Robinson.pdf 1.284 mM NNK PAMPA Membrane Retention at 18.5 hours

0.0084

0.0083

0.0082

0.0081 [NNK], M 0.008

0.0079

0.0078

NNK NNK + 0.13 mM NNK + 0.26 mM NNK + 0.521 mM

Menthol Menthol Menthol TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed 2010_TSRC38_Robinson.pdf Effective Permeability for 1.284 mM NNK, at 18.5 hours

4.00E-06

3.50E-06 3.00E-06 * 2.50E-06

2.00E-06 *

1.50E-06

1.00E-06 Effective Permeability, cm/s 5.00E-07

0.00E+00

NNK NNK + 0.13 mM NNK +0.26 mM NNK + 0.521 mM

Menthol Menthol Menthol TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed Conclusions

• Effective permeability of acrolein is influenced by 2010_TSRC38_Robinson.pdf menthol at concentrations 100x higher than present in smoke, but only at higher menthol concentrations and not consistently

• Addition of menthol to the PAMPA test system does not increase effective permeability of BaP

• Addition of menthol to the PAMPA test system at levels 100x higher than present in smoke increases effective permeability of NNK inconsistently

– Only at highest concentration at 6 hours

– Only at lower concentrations at 18.5 hours 19 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed Conclusions 2010_TSRC38_Robinson.pdf

• Addition of menthol has no statistically significant effect on membrane reservoir formation

• Data do not support a role of menthol in increasing human exposure to cigarette smoke constituents through changes in cell membrane permeation or membrane retention

• If menthol does not affect permeability at 100-

fold higher levels than delivered in smoke, it is

not expected to at realistic levels 20 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed

2010_TSRC38_Robinson.pdf Questions? TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed References 2010_TSRC38_Robinson.pdf

• Azzi, C.; Zhang, J.; Purdon,C.H.; Chapman, J.M.; Nitcheva, D.; Hebert, J.R.; Smith, E.W. 2006 , 27(1). 137–145.

• Kansy, M.; Senner, F.; Gubernator, K. Journal of Medicinal Chemistry 1998 , 41, 1007-1010.

• Squier, C.A.; Mantz, M.J.; Wertz, P.W. Nicotine & Tobacco Research 12 (7), 763–767. 2010, TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed TSRC2010(64) - Document not peer-reviewed by CORESTA 2010_TSRC38_Robinson.pdf Acrolein Effective Permeability 2010_TSRC38_Robinson.pdf

Analyte Effective Permeability at 6 hours, cm/s

Acrolein 2.18 x 10 -6 + 1.35 x 10 -6

Acrolein + 0.261 mM 2.97 x 10 -6 + 1.19 x 10 -6 Menthol

Acrolein + 0.521 mM 4.68 x 10 -6 + 1.16 x 10 -6 Menthol

Acrolein + 1.042 mM 3.66 x 10 -6 + 5.02 x 10 -7

Menthol 24 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed BaP Effective Permeability 2010_TSRC38_Robinson.pdf

Analyte Effective Permeability at 4 hours, cm/s

BaP 6.82 x 10 -8 + 5.22 x 10 -7

BaP + 0.13 mM menthol 3.86 x 10 -8 + 2.06 x 10 -7

BaP + 0.26 mM menthol 1.40 x 10 -7 + 4.69 x 10 -7

BaP + 0.52 mM menthol 6.70 x 10 -7 + 1.26 x 10 -7 25 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed NNK Effective Permeability 2010_TSRC38_Robinson.pdf

Analyte Effective Permeability at 18.5 hours, cm/s

NNK 2.56 x 10 -6 + 5.85 x 10 -8

NNK + 0.261 mM Menthol 3.32 x 10 -6 + 1.59 x 10 -7

NNK + 0.521 mM Menthol 2.99 x 10 -6 + 9.55 x 10 -8

NNK + 1.042 mM Menthol 2.55 x 10 -6 + 5.69 x 10 -8 26 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed Acrolein PAMPA Permeability, Donor Plate 2010_TSRC38_Robinson.pdf

1.80E-02 1.60E-02 1.40E-02 1.20E-02 1.00E-02 8.00E-03

[acrolein], M [acrolein], 6.00E-03 4.00E-03 2.00E-03 0.00E+00 0 20 40 60 80 100 120 140 160 180 200 Experiment duration, minutes

10 mM Acrolein 10 mM Acrolein/ 0.521 mM Menthol

10 mM Acrolein/ 0.26 mM Menthol 10 mM Acrolein/ 0.13 mM Menthol 27 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed Acrolein PAMPA Permeability, Acceptor Plate 2010_TSRC38_Robinson.pdf

2.50E-03

2.00E-03

1.50E-03

1.00E-03

[acrolein], M 5.00E-04

0.00E+00 0 20 40 60 80 100 120 140 160 180 200 -5.00E-04 Experiment Duration, minutes

10 mM Acrolein 10 mM Acrolein/ 0.521 mM Menthol

10 mM Acrolein/ 0.26 mM Menthol 10 mM Acrolein/ 0.13 mM Menthol 28 TSRC2010(64) - Document not peer-reviewed by CORESTATSRC2010(64) - Document not peer-reviewed