J. M. Fletcher, R. A. Hughes / Bioorg. Med. Chem. 17 (2009) 2695–2702
2701
1 ꢃ 10ꢁ5 M. Experiments were performed in triplicate, a total of
three or four times. Statistical analysis was performed using
GraphPad PrismTM software (version 4.0; GRAPHPAD Software Inc.).
For each experiment, data from triplicate well counts were aver-
aged and normalised to BDNF positive (set to 100%) and negative
(set to 0%) controls. Before grouping data from individual experi-
ments, parametric one-way analysis of variance (ANOVA) was car-
ried out to ensure variation of sample means between experiments
was not significant. Grouped data was then expressed as
mean standard error of the mean (SEM). One-way ANOVA, fol-
lowed by post-hoc Dunnett’s tests36 were used to assess significant
differences in neuronal survival between peptide treatments and
BDNF-treated positive controls. Pairs of concentration–response
curves were tested for significant difference by one-way ANOVA
followed by post-hoc Bonferroni’s multiple comparisons test.37
Data was deemed to be significantly different to controls when p
values were determined to be less than 0.05. For the purpose of
by measurement of trans-epithelial electrical resistance (TEER)
across each monolayer, and by measuring the passage of 14C-man-
nitol across control monolayers. Transport studies were performed
by adding peptide (200 lM in 400 lL HBSS) to the apical (inner)
chamber, and then measuring its accumulation in the basolateral
(outer) chamber (containing 1.5 mL HBSS with 30 g/L BSA (as rec-
ommended by39). At 30 min time points, for 150 min, 1 mL sam-
ples were removed from each basolateral compartment and
replenished with fresh buffer. Each sample was then prepared for
LCMS analysis as follows: (i) 250 lL of the peptide-containing solu-
tion was removed to a fresh 1.5 mL Clear-ViewÒ siliconized poly-
propylene tube; (ii) Peptides (and presumably BSA) were
acetylated by the addition of triethyl amine (25
lL) and acetic
anhydride (12.5 L) at room temperature for 1 h; (iii) The acetyla-
l
tion reaction mixture was diluted with 1 mL of water and lyophi-
lised; (iv) Dried, acetylated peptide-containing samples were
redissolved in 2% aqueous TFA (250 lL), sonicated (10 min) and
comparing peptide potency we used the parameter pEC20BDNF
—
placed on ice (10 min); (v) Samples were then centrifuged
which we define here as the negative log10 of the concentration
of each peptide that elicits a % normalised neuronal survival re-
sponse equivalent to 20% of that observed for BDNF positive
controls.
(10,000 RPM, 10 min) causing undissolved, acetylated BSA adducts
to collect as a solid pellet; (vi) 200 lL of supernatant was removed
and the presence of the single, bis-acetylated peptide species ana-
lysed by LCMS.
Full experimental details of membrane permeability experi-
ments are provided in the Supplementary data and are available
on the Web-based edition of this Journal.
5.4. Plasma stability assay
Peptides (0.2 mg) were incubated at 37 °C in a solution of plas-
ma prepared from a single mouse (50% in PBS, 200
lL) in a silicon-
Acknowledgments
ised 1.5 mL centrifuge tube. 4-Isopropylbenzyl alcohol (0.05% (v/
v)) was added as an internal standard,38 and the time-course of
peptide degradation monitored by RPHPLC. At the final time point
(24 h) the peak of interest was collected and the fraction analysed
by mass spectrometry to rule out the possibility of degradation
having occurred without a concomitant shift in RPHPLC retention
time. Samples were prepared for analysis as follows: At each time
J.M.F. was the recipient of a Melbourne Research Scholarship.
We would like to thank Dr. Colin House for assistance with LCMS
analysis.
Supplementary data
point, an aliquot (10
solution and placed immediately on ice. Each aliquot was then
treated with -lysine monohydrochloride (1000 equiv in 10 L) to
displace the peptide from possible plasma protein binding10, and
MeCN (60 L) causing plasma proteins to precipitate from solution.
Each aliquot was then centrifuged (4 °C, 10 min, 10,000 rpm) and
the supernatant (60 L) collected and transferred to a second
1.5 mL tube and diluted with water (100 L). Samples were then
lL) was removed from the peptide/plasma
Supplementary data associated with this article can be found, in
L
l
References and notes
l
1. Alberch, J.; Perez-Navarro, E.; Canals, J. M. Prog. Brain Res. 2004, 146, 195.
2. Huang, E. J.; Reichardt, L. F. Annu. Rev. Biochem. 2003, 72, 609.
3. Gentry, J. J.; Barker, P. A.; Carter, B. D. Prog. Brain Res. 2004, 146, 25.
4. The BDNF Study Group (Phase III). Neurology 1999, 52, 1427.
5. Poduslo, J. F.; Curran, G. L. Brain Res. Mol. Brain Res. 1996, 36, 280.
6. Ibanez, C. F. J. Neurobiol. 1994, 25, 1349.
7. O’Leary, P. D.; Hughes, R. A. J. Neurochem. 1998, 70, 1712.
8. Fletcher, J. M.; Hughes, R. A. J. Pept. Sci. 2006, 12, 515.
9. O’Leary, P. D.; Hughes, R. A. J. Biol. Chem. 2003, 278, 25738.
10. Fletcher, J. M.; Morton, C. M.; Zwar, R. A.; Murray, S. S.; O’Leary, P. D.; Hughes,
R. A. J. Biol. Chem. 2008, 283, 33375.
11. Ryden, M.; Murray-Rust, J.; Glass, D.; Ilag, L. L.; Trupp, M.; Yancopoulos, G. D.;
McDonald, N. Q.; Ibanez, C. F. EMBO J. 1995, 14, 1979.
12. Berger, H.; Fechner, K.; Albrecht, E.; Niedrich, H. Biochem. Pharmacol. 1979, 28,
3173.
13. Vermeirssen, V.; Deplancke, B.; Tappenden, K. A.; Van Camp, J.; Gaskins, H. R.;
Verstraete, W. J. Pept. Sci. 2002, 8, 95.
14. Lindmark, T.; Kimura, Y.; Artursson, P. J. Pharmacol. Exp. Ther. 1998, 284, 362.
15. Witt, K. A.; Gillespie, T. J.; Huber, J. D.; Egleton, R. D.; Davis, T. P. Peptides 2001,
22, 2329.
l
l
loaded onto a C18 ‘Rocket’ column (AlltechTM) and eluted using a
linear gradient ranging from 100% solution A (0.1% TFA in H2O)
to 100% solution B (0.1% TFA in MeCN) over 16 min. The amount
of peptide present at each time point was quantified as the ‘area
under the curve’ for each compound eluted. Integrated peak areas
for peptides were then expressed as a ratio of the internal standard
(4-isopropylbenzyl alcohol; 0.05% (v/v)), before being expressed as
a percentage of the ratio obtained at t = 0. Each experiment was
performed three times. Before grouping of data from each experi-
ment, parametric one-way analysis of variance (ANOVA) was car-
ried out to ensure variation of sample means between
experiments was not statistically significant. Grouped data was
then expressed as mean standard error of the mean (SEM) and
plotted against time.
16. Adessi, C.; Soto, C. Curr. Med. Chem. 2002, 9, 963.
17. Asada, H.; Douen, T.; Waki, M.; Adachi, S.; Fujita, T.; Yamamoto, A.; Muranishi,
S. J. Pharm. Sci. 1995, 84, 682.
18. Wong, A. K.; Ross, B. P.; Chan, Y. N.; Artursson, P.; Lazorova, L.; Jones, A.; Toth, I.
Eur. J. Pharm. Sci. 2002, 16, 113.
5.5. Membrane permeability experiments
19. Futaki, S.; Goto, S.; Sugiura, Y. J. Mol. Recognit. 2003, 16, 260.
20. Toth, I.; Malkinson, J. P.; Flinn, N. S.; Drouillat, B.; Horvath, A.; Erchegyi, J.; Idei,
M.; Venetianer, A.; Artursson, P.; Lazorova, L.; Szende, B.; Keri, G. J. Med. Chem.
1999, 42, 4010.
21. Egleton, R. D.; Mitchell, S. A.; Huber, J. D.; Janders, J.; Stropova, D.; Polt, R.;
Yamamura, H. I.; Hruby, V. J.; Davis, T. P. Brain Res. 2000, 881, 37.
22. Hadley, M. E.; al-Obeidi, F.; Hruby, V. J.; Weinrach, J. C.; Freedberg, D.; Jiang, J.
W.; Stover, R. S. Pigment Cell Res. 1991, 4, 180.
Peptides were examined for their ability to penetrate biological
membranes using the so-called ‘Caco-2 model’ of membrane pas-
sage.33 Experiments were based on those previously described.30
In brief, monolayers were prepared by seeding Caco-2 cells (pas-
TM
sage 82–96) onto Transwell polycarbonate membrane supports
(0.45
Monolayer integrity (i.e., tight junction formation) was confirmed
l
m pore size, 1.13 cm2) and cultivated for 24–25 days.
23. al-Obeidi, F.; Hruby, V. J.; Yaghoubi, N.; Marwan, M. M.; Hadley, M. E. J. Med.
Chem. 1992, 35, 118.