722 H. Brunner et al.
spectroscopy, elemental analysis and nuclear magnetic resonance
(
NMR).
1
H-NMR data. Compound 2·HCl (CD OD):
␦
0.88 (t, 3H, CH3),
.34 (m, 6H, CH ), 1.64 (m, 2H, CH ), 2.63 (AA of AA BB sys-
system, 2H, CH ), 4.0 (s, 2H,
3
1
Ј
Ј
Ј
2
2
tem, 2H, CH ), 2.84 (BB
Ј
of AA
Ј
BB
Ј
2
2
CH ), 4.24 (t, 2H, CH ).
2
2
Compound 3·HCl (CD OD):
␦
2.63 (AA
Ј of AAЈBBЈ system, 2H,
3
CH ), 2.84 (BBЈ of AAЈBBЈ system, 2H, CH ), 4.0 (s, 2H, CH ),
2
2
2
5
.1 (s, 2H, CH ), 7.37 (m, 5H, CH).
2
Compound 4·HCl (CD SOCD ):
␦ 1.33 (m, 6H, CH ), 1.76 (m,
3
3
2
4
H, CH ), 2.63 (AA
Ј
of AAЈBBЈ system, 2H, CH ), 2.84 (BBЈ of
2
2
AA
(
Ј
BB
broad peak, NH3).
Compound 5·HCl (CD SOCD ):
Ј
system, 2H, CH ), 4.0 (s, 2H, CH ), 4.68 (m, 1H, CH), 8.29
2
2
␦
0.87 (m, 6H, CH ), 1.15 (d,
3
3
3
3
H, CH ), 1.28 (m, 1H, CH), 1.55 (m, 2H, CH ), 2.55 (AA
Ј
of
3
2
AA
Ј
BB
Ј
system, 2H, CH ), 2.83 (BB
Ј
of AA
Ј
BB
Ј
system, 2H, CH2),
2
3
.93 (s, 2H, CH ), 4.68 (m, 1H, CH), 8.29 (broad peak, NH ).
2
3
Compound 6·HCl (CD OD):
␦
2.63 (AA
Ј
of AAЈBB
Ј
system, 2H,
3
CH ), 2.84 (BB
Ј
of AA
Ј
BB
Ј
system, 2H, CH ), 3.65 (s, 3H, CH ),
2
2
3
4
.0 (s, 2H, CH2).
Compound 7 (CD SOCD ):
␦
1.88 (s, 3H, CH ), 2.63 (AA
Ј
of
3
3
3
AAЈBBЈ system, 2H, CH ), 2.84 (BBЈ of AAЈBBЈ system, 2H, CH2),
2
3
.65 (s, 3H, CH ), 4.0 (s, 2H, CH ), 8.2 (m, 1H, NH).
3 2
Cell lines. Three human colonic adenocarcinoma cell lines with
various differentiation were used to represent the spectrum of cel-
lular changes seen in precancerous lesions and manifest tumors. A
colonic fibroblast cell line (CCD18) was added as the phenotype of
normal stroma. The cell lines CaCo (well differentiated) [G1–2]
and HT29 (moderately well differentiated) [G2] were maintained in
Dulbecco modified Eagle medium (Sigma Chemical Co., Deisen-
2
Figure 1. Structure of ALA 1 and synthesized ALA esters 2–7.
hofen, Germany) supplemented with 10% (CaCo ) or 5% (HT29)
2
fetal calf serum (FCS) (Sigma), and SW480 (low differentiated)
[G3–4] was maintained in Roswell Park Memorial Institute 1640
medium (Biochrom, Berlin, Germany) supplemented with 5% FCS,
lester 5. Reduced hydrophilicity of ALA esters (hydrochlo-
ride salts) could be achieved by protecting the amino group.
Such derivatives, being more lipophilic, should pass through
cell membranes easily. After cleavage of the protecting
group by unspecific amidases, they should induce high PPIX
levels. N-acetyl-ALA-methylester 7 and ALA-methylester 6
were synthesized to investigate these effects.
The present study examines the effects of ALA esters and
the effect of protecting the amino group on ALA-induced
PPIX accumulation in differently graded human colon car-
cinoma cell lines by increasing the lipophilicity. After a first
screening of compounds by measuring PPIX levels in ade-
nocarcinoma cells using flow cytometry, MTT tests were
performed to ensure that ALA derivatives showed no dark
toxicity. Compounds inducing high PPIX levels were further
investigated by measuring the concentration- and time-de-
pendent PPIX accumulation in differently graded adenocar-
cinoma cell lines and a human fibroblast cell line. Michae-
lis–Menten kinetics of concentration- and time-dependent
PPIX formation were determined.
1
%
L
-glutamine and 1% sodium pyruvate (GIBCO, Eggenstein, Ger-
many), and they were kept at 37 C in a humidified atmosphere con-
Њ
taining 5% carbon dioxide. The human colonic fibroblast cell line
CCD18 was maintained in modified Eagle medium, 10% FCS. Cells
were subcultured before reaching plateau growth, using trypsin–
ethylenediaminetetraacetic acid (0.1:0.04, %/%) (GIBCO) in phos-
phate-buffered saline (PBS) (Biochrom). Because of better simula-
tion of the in vivo situation, experiments were performed with po-
4
Ϫ2
stconfluent cells (cell density/growth period—SW480: 1
ϫ 10 cm /
4
Ϫ
2
4
Ϫ
2
7
days; HT29: 5
ϫ
10 cm /7 days; CaCo : 2.8
ϫ
10 cm /7 days;
ϫ 10 cm /8 days). Growth states were validated by
2
4
Ϫ2
CCD18: 1
flow cytometric DNA measurements.
Incubation. Stock solutions of ALA and ALA esters were pre-
pared in deionized water at a concentration of 0.6 mol/L and stored
at
Ϫ20ЊC. Depending on the prodrug concentration to be applied
(0.012–0.6 mmol/L), for each experiment the stock solution was
diluted in culture medium without FCS. Before addition of ALA
solutions the cell layer was rinsed with PBS to remove the remaining
FCS. Cells were incubated with incubation times of 5–180 min at
37ЊC in the dark, using a constant incubation volume. In subsequent
handling, care was taken to avoid exposure to light.
Determination of ALA-induced fluorescence by flow cytometry.
Following incubation with ALA, cells were trypsinized, removed
from 6-well plates and resuspended in medium without FCS to yield
MATERIALS AND METHODS
5
a cell concentration of about 5
ϫ
10 cells/mL. The cellular fluo-
Synthesis of ALA derivatives. All syntheses were carried out under
a nitrogen atmosphere. Chemicals were obtained from Merck
rescence was quantified by a FACScalibur cytometer (Becton-Dick-
inson, Heidelberg, Germany). Fluorescence of ALA-induced PPIX
was initiated by an argon ion laser emitting at 488 nm and collected
by a photomultiplier tube after passing through a 670 nm longpass
(Darmstadt, Germany). ALA esters 2–6 (Fig. 1) were prepared by
reacting ALA with the corresponding alcohol in the presence of
thionyl chloride, with the ALA esters as the hydrochloride salts (11).
An excess of the dry alcohol (20 mL) was cooled on ice, and 3.0
mL (41.4 mmol) of freshly distilled thionyl chloride was added drop-
wise. Then, ALA (0.5 g, 3 mmol) was added to the solution and
filter. The flow rate was adjusted to about 2000 events/s, and 2
ϫ
4
10 events were recorded for each sample. The same instrument
settings were used for all experiments. The stability of the cytometer
was maintained by weekly calibration using the AUTOCOMP soft-
ware (Becton-Dickinson). Data were recorded and analyzed with the
CellQuest program (Becton-Dickinson). Debris and cell aggregates
were excluded from analysis using forward and side scatter signals.
In order to compensate for the autofluorescence, the mean fluores-
cence of ALA- and ALA ester–incubated cells was determined by
dividing through the mean fluorescence of sham-treated control cells
in each experiment. In order to relate fluorescence intensities with
stirred at 60ЊC for 5 h. The excess of alcohol was evaporated in
vacuo. After recrystallization from methanol–diethylether at
Ϫ20ЊC
pure products were obtained as white hydrochloride salts.
N-acetyl-ALA-methylester 7 was synthesized according to the
method of A. Pfaltz (12). After purification by chromatography and
recrystallization from ether–pentane the pure product was obtained.
Identification of the synthesized compounds was based on mass